Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies

Marina Bisia; Carlos Alberto Montenegro-Quinoñez; Peter Dambach; Andreas Deckert; Olaf Horstick; Antonios Kolimenakis; Valérie R Louis; Pablo Manrique-Saide; Antonios Michaelakis; Silvia Runge-Ranzinger; Amy C Morrison

doi:10.1371/journal.pntd.0011591

. 2023 Aug 31;17(8):e0011591. doi: 10.1371/journal.pntd.0011591

Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies

Marina Bisia ¹, Carlos Alberto Montenegro-Quinoñez ^2,³, Peter Dambach ², Andreas Deckert ², Olaf Horstick ², Antonios Kolimenakis ¹, Valérie R Louis ², Pablo Manrique-Saide ⁴, Antonios Michaelakis ¹, Silvia Runge-Ranzinger ², Amy C Morrison ^5,^*

Editor: Andrea Morrison⁶

PMCID: PMC10499269 PMID: 37651473

Abstract

Background

After the unprecedented Zika virus (ZIKV) outbreak in the western hemisphere from 2015–2018, Aedes aegypti and Ae. albopictus are now well established primary and secondary ZIKV vectors, respectively. Consensus about identification and importance of other secondary ZIKV vectors remain. This systematic review aims to provide a list of vector species capable of transmitting ZIKV by reviewing evidence from laboratory vector competence (VC) studies and to identify key knowledge gaps and issues within the ZIKV VC literature.

Methods

A search was performed until 15^th March 2022 on the Cochrane Library, Lilacs, PubMed, Web of Science, WHOLIS and Google Scholar. The search strings included three general categories: 1) “ZIKA”; 2) “vector”; 3) “competence”, “transmission”, “isolation”, or “feeding behavior” and their combinations. Inclusion and exclusion criteria has been predefined and quality of included articles was assessed by STROBE and STROME-ID criteria.

Findings

From 8,986 articles retrieved, 2,349 non-duplicates were screened by title and abstracts,103 evaluated using the full text, and 45 included in this analysis. Main findings are 1) secondary vectors of interest include Ae. japonicus, Ae. detritus, and Ae. vexans at higher temperature 2) Culex quinquefasciatus was not found to be a competent vector of ZIKV, 3) considerable heterogeneity in VC, depending on the local mosquito strain and virus used in testing was observed. Critical issues or gaps identified included 1) inconsistent definitions of VC parameters across the literature; 2) equivalency of using different mosquito body parts to evaluate VC parameters for infection (mosquito bodies versus midguts), dissemination (heads, legs or wings versus salivary glands), and transmission (detection or virus amplification in saliva, FTA cards, transmission to neonatal mice); 3) articles that fail to use infectious virus assays to confirm the presence of live virus; 4) need for more studies using murine models with immunocompromised mice to infect mosquitoes.

Conclusion

Recent, large collaborative multi-country projects to conduct large scale evaluations of specific mosquito species represent the most appropriate approach to establish VC of mosquito species.

Author summary

The mosquitoes Aedes aegypti and Ae. albopictus are known to transmit Zika virus (ZIKV) but it is important to identify other potential secondary vectors. We conducted a systematic review of the literature to answer this question. We searched four databases (PubMed, Lilacs, Cochrane Library Web of Science), WHOLIS and Google Scholar using different combinations of Zika, Aedes, Culex, vector, or competence in the title/abstract, up to March 2022. Most of the studies reviewed were of high quality methodologically, but the methods were different making it hard to compare them. There is a need for standardization to better interpret these studies and make appropriate recommendations. Secondary vectors of ZIKV with evidence of low transmission rates comparable to primary vectors are Ae. japonicus, Ae. detritus and Ae. vexans at higher temperatures. Culex quinquefasciatus was not found to be a competent vector of ZIKV. Future research should focus on well-defined and established experimental approaches (midguts/bodies for infection, legs+wings/heads for dissemination and the use of murine models/other artificial feeding systems). Importantly, development of large collaborative multi-country projects are needed to conduct large scale evaluations of specific mosquito species with common protocols to appropriately address the inherent geographic variation in both mosquito and ZIKV strains.

Introduction

The emergence of Zika virus (ZIKV) transmission and associated neurological and congenital consequences in the western hemisphere in 2015 resulted in a World Health Organization declaration of a “Public Health Emergency of International Concern” for most of 2016 [1,2]. This emergency exposed significant knowledge gaps, not only about vector competence (VC) for “known” vectors Aedes aegypti and Ae. albopictus, but also on potential “unknown” secondary vectors. Following the Zika outbreak several review studies employing different search methodologies reported ZIKV isolations and laboratory-based VC studies for known and suspected mosquito vectors. In a scoping review Wadell and Grieg [3] included studies on 45 mosquito species of which 18 were positive for ZIKV from 1956 to 2015 in Africa and Asia. Epelboin et al. [4] included studies of 53 mosquito species across eight genera in a systematic review focused on vectors that included work published through August 2017. While these two studies were published about 18 months apart, the number of species tested for VC increased from 8 to 22. In a 2018 expert opinion review, Boyer et al. [5] summarized laboratory studies and cataloged the mosquito species from which Zika virus was isolated.

Vector competence is defined as the ability of a mosquito to become infected, allow virus amplification, and subsequently transmit a pathogen to another vertebrate host [6,7]. It represents all the intrinsic factors (genetic, physical, physiological, and immunological) underlying virus propagation in the mosquito; a virus’ journey through the mosquito that includes successful replication of the virus in the mosquito’s midgut epithelium, navigation across the midgut wall, dissemination to the salivary gland cells, and secretion into saliva. The success of a vector to transmit a pathogen also includes additional factors like longevity and host preferences, as well as extrinsic factors associated with behavioral and ecological characteristics of the species which increase exposure to mosquito bites; which together interplay and are defined as vectorial capacity [6]. Thus, vector competence is a necessary but not sufficient requirement to characterize definitively a vector species as epidemiologically relevant and that contributes significantly to the natural maintenance and transmission of a specific virus. For example, Ae. aegypti has many characteristics that enhance its vectorial capacity: larval development sites and adult resting sites strongly associated with human habitats in urban areas, highly anthropophilic biting behavior, females taking multiple blood meals during a single gonotrophic cycle, and diurnal feeding behavior [8–10].

Vector competence experiments generally include the following steps: 1) exposure to virus via an artificial blood feeder, or feeding on an infected live host (for Zika an immunocompromised mouse model) (number of engorged mosquitoes that were tested [#tested]), 2) testing of whole mosquito bodies, midguts, or carcasses to measure infection (the number of mosquitoes testing positive for virus or viral RNA are the #infected [#inf.]) 3) testing heads, legs+wings, salivary glands/ovaries to measure dissemination (the number samples testing positive for virus or viral RNA are mosquitoes with viral dissemination [#dissem.]), and 4) testing saliva or exposure of live animals to virus infected mosquitoes to measure transmission (number of samples testing positive for virus or viral RNA are mosquitoes that were able to transmit [#transm.]) [7]. Researchers present rates using two approaches where different denominators are employed: stepwise and cumulative [7]. Infection rate is the same for both approaches, #inf./#tested. Cumulative rates, the most informative for evaluating VC are #dissem./#tested and #transm./#tested for dissemination and transmission, respectively. Stepwise rates reveal where potential genetic barriers exist use smaller denominators for dissemination (#dissem./#inf) and transmission (#transm/#dissem). Terminology for these rates varies among authors and overtime but for the purposes of this review we use these recently proposed terms designed to establish minimum reporting standards for VC experiments [7]. The cumulative transmission rate alone implicates a species as a vector.

The challenges associated with assessment of VC studies for ZIKV are similar to those identified for dengue virus VC studies e.g., primarily the observation of significant variation in competence among geographically isolated vector populations [11,12]. The combination of virus and mosquito strains tested, assays used to detect virus (RNA compared to infectious virus), blood feeding and experimental methods, and parameters assessed (both stepwise and cumulative infection, dissemination, transmission rates) are all critical for assessment of VC [4,7,13].

Interest in ZIKV vectors has been characterized by an urgent response to outbreaks, first in the South Pacific [14–17] and then in the western hemisphere as described above. Prior to that, a forest cycle between ZIKV-non-human primates- canopy mosquitoes had been recognized since the first ZIKV was isolated in Uganda from a rhesus monkey and Ae. africanus in the 1940s [18]. Thus, much of our knowledge pre-dating the South Pacific epidemics originated from forest-based studies directed primarily at yellow fever (YF) [19–29]. During the peak of the ZIKV outbreak in the western hemisphere from 2015–2018, there was an increase of VC studies (particularly short communications) testing an array of mosquito species often from insectary-readily available colonies. During this period, some publications [30–32] implicated Cx. quinquefasciatus, a cosmopolitan species often associated with wastewater and abundant in urban settings, as a vector of ZIKV with potentially dramatic epidemiological consequences. Also important was the assessment of a variety of Aedes species, including the invasive Ae. japonicus and others with more restricted geographic distributions. After 2017, ZIKV transmission has decreased world-wide, and a period where VC studies with more complex and through study designs emerged.

After over 5 years since the peak of the Zika virus outbreak, there is an opportunity to evaluate and update all the evidence for possible secondary ZIKV vectors. There is now ampler and well-established evidence that Ae. aegypti and Ae. albopictus are vectors for ZIKV to humans [4,5,13], but still, no clear consensus about other possible vectors. We have assessed the latter in this systematic review and focused on laboratory VC studies, because they are a requirement to evaluate if a potential vector species can transmit an arbovirus, with the overall aim to identify key gaps/issues in the current literature to provide a concrete list of vector species capable of transmitting ZIKV.

Methods

Search strategy, databases, and search terms

Our research question formulated was “Is there evidence of vector competence for ZIKV for mosquito species in addition to Ae. aegypti and Ae. albopictus?”. Our review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [33]. Articles were identified by searching electronic databases until the 15^th of March 2022. The articles were extracted from: four databases, Cochrane Library (https://www.cochranelibrary.com/), Latin American and Caribbean of Health Sciences Information System- LILACS (https://lilacs.bvsalud.org/en/), PubMed (https://pubmed.ncbi.nlm.nih.gov/) and Web of Science (https://www.webofknowledge.com); one register, World Health Organization (WHO) library catalogue (WHOLIS, https://asksource.info/resources/wholis); and Google Scholar (https://scholar.google.com/). The search strings included three general categories: 1) “ZIKV”; 2) “vector”; 3) “competence” and their combinations using free text terms and medical subject headings (MeSH) terms when applicable (e.g., PubMed) for: Zika, Aedes, Culex, vector, or competence in the title/abstract. After piloting our initial search string and determined that some relevant articles based on previous reviews were not retrieved, we added the following search terms: transmission, isolation, and feeding behavior. Our principal search strings using MeSH terms are as shown in Box 1 and were adjusted to each search engine.

Box 1. Search string utilized to extract articles in Cochrane Library, Latin American and Caribbean of Health Sciences Information System (LILACS), PubMed, Web of Science, World Health Organization library catalogue (WHOLIS) and Google scholar. Medical subject headings (MeSH) terms were used when applicable (e.g., PubMed).

Zika*[Title/Abstract] AND [[[Aedes*[Title/Abstract] OR [Aedes*[MeSH Terms]] OR [[Culex*[Title/Abstract] OR [Culex*[MeSH Terms]] OR Vector*[Title/Abstract] AND Transmission*[Title/Abstract]

Zika*[Title/Abstract] AND [[Aedes*[Title/Abstract] OR [Aedes*[MeSH Terms]] OR [[Culex*[Title/Abstract] OR [Culex*[MeSH Terms]] OR Vector*[Title/Abstract] AND Isolation*[Title/Abstract]

Zika*[Title/Abstract] AND [[Aedes*[Title/Abstract] OR [Aedes*[MeSH Terms]] OR [[Culex*[Title/Abstract] OR [Culex*[MeSH Terms]] OR Vector*[Title/Abstract] AND [[Feeding behavior*[Title/Abstract] OR [behavior, feeding*[MeSH Terms]]

Zika*[Title/Abstract] AND [[Aedes*[Title/Abstract] OR [Aedes*[MeSH Terms]] OR [[Culex*[Title/Abstract] OR [Culex*[MeSH Terms]] OR Vector*[Title/Abstract] AND Competence*[Title/Abstract] OR Competence*[MeSH Terms] OR Transmission*[Title/Abstract] OR Isolation*[Title/Abstract] OR [Feeding Behavior*[Title/Abstract] OR [behavior, feeding*[MeSH Terms]]

We used Zotero (https://www.zotero.org/) to identify duplicate articles, and all the extracted articles were processed using the Rayyan platform (www.rayan.com) where two members of the research team (MB, CAMQ) screened each article title/abstract to identify ZIKV VC studies that represented primary research. The search was performed in English. All articles were examined by both researchers before inclusion and a third researcher and subject expert (ACM) was asked to assist with the final decision for articles without consensus. Exclusion criteria (Fig 1) were:

Systematic or literature reviews
Opinion papers
Studies focused on mathematical modelling
Vaccine development
Case reports
Field-based vector incrimination studies (isolation of virus from field-collected mosquitoes)
Papers regarding workshops
Meeting results

Inclusion criteria (Fig 1) were:

Laboratory evaluation of vector competence
Included vector species other than Ae. aegypti and Ae. albopictus
Published in a peer reviewed journal

Data were extracted (MB) and entered into extraction forms, including author, title, journal, publication date, and study design. Additional sections broadly followed content analysis methods, using categories as these emerged during analysis of the results [34], such as mosquito species and/or strains studied, type of mosquito used (colony or field), virus strains and doses administered, infection method used (blood feeding device, direct feeding on mouse), assays used to detect virus or RNA, strategy used to measure mosquito infection (positive bodies or midguts), dissemination (positive heads, legs, wings, salivary glands, ovaries, etc.), and most importantly, ability to transmit virus (saliva testing or transmission to mouse), as well as the number of days post-exposure that mosquitoes were processed or analyzed, results, limitation mentioned by authors, and conclusions. The data extraction forms were reviewed and checked for accuracy by ACM and CAMQ.

Quality assessment

We developed a grading tool to assess the quality of our evaluated articles, using a checklist developed from Reporting of Observational Studies in Epidemiology (STROBE) [35] and Strengthening of the Reporting of Molecular Epidemiology for Infectious Diseases (STROME-ID) [36] criteria (S1 Table). The STROBE checklist was enriched by the authors to be in accordance with the procedures followed in the papers attempting to include score categories for all relevant STROBE and STROME-ID criteria (S1 Table). The checklist was finally composed of 33 items with a possible maximum of 38 points. The evaluation, however, was heavily weighted on 20 items evaluating study methodology and a “methodology score” was additionally computed that comprised a maximum of 24 possible points. The items included a clear description of the virus and mosquito strains used in the experiments, the type of assay used to detect Zika virus or RNA in the mosquitos, if infection, dissemination, and transmission were appropriately measured, and the number of mosquitoes used to evaluate these parameters and if replicate experiments were conducted. Some items that comprised this portion of score had higher maximum points. The remaining items, not part of the methodology score emphasized proper reporting and included the term “vector competence” in the title, clear and accurate abstract and objectives, presentation of key study results in relation to study objectives, appropriate description and use of statistical analysis, transparent discussion of study limitations, and appropriate interpretation and generalizability of the study results. Two researchers (MB and ACM) scored each of the 45 included articles independently. If the scores did not match, all 38 scores were compared and where differences were observed discussed between the two researchers. For most scores that were not concordant, the article was reviewed to confirm that each researcher had correctly extracted the information necessary to provide a score (e.g., type of laboratory assay, mosquito colony history, virus passage history, number of virus or mosquito strains used). Scores were adjusted based on careful review of the manuscript and the score justification provided by each researcher. For some scores that were more subjective (e.g., clearly written abstract, adequate discussion of study limitation) the researchers discussed their scores, compared them to other articles to standardize their approach to each question. Each score was discussed until both researchers agreed on a single score. No studies were excluded after the quality assessment, but the analysis and the report of the results consider this quality assessment.

Results

Descriptive results

We retrieved 8,986 articles from four databases, one register, and one searchable website. We identified 6,637 as duplicates that were removed prior to screening (Fig 1). We screened the title and abstract of the remaining 2,349 articles and from those 2,246 were excluded based on the inclusion/exclusion criteria: of the 103 articles retrieved, 58 only evaluated Ae. aegypti or Ae. albopictus, finally leaving 45 papers that were included in our analysis of secondary vector species for ZIKV.

The 45 included peer-reviewed articles, summarized in Table 1, came from 20 journals, most with a focus on emerging infections (Emerging Infectious Diseases [3], Emerging Microbes and Infections [5], Eurosurveillance [3]), infectious and tropical diseases (American Journal of Tropical Medicine and Hygiene [2], BMC Infectious Diseases [1], Mem. Insti. Oswaldo Cruz [1], PLOS Neglected Tropical Diseases [9], Parasites and Vectors [4], Vector-borne and Zoonotic Diseases [1]), virology or microbiology (Frontiers in Microbiology [1], Journal of General Virology [1], Mbio [1], MDPI-pathogens [2], Virology Journal [1], Viruses [2]), entomology (Journal of the American Mosquito Association [1], Journal of Medical Entomology [3]), and three general journals (Nature [2], PeerJ [1], Proceedings B [1]).

Table 1. Evidence table, in chronological order from 2014 to 2021.

ZIKV = Zika virus, USUV = Usutu virus, VC = vector competence, VD = Virus/RNA detection in field collected mosquitoes, IT = Intrathoracic inoculations (examining upstream barriers to transmission), dpi = days post infection (includes manuscripts reporting dpe = days post exposure), RT = Room temperature. Infection Rate (IR, [#inf./#tested]) is defined as the percentage of mosquitoes containing virus in bodies or midguts (number positive/number tested). Terminology used for dissemination and transmission are expressed as cumulative (C) or stepwise (S) rates depending on the experimental design. For dissemination, we use cumulative dissemination rate (CDR, [#dissem./#tested]) or stepwise dissemination rate (SDR, [#dissem./#inf]), defined as the percentage of mosquitoes containing virus in head, legs+wings, or salivary glands/ovaries (number positive/number of engorged mosquitoes tested for infection [CDR] or number positive /number of infected mosquitoes [SDR]). For transmission, we use cumulative transmission rate (CTR, [#transm./#tested]) or stepwise transmission rate (STR, [#transm./#dissem.]), defined as the number of mosquitoes with virus in saliva or transmitting ZIKV to a mouse (number positive/number of engorged mosquitoes tested [CTR] or number positive/number disseminated infections [STR]). For authors that emphasized salivary gland testing, CSGR = cumulative salivary gland positivity rate is used. For the quality assessment TS = Total Score, MS = Methodology Score, QAS = percentile in quantile analysis among all articles scored.

	Reference (Article Type)	Study Objectives	Mosquitoes/Virus/ Temperature	Results	Quality Assessment (QAS)
[37]	Lederman JP et al. PLoS NTD 2014 doi:10.1371/journal.pntd.0003188 Aedes hensilli as a potential vector of Chikungunya and Zika viruses (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) VD Outbreak Investigation	Ae. hensilli (F12-15) MR766 4.9 log₁₀ PFU/ml 5.7 log₁₀ PFU/ml 5.9 log₁₀ PFU/ml 28°C	Results of a late outbreak investigation on YAP island. Carried out 1 week of diurnal and nocturnal collections including larval surveys and adult mosquitoes using light and gravid traps, as well as household aspirations. Ae. hensilli was the most abundant species, followed by Cx. quinquefasciatus, but no virus was isolated from field caught mosquitos. A colony of Ae. hensilli was established. F_12-15 mosquitoes were orally infected with MR766 ZIKV strain at three doses. Infection (bodies) and dissemination (heads) were evaluated at 8 dpi. IR at lowest dose was 7% (1/14) compared to higher doses = 84% (47/56). SDR was observed only at the two higher doses (19%, 9/47).	QAS: < 25% (TS = 20, MS = 11) Strengths: Dose Response evaluated, locally derived colony. Weakness Low sample sizes, only evaluated 8 dpi, rather than the standard 14 dpi.
[38]	Diagne CT et al. BMC Infect Dis 2015;15: 492. doi:10.1186/s12879-015-1231-2 Potential of selected Senegalese Aedes spp. mosquitoes (Diptera: Culicidae) to transmit Zika virus (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) Forest cycle	Ae. unilineatus, Ae. vittatus, Ae. luteocephalus Virus: ArD 128000, ArD 132912, ArD 157995, ArD 165522 (mosquito origin); HD 78788, MR766 (human origin) 6–7 log₁₀ PFU/ml 27±1°C, 80±5% RH	Orally infected F₁ Ae. aegypti, Ae. unilineatus, Ae. vittatus, and Ae. luteocephalus mosquitos with 5 ZIKV strains (3 and 2 of mosquito and human origin, respectively) at doses ranging from 6–7 log₁₀ PFU/ml. IR (bodies), SDR (heads), and STR (saliva) were evaluated at 5, 10, and 15 dpi using qPCR. VC parameters varied with virus strain used for each species evaluated. Across strains and doses Ae. unilineatus (IR = 56/300, SDR = 3/56, STR = 0/3) was unable to transmit ZIKV whereas Ae. vittatus, (IR = 37/56, SDR = 10/37, STR = 2/10) and Ae. luteocephalus (IR = 45/60, SDR = 19/45, STR = 50%) were able to transmit virus strains isolated from monkey/human sera (MR766, HD78788).	QAS: > 50% (TS = 32, MS = 20) Strengths: used F1 mosquitoes and multiple ZIKV strains, range of dpi evaluated. Weakness: Used PCR only to test mosquitoes. Saliva testing for Ae. luteocephalus not clear.
[39]	Aliota MT et al. Emerg Infect Dis 2016;22:1857–1859. http://dx.doi.org/10.3201/eid2210.161082 Culex pipiens and Aedes triseriatus mosquito susceptibility to Zika virus (Letter to Editor)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) Murine Model	Cx. pipiens, Ae. triseriatus, Ae. aegypti, Ae. albopictus Asian linage PRVABC59 4.7 log₁₀ PFU/ml 6.0 log₁₀ PFU/ml 6.8 log₁₀ PFU/ml No rearing temperature provided.	Laboratory studies, using ZIKV Ifnar-/- mice, Asian linage PRVABC59, at 14 dpi (3 replicates), Cx. pipiens, Ae. triseriatus, Ae. aegypti, Ae. albopictus (all from colonies, F>>10) collected bodies, legs, saliva, to evaluate IR, CDR, and CTR. Cx. pipiens was refractory to infection. Ae. triseriatus (IR_{6.8log10 PFU/ml} = 4/13) became infected at high titers but showed no dissemination or transmission. Ae. albopictus infection, dissemination, and transmission was low compared to Ae. aegypti and dose dependent. Ae. aegypti evaluated at highest dose had IR = 17/17, CDR = 12/17, and CTR = 4/17.	QAS: < 25% (TS = 24, MS = 13) Strengths: All samples screened by plaque assay (infectious assay). Infected via animal feeding. Range of doses. Weakness: Laboratory mosquito strains (>15 yrs), single virus strain; short format. Rearing temperature not reported.
[40]	Amraoui F. et al. Euro Surveill 2016;21. doi:10.2807/1560-7917.ES.2016.21.35.30333 Culex mosquitoes are experimentally unable to transmit Zika virus. (Rapid Communication)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) Temperate species	Cx. quinquefasciatus (San Joaquin Valley, CA, 1950) Cx. pipiens (Tunisia, 2010) NC201-5132, New Caledonia 2014 7.2 log₁₀ PFU/ml 28°C	VC assays using laboratory strains of Cx. quinquefasciatus and Cx. pipiens orally infected with single ZIKV strain at a single dose, evaluated at 3, 7, 14, and 21 dpi. The number of virus particles ingested were titrated in recently engorged mosquitoes were 4.8 log₁₀ PFU/ml for Cx. pipiens, and 5.0 log₁₀ PFU/ml for Cx. quinquefasciatus. IR for Cx. pipiens was 2% (1/48) at 3 dpi, 6% (3/47) at 7 dpi, 0% (0/47) at 14 dpi, and 13% (6/46) at 21 dpi. In contrast, for Cx. quinquefasciatus IR was 0% (0/42) at 3 dpi, 2% (1/47) at 7 dpi, 17% (7/41) at 14 dpi, and 13% (5/40) at 21dpi. Only Cx. quinquefasciatus was able to disseminate the virus (CDR_14dpi = 1/41, CDR_21dpi = 3/40) and neither species was able to transmit ZIKV up to 21 dpi.	QAS: 25% (TS = 26, MS = 14) Strengths: All samples screened by plaque assay (infectious assay); range dpi evaluated; intrathoracic injections provided additional support for Culex not being a vector. Weakness: Single virus strain, use of old laboratory colonies. No Ae. aegypti control
[41]	Boccolini D et al. Euro Surveill 2016;21. doi:10.2807/1560-7917.ES.2016.21.35.30328 Experimental investigation of the susceptibility of Italian Culex pipiens mosquitoes to Zika virus infection (Rapid Communication).	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) Temperate species	Ae. aegypti Cx. pipiens ZIKV H/PF/2013 (Origin human French Polynesia 2013) 6.46 log₁₀ PFU/ml 26±1°C; 70% RH 14 h:10 h L:D	Culex pipiens, collected from Rome, Italy in 2015 (generation not provided) and Ae aegypti, from a laboratory colony from Reynosa, Mexico (1998, F>>10) were orally infected with ZIKV at a single dose and evaluated at 0, 3, 7, 10, 20, 24 dpi. Aedes aegypti, IR: 0 dpi = 8/8, 7 dpi = 6/12, 14 dpi = 4/8, 20d = 4/10; CDR: 7 dpi = 6/12, 14 dpi = 4/8, 20 dpi = 3/10; CTR: 7 dpi = 2/12, 14 dpi = 3/8, 20 dpi = 3/10. Cx. pipiens were 100% refractory.	QAS: 25% (TS = 26, MS = 14) Strengths: Evaluated range of dpi, used local Culex strain. Weakness: Used PCR only to test mosquitoes, single ZIKV strain and dose, single Culex strain.
[42]	Fernandes RS et al. PLoS NTD 2016 doi:10.1371/journal.pntd.0004993 Culex quinquefasciatus from Rio de Janeiro is not competent to transmit the local Zika virus (Research article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) Site with active ZIKV transmission	Ae. aegypti, Cx. quinquefasciatus Rio-U1 (KU926309), Rio-S1 (KU92630) (Local) 6 log₁₀ PFU/ml 26°C, 70±10% RH 12 h:12 h L:D	Orally infected 4 strains (F1-F3) of Cx. quinquefasciatus from 4 districts, Rio de Janiero, Manguinhos (MAN), Triagem (TRI-colony), Copacabana (COP), Jacarepagua (JAC); Ae. aegypti Urca (URC), Paqueta (PAQ-F2), with 2 local Brazilian strains of ZIKV. Infection (bodies), dissemination (heads), and transmission (saliva) were evaluated at 7, 14, 21 dpi using plaque assay and qPCR. Aedes aegypti infection rates varied with mosquito strain. IR: 7 dpi = 80%, 14–21 dpi = 90–100%; SDR: 7 dpi = 40%, 14–21 dpi = 85–100%; STR_14dpi = 72–97%, CTR_14dpi = 61–93%. For all Culex strains, IRs were negligible to null, and no dissemination was observed.	QAS: ≥ 99% (TS = 35, MS = 22) Strengths: Used multiple mosquito and virus strains, infectious assay, and evaluated a good dpi range. Included Ae. aegypti control. Mosquitoes were recently collected from field. Weakness: none noted
[30]	Guo XX et al. Emerg Microbes Infect 2016;5:e102. doi: 10.1038/emi.2016.102 Culex pipiens quinquefasciatus: a potential vector to transmit Zika virus (Research article)	VC: #inf./#tested (IR) #SG+/#tested (CSGR) #transm./#dissem. (STR) #transm./#tested (CTR) Transmission to neonatal mice in addition to saliva testing	Cx. quinquefasciatus SZ01 (human traveler from Samoa 2016) 5.5 log₁₀ PFU/ml 26±1°C, 75±5% RH 14 h:10 h L:D	Orally infected Cx. quinquefasciatus laboratory strain (Hainan province 2014, generation unknown) with single low passage ZIKV strain at a single dose. Infection (midguts), dissemination (salivary glands/ovaries), and transmission (ZIKV RNA + saliva over number of mosquitoes with disseminated infection), and CTR (ZIKV RNA + saliva over number of blood fed mosquitoes tested). ZIKV exposed mosquitoes allowed to feed on mice. Evaluations at 2,4,5,8,12,16,18 dpi using PCR. IR, 80% at 2 dpi, then oscillates between 10–40%. Salivary gland infection (CSGR) first observed at 2 dpi, peaks at 8 dpi, then decreases. Detection of ZIKV RNA in saliva peaks at 8 dpi, then decreases on 12 and 18 dpi. CTR, 6 dpi = 0%, 8 dpi = 80%, 12 dpi = 10%, 16 dpi = 0%. One day 10 post-exposure to ZIKV infected mosquitos, 8 of 9 infant mice had viral RNA in their brain.	QAS: < 25% (TS = 22, MS = 12) Strength: Transmission to mice is convincing but appears transient. Weakness: No infectious assay, colony material, single virus and mosquito strains. Poor discussion of possible explanation of results. No Ae. aegypti control. Results are inconsistent with biology, looks like transient infections no persistent infection which would be a new paradigm.
[43]	Hall-Mendelin S. et al. PLoS NTD 2016; 10:e0004959 doi:10.1371/journal.pntd.0004959 Assessment of local mosquito species incriminates Aedes aegypti as the potential vector of Zika virus in Australia (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #dissem/#tested (CDR) #transm./#dissem. (STR) #transm./#tested (CTR) Local species Australia	Ae. aegypti, Ae. vigilax, Ae. procax Ae. notoscriptus, Cx. quinquefasciatus, Cx. annulirostris, Cx. sitiens MR766 Dose: 6.5–6.9 log₁₀ TCID₅₀/ml 26°C, 12 h:12 h L:D	Vector competence studies on potential Australian ZIKV vectors: Ae. vigilax, Ae. procax, Cx. annulirostris, Cx. sitiens (F1), Ae. aegypti F4, Townsville; Ae. notoscriptus, Cx. quinquefasciatus F1 (All from Queensland, Australia). Carried oral infections with MR766 prototype virus at a single dose. Ae. notoscriptus, IR_7dpi = 18/25, IR_14dpi = 34/60, SDR_7dpi = 2/18, SDR_14dpi = 6/16, CDR_7dpi = 2/25, CDR_14dpi = 6/60. Ae. procax, IR_14dpi = 2/6, SDR_14dpi = 1/6, CDR_14dpi = 1/2. Ae. vigilax, IR_14dpi = 17/30, SDR_14dpi = 8/30, SDR_14dpi = 8/17. Cx. quinquefasciatus, IR_14dpi = 2/30, SDR_14dpi = 0/30. Cx. annulirostris and Cx. sitiens were completely refractory. No evidence of ZIKV transmission was observed in any species other than Ae. aegypti, the species with infected saliva. Ae. aegypti STR_10dpiI = 3/25, STR_14dpi = 8/30, CTR_10dpiI = 3/7, STR_14dpi = 8/12.	QAS: ≥ 75% (TS = 33, MS = 20) Strengths: Used F1 mosquitoes collected from the field, established infecting dose. Weakness: Used a single ZIKV strain and tested mosquitoes by PCR rather than infectious assay.
[44]	Huang YJ et al. Vector Borne Zoonotic Dis 2016. doi:10.1089/vbz.2016.2058 Culex species mosquitoes and Zika virus (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR)	Cx. pipiens, Cx. quinquefasciatus (Colonized < 2yrs) PRVABC59 7.2 log₁₀ TCID₅₀/ml 28°C, 16 h:8 h L:D	Orally infected 2 laboratory strains of Cx. pipiens from California (F15) and New Jersey (F7) and one strain of Cx. quinquefasciatus (Vero Beach, F7) with ZIKV strain PRVABC59 (low passage) at a single dose. Evaluated infection (bodies) and dissemination (heads) at 7 and 14 dpi. Screened by TCID₅₀ followed by confirmation by PCR. All strains were refractory to infection.	QAS: < 25% (TS = 24, MS = 15) Strengths: Used low passage virus strain. Used infectious assay. Weakness: No Ae. aegypti control, mosquito colonies, single virus strain.
[45]	Richard V et al. PLoS NTD 2016;10:e0005024 doi:10.1371/journal.pntd.0005024 Vector competence of French Polynesian Aedes aegypti and Aedes polynesiensis for Zika Virus (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) VD Evaluated EIP/Kinetics	Ae. aegypti Ae. polynesiensis PFI/251013, 3 passages 7 log₁₀ TCID₅₀/ml 27°C, 75% RH 12 h:12 h L:D	Aedes aegypti and Ae. polynesiensis were suspected to be ZIKV vectors in addition of Ae. aegypti. Laboratory colonies (F16 to F18) were orally infected with a French Polynesian ZIKV strain at a single dose and evaluated at 2, 6, 9, 14, 21 d dpi. Infection (bodies), dissemination (legs), and transmission (saliva) evaluated by TCID₅₀ in C6/36 cells. Ae. aegypti, IR = 85–93% starting 6 dpi, CDR = 18% at 6 dpi, 75% 9 dpi, 85% 14 dpi and 93% at 21 dpi, CTR = 36% at 14 dpi, 73% at 21 d. Ae. polynesiensis, IR_6dpi = 11% (10/95), IR_9dpi = 20% (18/89), IR_9dpi = 36% (24/66); CDR_9dpi = 3% (3/89), CDR_14dpi = 18% (12/66), CTR = 0%. Overall, Ae. aegypti was more susceptible to ZIKV and has more favorable kinetics than Ae. polynesiensis	QAS: > 50% (TS = 31, MS = 19) Strengths: Tested two possible vector strains from French Polynesian ZIKV outbreak, evaluated EIP/Kinetics. Weakness: Used a single virus strain
[46]	Weger-Lucarelli J et al. PLoS NTD 2016; 10:e0005101 doi:10.1371/journal.pntd.0005101 Vector competence of American mosquitoes for three strains of Zika virus (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) Evaluation of freeze thaw cycles. In-Vitro replication, competitive fitness	Ae. aegypti, Cx. tarsalis, Cx, pipiens, Cx. quinquefasciatus PRVABC59 (4 passages) MR766 (149 passages) 41525 (isolated Aedes, 1 passage C6,36; 4 vero cells) 7.2 log₁₀ PFU/ml 6.7 log₁₀ PFU/ml 28°C, 70% RH 14 h:10 h L:D	Aedes aegypti Mexico (F11-13), Cx. quinquefasciatus (Sebring County FLA 1988), Cx. pipiens (Pennsylvania 2002), Cx. tarsalis (California 1953) were orally infected with the epidemic American strain PRVABC59, and evaluated at 7 and 14 d dpi. Plaque assay was used to detect virus. Compared frozen versus fresh virus, African strains had a fitness advantage in vitro, Freezing decreased IR, SDR, STR, and CTR in Ae. aegypti. In fitness experiments West (Senegal) and East (MR766) African strains out competed the American strain (PRVAC59). Except for infectious virus detected in one Cx. quinquefasciatus mosquito exposed to frozen virus, no Culex mosquito became infected. VC of Ae. aegypti varied by virus strain, with STR ranged from 60–80% and transmission efficiency (CTR) ranged from 20–70%.	QAS: ≥ 50% (TS = 30, MS = 18) Strengths: Multiple virus strains, infectious virus assays. Weakness: Used laboratory colonies and only a single virus infecting dose.
[47]	Dibernardo A et al. J Am Mosq Control Assoc. 2017;33:276–281 https://doi.org/10.2987/17-6664.1 Vector competence of some mosquito species from Canada for Zika virus. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#tested (CTR) IT—used to estimate transmission. Temperate species southern Manitoba, Canada	Ae. cinereus, Ae. euedes, Ae. fitchii, Ae. sticticus, Ae. vexans, Coq. perturbans, Cx. restuans, and Cx. tarsalis PRVABC59 KF993678 (Canadian traveler infected in Thailand) 5.4 log₁₀ PFU/ml 25°C, 70–80% RH 18 h:6 h L:D	Field collected mosquitoes held at 25°C. Few of the mosquitoes became infected. Although none of the Ae. euedes, Ae. fitchii, Ae. sticticus, Cx. tarsalis, Coquillettidia perturbans contained ZIKV RNA after oral exposure. Ae vexans contained ZIKV RNA (IR = 4/131, SDR = 2/4). All other species tested were refractory. To detect the presence of a salivary gland barrier, that had either developed a disseminated infection after oral exposure or had been inoculated with ZIKV tested saliva. All the species tested had evidence of a salivary gland barrier. However, but low numbers of the Ae. vexans, Coq. perturbans and Cx. restuans had evidence of disseminated infection. Two Ae. vexans with disseminated infections also had ZIKV RNA + saliva.	QAS: ≥ 75% (TS = 33, MS = 20) Strengths: Use of field collected mosquitoes. Multiple virus strains. Confirmation of positive samples with infectious assay. Weakness: Single viral dose, reporting error in original manuscript for IR. Errors and inconsistencies in reported data.
[48]	Duchemin JB et al. Virol J. 2017;14 doi:10.1186/s12985-017-0772-y Zika vector transmission risk in temperate Australia: a vector competence study (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) Local species Australia	Ae. (Och.) camptorhynchus, Ae. (Ram.) notoscriptus, Ae. aegypti Ae. albopictus, Cx. annulirostris Cx. quinquefasciatus Cambodia 2010 (GenBank KU955593) 5.8 log₁₀ TCID₅₀/ml 25°C, 65% RH 14 h:10 h L:D	Field collected mosquitoes held at 25°C. IR (midguts) and DE (carcass containing ovaries and exoskeleton) were tested by PCR, whereas saliva was tested by TCID₅₀ at 14 dpi. Aedes aegypti was the most efficient vector (IR = 40/48), CDR = 39/47, CTR = 33/38). Aedes albopictus (IR = 19/26, CDR = 19/26, CTR = 20/26). Ae. notoscriptus (IR = 12/35; CDR = 2/59, CTR = 24/57). Ae. camptorhynchus: (IR = 5/18, CDR = 5/40, CTR = 5/37). Cx. quinquefasciatus (IR = 0/32, CDR = 032, CTR = 0/32) and Cx. annulirostris (IR = 0/20, CDR = 0/20, CTR = 0/20) were refractory to ZIKV.	QAS: ≥90% (TS = 34, MS = 21) Strengths: Used field collected mosquitoes, infectious assay to test saliva. Comparison to local Ae. aegypti and Ae. albopictus. Weakness. Used single strain of virus, and questionable method to assess dissemination.
[49]	Fernandes RS et al. Mem Inst Oswaldo Cruz. 2017;112: 577–579. doi: 10.1590/0074-02760170145 Culex quinquefasciatus from areas with the highest incidence of microcephaly associated with Zika virus infections in the Northeast Region of Brazil are refractory to the virus. (Short Communication)	VC: #inf./#tested (IR) #dissem/#tested (CDR) Site with active ZIKV transmission	Cx. quinquefasciatus F₁ Recife, Campina Grande, and Rio de Janeiro >F10 Cx. quinquefasciatus and Ae. aegypti colonies. (Local Brazilian virus strains) ZIKVPE243 (6.4 log₁₀ PFU/ml) ZIKVSPH (7.2 log₁₀ PFU/ml) ZIKVU1 (6.6 log₁₀ PFU/ml) No rearing temperature provided	Four Cx. quinquefasciatus strains were refractory to ZIKV regardless of 3 viral strains tested. Most mosquito-virus pairs evaluated at 7, 14, 21 dpi. Screening for ZIKV done with plaque assay then confirmed with PCR. Saliva was collected for testing if infection and dissemination was observed. Only one of 20 bodies of Cx. quinquefasciatus from Recife challenged with the ZIKV Rio-U1 was feebly positive at 7 dpi and the virus did not disseminate in this individual, as shown by the head repeatedly testing negative. As the virus did not disseminate in any Cx. quinquefasciatus, the saliva was not examined. For Ae. aegypti IR = 65–75% at 7 dpi and 68–100% at 14 dpi; CDR = 86–100% at 14 dpi.	QAS: ≤ 50% (TS = 30, MS = 18) Strengths: Multiple virus and mosquito strains tested from Brazil where ZIKV transmission occurred. Infectious Assay to detect virus. Weakness: Single viral dose, per strain. Did not test saliva of Ae. aegypti. Short format. Calculation of DE not clearly defined. Rearing temperature not included.
[50]	Gendernalik A et al. Am J Trop Med Hyg. 2017;96: 1338–1340 doi:10.4269/ajtmh.16-0963 American Aedes vexans mosquitoes are competent vectors of Zika virus. (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) Temperate species	Ae. vexans F1 from N. Colorado PRVABC59 6.8 log₁₀ PFU/ml 7.1 log₁₀ PFU/ml 7.2 log₁₀ PFU/ml 26°C, 80% RH, 16h: 8 h L:D	Conducted 3 biological replicates of mosquitoes. Infection (midguts), dissemination (legs) and transmission (saliva) were tested with Plaque assay and positives confirmed by PCR. IR ranged from 66–84% (118/148, mean 80%). CDR ranged from 3–25% (24/148, mean 16%). CTR ranged from 2–7% (7/148, mean = 5%) Wild-caught Ae. vexans from northern Colorado were highly susceptible to infection by ZIKV, however, dissemination and transmission were relatively low, indicating the existence of a moderately strong midgut escape and salivary gland barriers.	QAS: ≤ 50% (TS = 30, MS = 17) Strengths: Infectious assay, biological replicates, F1 mosquitoes. Weakness: single virus strain, single dpi evaluated and not clearly specified.
[31]	Guedes RD et al. Emerg Microbes Infect. 2017;6: 1–11 https://doi.org/10.1038/emi.2017.59 Zika virus replication in the mosquito Culex quinquefasciatus in Brazil (Research Article)	VC: #inf./#tested (IR) #SG+/#tested (CSGR) VD Electron microscopy Site with active ZIKV transmission	Ae. aegypti, (F1-F2, colony 1996) Cx. quinquefasciatus (colony since 2009) ZIKV BRPE243/2015 4 log₁₀ PFU/ml 6 log₁₀ PFU/ml 26±2°C, 65–85% RH 12 h:12 h L:D	Testing of midguts (infection), salivary glands (dissemination), and FTA cards (transmission), evaluated at 3, 7 and 15 dpi at two doses. PCR used to detect ZIKV RNA. At 6 log₁₀ PFU/ml, Cx. quinquefasciatus, IR at 7 dpi (10/12) and 15 dpi (7/18), CSGR at 7 dpi (12/12), at 15 dpi (5/18) compared to Ae. aegypti IR = at 7 dpi (18/20), at 15 dpi (6/16), CSGR at 7 dpi (12/20) and 15 dpi (6/16). At 6 log₁₀ PFU/ml, Cx. quinquefasciatus, IR at 7 dpi (9/25) and 15 dpi (2/19), CSGR 7 dpi (2/25) and 15 dpi (0/19) compared to Ae. aegypti IR 7 dpi (9/20), 15 dpi (9/18), CSGR at 7 dpi (2/18), 15 dpi (9/18). To confirm ZIKV-infective particles in salivary glands, two Ae. aegypti RecLab and two Cx. quinquefasciatus-positive samples collected at 7 dpi were inoculated in VERO cells for 10 days. Virus particles in salivary glands were observed by Electron Microscopy. From 270 pooled samples of adult female Cx. quinquefasciatus and 117 pools of Ae. aegypti mosquitoes assayed by RT-qPCR, three Cx. quinquefasciatus and two Ae. aegypti pools were positive for ZIKV.	QAS: < 25% (TS = 27, MS = 17) Strengths: Tested two doses of virus and multiple dpi. Weakness: Used colonized mosquito lines, did not use infectious assays but only PCR, FTA card data presumable negative, low sample size. Electron microscopy showed virus particles but could not identify them.
[51]	Hart CE et al. Emerg Infect Dis 2017; 23:559–560. http://dx.doi.org/10.3201/eid2303.161636 Zika Virus Vector Competency of Mosquitoes, Gulf Coast, United States (Research Letter)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) Compare artificial feeder and murine model	Cx. quinquefasciatus Ae. taeniorhynchus FSS13025 DAKAR41525 MEX1–7 MEX1–44 PRVABC59 4–7 log₁₀ FFU/ml 27°C, 80% RH	Cohorts of 50 Cx. quinquefasciatus from laboratory colony (no history provided) and the field in Houston (F2) were infected by feeding on interferon type I receptor knockout mice (4–7 log₁₀ FFU/ml, FSS13025). Experiment using artificial feeders infected Cx. quinquefasciatus and Ae. taeniorhynchus (>F10) with 3 strains of ZIKV (FSS13025 [2010 Cambodia related American strains], DAKR41525 [1985 Senegal], PRVABC59, MEX1-7 [2015 outbreak]) at a dose of 4–6 log₁₀ FFU/ml. At 3, 7 14 dpi, heads and legs were tested and at day 7, 14 saliva was tested by FFA. Murine feeds at 4, 7, 6 FFU/ml tested bodies at day 7 and 14 and legs, saliva on day 14 dpi, Ae. taeniorhynchus fed 6 logs of Mex strain, salivary glands, legs, midguts dissected and screened by FFA was refractory to ZIKA virus.	QAS: < 25% (TS = 25, MS = 13) Strengths: used multiple virus strains, included field collected Culex, used infectious assay and a murine infection model. Weakness: Pulled multiple experiments together into a single paper, the methodology was not consistent. Did not include Aedes controls.
[52]	Heitmann A et al. Euro Surveill. 2017;22. Doi:10.2807/1560-7917.ES.2017.22.2.30437 Experimental transmission of Zika virus by mosquitoes from central Europe (Research Article)	VC: #inf./#tested (IR) #transm./#inf. (STR) Role of rearing temperature Temperate species	Ae. aegypti Ae. albopictus Cx. p. molestus Cx. p. pipiens Cx. torrentium FB-GWUH-2016 7 log₁₀ PFU/ml 18°C and 27°C	Ae. aegypti (Bayer company); Cx. p. molestus (Heidelberg, GER) in colony since 2011, Cx p. pipiens (F0, collected in Hamburg, Germany summer 2016), Cx torrentium (F0, Hamburg, GER), Ae albopictus (F7, Freiburg GER), and Ae albopictus (Calabria, Italy) were infected a single low passage ZIKV strain) and evaluated IR (bodies) and TR (saliva) at 14 and 21 dpi at 18°C and 27°C, by PCR with confirmation by Plaque Assay. Cx. p. molestus IRs at 14 dpi were 12/41 at 18°C and 7/29 at 27°C and at 21 dpi were 2/32 at 18°C and 12/38 at 27°C. Cx. p. pipiens IRs at 14 dpi were 16/34 at 18°C and 3/37 at 27°C and at 21 dpi were 3/32 at 18°C and 0/35 at 27°C. Cx. torrentium IRs at 14 dpi were 11/35 at 18°C and 4/36 at 27°C and at 21 dpi were 1/38 at 18°C and 0/34 at 27°C. For Ae. aegypti and Ae. albopictus transmission (saliva) was observed only at 27°C ranging from 13–33%. No ZIKV was detected in any Culex species tested.	QAS: < 25% (TS = 24, MS = 14) Strengths: Evaluated temperature of incubation, multiple mosquito strains, recent field material included. Weakness: Selection of mosquito strains appeared to be fishing expedition. Transmission measured at # saliva+/i# infected.
[53]	Kenney JL et al. Am J Trop Med Hyg 2017; 96: 1235–1240. doi:10.4269/ajtmh.16-0865 Transmission incompetence of Culex quinquefasciatus and Culex pipiens pipiens from North America for Zika virus (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) IT Temperate species	Cx. quinquefasciatus Cx. pipiens Ae. aegypti (>>>F10) MR766 PRVABC59 R103451-Honduras 2016 4–7.1 log₁₀ PFU/ml 28°C, 70–75% RH	Vector competence studies for laboratory strains (>>>F10) of Cx. quinquefasciatus (Sebring, FL 1998), Cx. pipiens (Chicago 2010), and Ae. aegypti (Poza Rica, Mexico). Mosquitoes were orally infected with 3 strains of ZIKV (MR766, PRVABC59, and R103451-Honduras 2016) at doses ranging from 4–7.1 log₁₀ PFU/ml and held at 28°C. with infection (bodies), dissemination (legs+wings) and transmission (saliva) evaluated at 14 dpi using plaque assay and qPCR. IT inoculations and in vitro growth in mosquito cell lines were examined. Ae. aegypti, IR = 100%, CTR = 67%, Cx. quinquefasciatus, IR = 0–1% (1/108), CTR = 0%; Cx. pipiens; IR = 5–10% (5/58), CTR = 0, For IT inoculated mosquitoes, IR = 15–70% in Cx. quinquefasciatus, 61% in Cx. pipiens, and 100% in Ae. aegypti, but saliva was positive only for Ae. aegypti (67%), In vitro experiments showed significant growth restriction in Culex cells.	QAS: ≤ 50% (TS = 30, MS = 18) Strengths: IT inoculations provide additional evidence against Culex vectoring ZIKV. Infectious assay. In vitro studies. Inclusion of Ae. aegypti. Multiple virus strains. Weakness: Using mosquito laboratory colonies.
[54]	Liu Z et al. Emerg Infect Dis. 2017;23: 1085–1091 https://dx.doi.org/10.3201/eid2307.161528 Competence of Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus mosquitoes as Zika virus vectors, China. (Research Article).	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) Local species China	Ae. aegypti Ae. albopictus Cx. quinquefasciatus (Colonies > 20 years) Asian lineage (KU820899.2) Human origin China 2016 5.45 ± 0.38 log₁₀ copies/μl 27±1°C, 70–80% RH 16 h: 8 h L:D	18–30 mosquitoes were examined at 0, 4, 7, 10, and 14 dpi. Tested midguts, heads, and salivary glands. Cx. quinquefasciatus IR was 22/138 (15.9%), with no dissemination or transmission; Ae. aegypti IR = 124/138 (90%), SDR = 91/124 (73.4%), STR = 78/124 (63%), CTR = 78/138 (57%); Ae. albopictus IR = 121/138 (88%), SDR = 51/121 (42%), STR = 29/121 (24%), CTR = 29/138 (21%). No transmission rates were calculated based on salivary glands incorrectly. SDRs were observed as early as 4 dpi, increasing rapidly to 100% at 7 dpi. For Ae. albopictus mosquitoes’ dissemination was first detected at 7 dpi but was lower overall than for Ae. aegypti at the same time points. Zika virus was not detected in the head tissues of Cx. quinquefasciatus mosquitoes.	QAS: < 25% (TS = 25, MS = 14) Strengths: Monitored the kinetics of infection from 0 to 14 dpi. Weakness: Single virus strain, old laboratory mosquito strains, did not measure transmission appropriately directly testing salivary glands, tested only with PCR.
[55]	Lourenço-de-Oliveira R et al. J Gen Virol. 2018;99: 258–264. Doi:10.1099/jgv.0.000949 Culex quinquefasciatus mosquitoes do not support replication of Zika virus. (Short Communication)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) Role of Wolbachia endosymbionts on VC.	Cx. quinquefasciatus containing or free of Wolbachia NC-2014-5132 7 log₁₀ TCID₅₀/ml Rearing temperature not provided	All Cx. quinquefasciatus lines challenged with ZIKV were refractory to the virus whether they contained Wolbachia or not. Used plaque assays to detect virus. No infection, dissemination or transmission was detected in any of the mosquito lines at 7 and 14 dpi. ZIKV does not replicate to detectable levels in mosquito cells following a blood meal, in line with the infectivity data described above.	QAS: < 25% (TS = 25, MS = 13) Strengths: Used infectious assay. Weakness: Single virus strain, but authors were more oriented in finding how Wolbachia might account for refractoriness of Culex. Rearing temperature was not reported.
[56]	O’Donnell KL. Et al. J Med Entomol. 2017;54:1354–1359. Doi:10.1093/jme/tjx087 Potential of a Northern population of Aedes vexans (Diptera: Culicidae) to transmit Zika virus. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #dissem./#tested (CDR) IT Temperate species Upper Great Plains	Ae. vexans (F1), Ae. aegypti (F39) Unspecified from Puerto Rico 2016 Fresh: 5.3 log₁₀ PFU/ml Frozen: 7.0 log₁₀ PFU/ml 28°C, 16 h: 8 h L:D	Infection (bodies), dissemination (legs) and transmission, through IT inoculation were assessed. Mosquitoes were challenged with thawed frozen and fresh virus from cell culture and held at 28°C. For Ae. vexans challenged with thawed virus, IR = 29% (8/28) and CDR = 12% (1/8) compared to those fresh virus IR = 28% (9/32) and CDR = 3% (1/32). For Ae. aegypti challenged with thawed virus, the IR = 61% (11/18), SDR = 36% (4/11). Saliva tested in mosquitoes infected IT on 16–17 dpi. Ae. aegypti had significantly higher rates of viral infection and dissemination than Ae. vexans. All 47 inoculated Ae. vexans and 22 of 23 inoculated Ae. aegypti were positive for Zika virus.	QAS: > 50% (TS = 31, MS = 19) Strengths: Used field captured mosquitoes. Compare frozen and fresh virus preps. Weakness: PCR only for detection of viral RNA, single virus strain.
[57]	Calvez E et al. PloS NTD 2018;12:e0006637 https://doi.org/10.1371/journal.pntd.0006637 Zika virus outbreak in the Pacific: vector competence of regional vectors (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) Local species French Polynesia	Ae. aegypti (3 populations from French Polynesia, New Caledonia, Samoa (F1-F3) Ae. polynesiensis (2 populations from French Polynesia, Wallis and Futuna F1-F3) NC-2014-5132 7 log₁₀ TCID₅₀/ml 28°C, 80% RH, 16h: 8 h L:D	Examined distinct local Ae. aegypti and Ae. polynesiensis populations across the south pacific at 6, 9, 14, and 21 dpi. After oral challenge, mosquito infection (bodies), dissemination (heads) and transmission (saliva) were evaluated by plaque assay for ZIKV. For French Polynesian population of Ae. polynesiensis: IR 6 dpi (34/68), 9 dpi (64/86), 14 dpi (84/108), 21 dpi (71/92); SDR 6 dpi (6/33), 9 dpi (10/57), 14 dpi (32/71), 21 dpi (36/60); STR 14 dpi (1/32), 21 dpi (2/36); CTR at 14 dpi (1/72), 21 dpi (2/69). Ae. aegypti populations had significant heterogeneity in VC parameters and low competence overall among the three mosquito populations tested CTR was 0%, 6%, and 17%.	QAS: ≥ 99% (TS = 35, MS = 21) Strengths: Used Infectious assay for detection of ZIKV, provided raw data in supplementary data, geographically diverse mosquitoes strains recently from field, range of dpi studied. High sample sizes. Weakness: Single virus strain.
[58]	Dodson BL & Rasgon JL Peer J 2017 doi: 10.7717/peerj.3096 Vector competence of Anopheles and Culex mosquitoes for Zika virus (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR)	An. gambiae, An. stephensi Cx. quinquefasciatus (>F10) PRVABC59 MR766 4.6–7.7 log₁₀ PFU/ml 27±1°C, 12 h: 12 h L:D	Vector competence studies carried out for laboratory strains (>F10) of An. gambiae, An. stephensi (Liston strain, Johns Hopkins University) and Cx. quinquefasciatus (Wadsworth Center, originally from Benzon Research) infected with ZIKV (MR766 Uganda prototype and PRVABC59) at 6 doses, by orally infecting mosquitoes on artificial feeder and human blood. Mosquitoes were held at 27°C. Infection (bodies), dissemination (legs), and transmission (saliva) were evaluated by Plaque assay at 5, 7, 14 dpi. No species were infected at any time point.	QAS: < 25% (TS = 27, MS = 15) Strengths: Used multiple virus strains and infectious doses and evaluated competence parameters at 3 time points. Weakness: Used highly passaged frozen virus, no Ae. aegypti control, and colonized mosquitoes.
[59]	Jansen S et al. Emerg Microbes Infect. 2018;7: 192 doi:10.1038/s41426-018-0195-x Experimental transmission of Zika virus by Aedes japonicus japonicus from southwestern Germany (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) Temperate species Role of rearing temperature	Ae. japonicus japonicus (F1) ZIKV_FB-GWUH-2016 (KU870645) 7 log₁₀ PFU/ml 21°C, 24°C, and 27°C, 80% RH	Groups of 20 females, previously screened by pan-Flavi-, pan-Bunya- and pan-Alphavirus by PCR were exposed to blood meal with ZIKV and bodies, legs, and saliva were evaluated at 14 dpi by PCR except for saliva, also tested by plaque assay. IR = 10% (3/30) at 21°C, 24% (7/29) at 24°C, 66.7% (14/21) at 27°C. SDR, 0% at 21°C, ~26% at 24°C, ~10% at 27°C, but RNA copies were higher at 27°C. STR, 14% (2/14) at 27°C and CTR of 9.5% (2/21) at 27°C.	QAS: > 50% (TS = 31, MS = 19) Strengths: Mosquitoes from field, used infectious assay to assess transmission. Role of temperature. Weakness: single virus strain and titer. Difficult to extract metadata, especially positive legs.
[60]	Karna AK et al. Viruses. 2018; 10:434 doi:10.3390/v10080434 Colonized Sabethes cyaneus, a sylvatic New World mosquito species, shows a low vector competence for Zika virus relative to Aedes aegypti. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) Forest species Mouse model	Sa. cyaneus (since 1988), Ae. aegypti (F7) ZIKV MEX 1–7 (isolated from Ae. aegypti 2015) 5 log₁₀ PFU/ml Infected infr/- mouse 27±1°C, 80% RH, 16 h: 8 h L:D	After oral exposure to virus infected blood, feeding rates for Sa. cyaneus were low, with 21% feeding on mice at 1 dpi and 28% feeding on mice at 2 dpi; in contrast, more than 85% of Ae. aegypti fed on mice on each day. Of 69 engorged Sa. cyaneus, ZIKV was detected in only one individual, albeit in all body compartments sampled (body, legs, and saliva) at 21 dpi This mosquito had fed on a mouse at day 2 dpi; titers increased in the mice by approximately tenfold between day 1 and day 2 dpi. In contrast, Ae. aegypti showed high levels of ZIKV infection, dissemination, and transmission.	QAS: < 50% (TS = 28, MS = 14) Strengths: Infectious assay to detect ZIKV, wide range of dpi, Ae. aegypti comparator. Weakness: Old colony, did not blood feed well sample size, single specimen became infected, showed dissemination and transmission.
[61]	Main BJ et al. PloS NTD 2018;12: e0006524 https://doi.org/10.1371/journal.pntd.0006524 Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus. (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) Temperate species California USA Mouse model	Ae. aegypti (F6), Cx. quinquefasciatus(F5), Cx. tarsalis (>F10) PRVABC59 (KX601168), MA66, P6-740 (KX601167.1), SPH2015 (KU321639); 5 log₁₀ PFU/ml 26°C, 80% RH, 12 h: 12 h L:D (Ae. aegypti and Cx. tarsalis) 22°C, 33% RH (RT) (Cx. quinquefasciatus)	Ae. aegypti and Cx. tarsalis held at 26°C whereas Cx quinquefasciatus were held at 22°C and challenged with PR15 5.4–6.4 log₁₀ PFU/ml. IR = 4% (2/46) at 14 dpi, 30% (6/20) at 21 dpi; CDR 4% (2/46) at 14 dpi, and 5% (1/20) at 21 dpi, CTR = 0% at 14 and 21 dpi. Cx. quinquefasciatus were refractory at 14 and 21 dpi. In contrast, Ae. aegypti: challenged with ZIKV MA66: IR = 86% (73/85) at 14 dpi and 96% (22/23) at 21 dpi; CDR = 79% (69/85) at 14 dpi and 91% (21/23) at 21 dpi; CTR = 53% (45/85) at 14 dpi and 87% (20/23). For ZIKV PR15, the infection, dissemination, and transmission rates on 14 dpi were 85%, 78%, and 65%, respectively. For ZIKV BR15 harvested 15 dpi had infection, dissemination, and transmission rates of 90%, 90%, and 75%, respectively.	QAS: ≥ 90% (TS = 34, MS = 21) Strengths: Cx. quinquefasciatus colony F5, Ae. aegypti comparator, confirmation of infectious virus. Range doses with Cx. tarsalis. Good sample size. Weakness: Cx. tarsalis old colony, single strain of virus, retrospective confirmation of infectious virus.
[32]	Smartt TC et al. Front Microbiol. 2018;9: 768. doi: 10.3389/fmicb.2018.00768 Culex quinquefasciatus (Diptera: Culicidae) from Florida transmitted Zika virus. (Research Article)	VC: #inf./#tested (IR) Transmission verified through presence of ZIKV in saliva eluted from FTA cards	Cx. quinquefasciatus (>F10) Asian lineage, Gen Bank KU501215.1 5.7 log₁₀ PFU/ml 28°C, 80% RH	IR (bodies) at 16 dpi revealed 9 female mosquito bodies with ZIKV RNA. Analysis of RNA in saliva eluted from the filter paper at 16 dpi revealed an average titer of 5.6 ± 4.5 log₁₀ ZIKV PFU/ml per card and there was no significant difference in the titers per cage. The mosquitoes from the cages revealed positive bodies in cages 1 and 2 (IR = 55%). Plaque assays of the saliva samples eluted from the filter paper cards were positive for ZIKV infectious virus.	QAS: < 25% (TS = 21, MS = 12) Strength: Infectious virus confirmation of ZIKV in FTA card showing transmission. Weakness: Difficult to calculate rates, poor presentation of experiment metadata.
[62]	Ben Ayed W et al. J Med Entomol. 2019; 56:1377–1383. Doi: 10.1093/jme/tjz067 A survey of Aedes (Diptera: Culicidae) mosquitoes in Tunisia and the potential role of Aedes detritus and Aedes caspius in the transmission of Zika virus. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) (Tunisia) Entomology survey	Ae. caspius (F1) Ae. detritus (F1) NC-2014-5132 7.2 log₁₀ PFU/ml 28±1°C, 80% RH, 16 h: 8 h L:D	Aedes detritus: Owing to the low survival rate in the laboratory and low feeding rates in BSL-3 conditions, few mosquitoes were examined at 14 dpi. Of these, IR = 75% (3/4), SDR = 0%, STR = 0%. Aedes caspius: IR 4% (1/24) and SDR = 0% at 7 dpi. At 14 dpi, IR = 10% (2/20), SDR = 100%l (2/2) and STR = 0%. Thus, neither species were competent to transmit ZIKV.	QAS: ≤ 50% (TS = 30, MS = 18) Strength: Infectious assay for bodies and heads. F1 mosquito strains. Weakness: Single virus strain, low numbers, PCR only to detect virus.
[63]	Elizondo-Quiroga D et al. Sci Rep 2019;9: 16955. https://doi.org/10.1038/s41598-019-53117-1 Vector competence of Aedes aegypti and Culex quinquefasciatus from the metropolitan area of Guadalajara, Jalisco, Mexico for Zika virus. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #dissem./#tested (CDR) #transm./#dissem. (STR) #transm./#tested (CTR) Site with active ZIKV transmission	Ae. aegypti (F0) Cx. quinquefasciatus. (F0) 3 virus strains from Mexico 4.7 log₁₀, 5.2 log₁₀, 5.6 log₁₀, and 6.4 log₁₀ TCID₅₀/ml 28±1°C, 80% RH, 12 h: 12 h L:D	Bodies, heads, and saliva evaluated at 14 dpi and different virus titers. Cx. quinquefasciatus were refractory at all virus concentrations. Ae. aegypti infection did not occur at low (4.7 log₁₀ TCID₅₀/ml) virus concentration, but at medium concentrations (5.2–5.6 log₁₀ TCID₅₀/ml), IR = 38%, SDR = 24%, STR = 32% and CTR = 7.7%. At high virus concentration (6.4 log₁₀ TCID₅₀/ml), IR = 93%, SDR = 72%, STR– 19.3%, and CTR = 14%.	QAS: ≥ 75% (TS = 33, MS = 19) Strength: Infectious assay, F1 mosquito strains, range of virus doses. Multiple virus strains. Weakness: Poor metadata on virus strain.
[64]	Fernandes RS et al. Sci Rep. 2019;9: 20151. https://doi.org/10.1038/s41598-019-56669-4 Low vector competence in sylvatic mosquitoes limits Zika virus to initiate an enzootic cycle in South America. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) IT Forest species.	Haemagogus leucocelaenus, Ae. terrens, Ae. scapularis, Sa. identicus, Sa. albiprivus (field) Rio-U1 Rio-S1 Oral dose: 6.0 log₁₀ PFU/ml IT dose: 6.5 log₁₀ PFU/ml 28±1°C, 80±10% RH 12 h: 12 h L:D	30 mosquitoes examined (bodies, heads, saliva) at 7, 14, and 12 dpi. Hg. leucocelaenus infected with ZIKV Rio-S1 had IRs at 7 dpi (4/20), 14 dpi (7/21), and 21 dpi (12/30) and SDR at 7 dpi (1/4), 14 dpi (1/7), and 21 dpi (1/12), but no transmission was observed. In contrast, when challenged with ZIKV Rio-U1 IR was lower at 14 dpi (4/30) and neither dissemination nor transmission was observed. Sa. albiprivus was refractory to ZIKV Rio-U1 at 7, 14, and 21 dpi, but with ZIKV Rio-S1 had low IR (1/32) only at 14 dpi with no infection at 7 or 21 dpi. No further dissemination was observed. Ae. scapularis challenged only with ZIKV Rio-S1 had low IR (1/42) but no further dissemination. Ae. terrens and Sa. identicus were completely refractory to ZIKV challenge. Transmission (virus present in saliva) was not detected in any mosquito orally challenged with ZIKV, regardless of viral isolate and incubation time. Dissemination and transmission were observed in Hg. leucocelaenus, Sa. albiprivus, and Sa. identicus after IT inoculation.	QAS: ≥ 75% (TS = 33, MS = 20) Strengths: Use of field collected mosquitoes; Use of infectious virus assay, >1 virus strain, infectious assays to detect ZIKV. Weakness: Low numbers for some species.
[65]	Gutiérrez-López R et al. Emerg Infect Dis. 2019;25: 346–348 https://doi.org/10.3201/eid2502.171123 Vector competence of Aedes caspius and Ae. albopictus mosquitoes for Zika virus, Spain. (Dispatches)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) Temperate species (Spain)	Ae. albopictus (F2) Ae. caspius (F0) Ae. aegypti (F8) CAM (JN860885): PR (KU501215) 7.6 log₁₀ PFU/ml No rearing temperature provided	Bodies, legs, saliva tested by PCR at 7, 14, and 21 dpi. Aedes caspius had IRs at 7 dpi (21.4% [3/14]), 14 dpi (40% [10/25]), and 21 dpi (18.5% [5/27]), no virus dissemination or transmission was detected at any point with Puerto Rican Virus strain. Aedes albopictus Cambodia strain. IR = 90.5% at 7 dpi, 82% at 14 dpi and 94.4% at 21 dpi; SDR = 42% at 7 dpi, 82% at 14 and 21 dpi; STR = 10.5% at 7 dpi, 9% at 14 dpi, 23.6% at 21 dpi. Puerto Rico Strain: IR = 97% at 7 dpi, 93% at 14 dpi, 96% at 21 dpi; SDR = 31% at 7 dpi, 68% at 14, 96% at 21 dpi; STR = 0% at 7 and 14 dpi, 36% at 21 dpi. Aedes aegypti Cambodia, IR = 24% at 7 dpi, 23% at 14 dpi and 36% at 21 dpi; SDR = 75% at 7 dpi, 71% at 14 and 100% at 21 dpi; STR = 12.5% at 7 dpi, 14.3% at 14 dpi, 40% at 21 dpi. Puerto Rico Strain: IR = 62% at 7 dpi, 45% at 14 dpi, 56% at 21 dpi; SDR = 38% at 7 dpi, 78% at 14, 89% at 21 dpi; STR = 0% at 7, 16.7% at 14 dpi, 39% at 21 dpi.	QAS: ≥ 75% (TS = 33, MS = 21) Strengths: Mosquito strains from field or recently colonized, > 1 virus strain (only 1 for Ae. caspius). Weakness: Single virus strain for Ae. caspius, PCR only for virus detection. No rearing temperature reported.
[66]	Hery L et al. Emerg Microbes Infect. 2019;8:699–706. https://doi.org/10.1080/22221751.2019.1615849 Transmission potential of African, Asian and American Zika virus strains by Aedes aegypti and Culex quinquefasciatus from Guadeloupe (French West Indies). (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #dissem./#tested (CDR) #transm./#dissem. (STR) #transm./#tested (CTR) Site with active ZIKV transmission	Ae. aegypti (F1) Cx. quinquefasciatus. (F0) Senegal (KU955592) Martinique (KU647676) Malaysia (KX694533) 7 log₁₀ TCID₅₀/ml 27±1°C, 70% RH, 12 h: 12 h L:D	For Cx. quinquefasciatus no infection, nor dissemination nor transmission was detected for any of the ZIKV strains at 7, 14 or 21 dpi. For Ae aegypti Senegal strain, IR = 90% at 7 dpi, 92% at 14 dpi, and 84.6% at 21 dpi; CDR = 96.3% at 7 dpi, 91.3% at 14 dpi, 95.5% at 21 dpi, CTR 42.3% at 7 dpi, 62% at 14 dpi, 76.2% at 21 dpi. For Malaysia strain, IR = 23.3% at 7 dpi, 23.3% at 14 dpi, 16.7% at 21 dpi; CDR = 28.6% at 7 dpi, 71.4% at 14 dpi, 80% at 21 dpi, CTR 0% at 7 dpi, 20% at 14 dpi, 75% at 21 dpi. Martinique strain, IR = 23.3% at 7 dpi, 37% at 14 dpi, 27% at 21 dpi; CDR = 29% at 7 dpi, 54.5% at 14 dpi, 62.5% at 21 dpi, TR 0% at 7 and 14 dpi, 80% at 21 dpi.	QAS: ≥ 90% (TS = 34, MS = 22) Mosquito strains from field, multiple virus strains, range of dpi, infectious assays. Weakness: Single virus dose.
[67]	Núñez AI et al. Parasit Vectors. 2019;12: 363. https://doi.org/10.1186/s13071-019-3620-7 European Aedes caspius mosquitoes are experimentally unable to transmit Zika virus. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) Temperate species (Spain)	Ae. caspius (F0) Ae. aegypti (since 1994) Suriname (EVAg no. 011V-01621; Asian lineage), MR766 (African I lineage) 7 log₁₀ TCDI₅₀/ml 26/22°C (day/night), 80% RH 14 h: 10 h L:D	After virus challenge, infection (bodies), dissemination (legs), and transmission (saliva) evaluated at 7, 14, and 21 dpi. Screened by PCR, confirmation with plaque assay. Aedes caspius Suriname, 0% IR, SDR, and STR at all time points. ZIKV RNA was detected at low levels at 14 and 21 dpi. MR766 (African I lineage) 0% IR, SDR, and STR at all time points, also with low levels of ZIKV detection by RT-qPCR. Aedes aegypti Suriname, IR = 85% at 7 dpi, 100% at 14 dpi, 95% at 21 dpi, SDR = 45% at 14 dpi, 84% at 21 dpi, STR = 33% at 14 dpi, 6.2% at 21 dpi; MR766, IR = 10% at 7 and 14 dpi, 5.2% at 21 dpi, but no dissemination or transmission	QAS: ≤ 50% (TS = 29, MS = 18) Strengths: Use of field collected mosquitoes; Use of infectious virus assay, >1 virus strain. Weakness: Low numbers for DR and TR assessments, single virus dose.
[68]	Abbo SR et al. PloS NTD 2020; 14: e0008217. Doi: https://doi.org/10.1371/journal.pntd.0008217 The invasive Asian bush mosquito Aedes japonicus found in the Netherlands can experimentally transmit Zika virus and Usutu virus. (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) IT RNA replicative intermediates Entomology survey Temperate species	Ae. japonicus (F0) Ae. aegypti (control, Rockefeller) Suriname 2016 USUV (MH891847.1) 7.2 log₁₀ TCID₅₀/ml 28°C, 12 h: 12 h L:D	Assessed infection (bodies), dissemination (legs+wings), and transmission (saliva) at 14 dpi at 28° C for both ZIKV and Usutu (USUV). IT inoculations were also performed. For ZIKV-blood fed mosquitoes, IR = 10% (6/62), CDR = 8% (5/62), and CTR = 3% (2/62). For USUV-blood fed mosquitoes, IR = 13% (4/30), CDR = 13% (4/30), and CTR = 13% (4/30). Of the intrathoracically injected mosquitoes, 96% (ZIKV) and 88% (USUV) showed virus-positive saliva at 14 dpi. Small RNA deep sequencing of orally infected mosquitoes confirmed active replication of ZIKV and USUV, as demonstrated by potent small interfering RNA responses against both viruses. Additionally, de novo small RNA assembly revealed the presence of a novel narnavirus in Ae. japonicus. Ae. aegypti controls IR = 100%, CDR = 100%, CTR = 80%)	QAS: > 50% (TS = 32, MS = 21) Strengths: Use of field collected mosquitoes; Intrathoracic injections to identify mechanistic barriers to transmission. Use of infectious virus assay. Weakness: No statistical analysis or description or group replicates.
[69]	Abbo SR et al. Viruses. 2020;12;659 doi:10.3390/v12060659 Forced Zika virus infection of Culex pipiens leads to limited virus accumulation in mosquito saliva. (Research Article)	VC: #inf./#tested (IR) #transm./#tested (CTR) IT Temperate species	Cx. p. molestus, Cx. p. pipiens (colony) Suriname 2016 USUV (MH891847.1) 7.0 log₁₀ TCID₅₀/ml 28°C	Infection (bodies) and transmission (saliva) determined at 14 dpi. ZIKV IR for Cx. pipiens (2/133) but no transmission (0/133) observed after an infectious blood meal with ZIKV titers in Cx. pipiens observed to be low. ZIKV IR for Ae. aegypti was 100% (121/121) and CTR was 65% (79/121). Cx. molestus was refractory to ZIKV. The infection and transmission of potential of ZIKV-injected Cx. pipiens was dependent on the viral dose provided. Viral dissemination into the saliva of Cx. pipiens does not always correlate with a high viral titer in the mosquito body. Cx. pipiens is an inefficient vector for ZIKV.	QAS: < 25% (TS = 27, MS = 17) Strengths: Infectious assay to detect ZIKV. Weakness: Used colonized mosquito lines with poorly described history.
[70]	Blagrove MSC et al. Proc Biol Sci 2020;287:2020019 http://dx.doi.org/10.1098/rspb.2020.0119 Potential for Zika virus transmission by mosquitoes in temperate climates. (Research Article)	VC: #inf./#tested (IR) #transm./#tested (CTR) Temperate species Role of rearing temperature Modeling	Ae. albopictus (Verano F3) Ae. detritus (F0) (reported as Oc. detritus in original article) PE243 (Brazil) 6 log₁₀ PFU/ml 17°C, 19°C, 21°C, 24°C, 27°C and 31°C 70% RH, 12 h: 12 h L:D	Mortality and competence of wild-obtained Ae. detritus and colony Ae. albopictus were tested at six different temperatures: 17°C, 19°C, 21°C, 24°C, 27°C and 31°C. Adult females were sacrificed at eight time points: 0, 5, 7, 10, 14-, 17-, 21- and 28-days dpi. Zika RNA was detected in the saliva of both Ae. albopictus (Verano colony) and Ae. detritus at all temperatures from 19°C to 31°C, as early as 7 dpi at 31°C for Ae. albopictus and 10 dpi at 27°C and 31°C for Ae. detritus. ZIKV titers in Ae. albopictus saliva were 3.8x higher than in Ae. detritus. Ae. detritus detected starting at 7 dpi and 19°C until 28 dpi and 31°C (IR at 19°C: 14 dpi = 1/12, 17 dpi = 2/19, 21 dpi = 3/16, 28 dpi = 3/18; CTR at 19°C: 17 dpi = 1/19, 28 dpi = 1/18; IR 21°C: 14 dpi = 5/20, 17 dpi = 4/15, 21 dpi = 3/16, 28 dpi = 4/16; CTR at 21°C: 14dpi = 4/20, 17 dpi = 1/15, 21 dpi = 1/16, 28 dpi = 2/16; IR 24°C: 10 dpi = 1/9, 14 dpi = 1/17, 17 dpi = 3/21, 21 dpi = 5/16, 28 dpi = 3/17; CTR at 24°C: 14dpi = 1/17, 17 dpi = 1/21, 21 dpi = 3/16, 28 dpi = 3/17; IR 27°C: 10 dpi = 2/10, 14 dpi = 3/21, 17 dpi = 3/17, 21 dpi = 2/15; CTR at 27°C: 10 dpi = 1/10, 14dpi = 2/21, 17 dpi = 3/17, 21 dpi = 2/15; IR at 31°C: 10 dpi = 2/9, 14 dpi = 2/13, CTR at 31°C: 10 dpi = 1/9, 14 dpi = 2/13. Ae. albopictus detected starting 7 dpi.	QAS: > 25% (TS = 27, MS = 16) Strengths: Examined a range of temperatures. Weakness: Used PCR assays to detect ZIKV RNA. Single dose and virus strain.
[71]	Chan KK et al. Parasit Vectors 2020; 13:188. https://doi.org/10.1186/s13071-020-04042-0 Vector competence of Virginia mosquitoes for Zika and Cache Valley viruses. (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR) IT Temperate species (Virginia)	Field collected (F1) Cx. pipiens, Cx. restuans, Ae. albopictus, Ae japonicus, Ae. triseriatus. Ae. aegypti (Colony) PRVABC59 Oral dose (Aedes) 6.5–7.7 log₁₀ PFU/ml IT dose (Aedes) 4.7–5.3 log₁₀ PFU/ml Oral dose (Culex) 6.7–7.5 log₁₀ PFU/ml 24°C, 75% RH, 16h: 8 h L:D	IR (bodies), CDR (legs and wings), and CTR (saliva) determined at 14 dpi by plaque assay. Mosquitoes reared at 24°C. Ae. aegypti: IR = 17/25, CDR = 15/25, CTR = 12/25. Ae. albopictus: IR = 18/37, CDR = 15/37, CTR = 9/37. Ae. japonicus: IR = 15/73, CDR = 7/73, CTR = 2/73). Ae. triseriatus: IR = 7/28, CDR = 0/28, CTR = 0/28. Cx. pipiens and Cx. restuans were completely refractory to ZIKV infection. Transmission rates after IT inoculation: Ae. albopictus (63%, 12/19), Ae. japonicus (19%, 4/21), Ae. aegypti (71%, 15/21) No virus detected in the saliva of Ae. triseriatus from either orally (0/28) or parenterally (0/23) infected groups. Study also examined VC to Cache Valley virus (CVV). CVV was detected in the saliva of Ae. albopictus (high titer: 68%, low titer: 24%), Ae. triseriatus (high titer: 52%, low tier: 7%), Ae. japonicus (high titer 22%, low titer: 0%) and Ae. aegypti (high titer: 10%; low titer: 7%). Culex pipiens and Cx. restuans were also refractory to CVV.	QAS: > 50% (TS = 32, MS = 19) Strengths: Infectious assay to detect ZIKV. Mosquitoes from field Weakness: Single virus strain, experimental metadata difficult to extract.
[72]	Fernandes SR et al. Pathogens. 2020; 9: 575. Doi:10.3390/pathogens9070575 Vector competence of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Brazil and New Caledonia for three Zika virus lineages. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) Sites with active ZIKV transmission	2 strains Ae. aegypti (F1) Cx. quinquefasciatus (F0- New Caledonia) 5 strains Ae. aegypti (F2–F4- Brazil), Ae. albopictus (F2–F4 Brazil) MRS_OPY_Martinique_PaRi_2015, DAK 84, MASS 66 7 log₁₀ TCID₅₀/ml 28±1°C, 70–80% RH 12 h: 12 h L:D	After mosquito were challenged with ZIKV infected blood IR (bodies), dissemination (heads), and transmission (saliva) at 7, 14, and 21 dpi tested by Plaque assay, comparing Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus with 3 strains of ZIKV. Cx. quinquefasciatus only at Dumbea, were refractory. Ae. aegypti: At 7 dpi IR = 30–100%, SDR = 0–100%, STR = 0–85%, CTR = 0–85%; at 14 dpi IR 60–100%, SDR = 25–100%, STR = 14–100%, CTR = 3–100%; at 21 dpi, IR = 45–100%, SDR = 19–100%, STR = 0–96%, CTR = 0–90%. Aedes albopictus: At 7 dpi IR = 7–73%, SDR = 0–41%, STR = 0–57%, CTR = 0–13%; at 14 dpi IR 7–80%, SDR = 0–83%, STR = 0–100%, CTR = 0–50%; at 21 dpi, IR = 7–80%, SDR = 10–100%, STR = 0–100%, CTR = 0–67%. There was high variability in VC based on virus strain-mosquito combinations.	QAS: > 50% (TS = 32, MS = 19) Strengths: Infectious assay to detect ZIKV. Comparing VC numerous sites. Multiple virus strains. Mosquitoes from field Weakness: Single site for Cx. quinquefasciatus, low sample sizes for dissemination and transmission experiments.
[73]	Glavinic U et al. Parasit Vectors 2020; 13:479. Doi: https://doi.org/10.1186/s13071-020-04361-2 Assessing the role of two populations of Aedes japonicus japonicus for Zika virus transmission under a constant and a fluctuating temperature regime. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) #transm./#tested (CTR) Including fluctuating vs constant temperature rearing schemes.	Ae. japonicus (2 sites) Zurich Steinbach DAK84 Senegal 7.1 log₁₀ TCID₅₀/ml Constant temperature: 27°C, 85% RH Fluctuating temperature: 14–27°C (mean = 23°C), 45–90% RH 16h: 8 h L:D	Infection (Abdomen/thorax), dissemination (heads), and transmission (saliva) were determined by plaque assay at 7, 14, and 21 dpi in two mosquito strains (Zurich, Steinbach) reared at constant and fluctuating temperature. For the Zurich strain held at a constant temperature: IR at 7 dpi (25/30), at 14 dpi (28/30), at 21 dpi (26/30), SDR at 7 dpi (10/25) at 14 dpi (9/28), 21 dpi (3/26), CTR at 7 dpi (7/30), at 14 dpi (6/30), 21 dpi (2/30). For the Steinbach strain held at a constant temperature: IR at 7 dpi (20/30), at 14 dpi (30/30), at 21 dpi (20/30), SDR at 7 dpi (8/20) at 14 dpi (6/30), 21 dpi (5/20), CTR at 7 dpi (2/30), at 14 dpi (3/30), 21 dpi (2/30). For the Zurich strain held at a fluctuating temperature: IR at 7 dpi (14/30), at 14 dpi (5/30), at 21 dpi (23/30), SDR at 7 dpi (10/14) at 14 dpi (3/5), 21 dpi (6/23), CTR at 7 dpi (3/30), at 14 dpi (0/30), 21 dpi (3/30). For the Steinbach strain held at a fluctuating temperature: IR at 7 dpi (22/30), at 14 dpi (18/30), at 21 dpi (14/30), SDR at 7 dpi (7/22) at 14 dpi (11/18), 21 dpi (9/14), CTR at 7 dpi (3/30), at 14 dpi (8/30), 21 dpi (1/30). SDR decreased over time regardless of the incubation temperature regimes. Saliva was positive at 7 dpi for all populations independent of incubation conditions.	QAS: < 50% (TS = 29, MS = 17) Strengths: Infectious assay to detect ZIKV. Field collected mosquitoes. Comparison of different mosquito populations. Impact of fluctuating temperature. Weakness: Single virus strain and dose.
[74]	Gomard Y et al. Parasit Vectors 2020;13: 392. https://doi.org/10.1186/s13071-020-04267-z Contrasted transmission efficiency of Zika virus strains by mosquito species Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from Reunion Island. (Research Article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) #transm./#dissem. (STR) Sites with active ZIKV transmission.	Ae. albopictus (2 strains, F1) Ae. aegypti (1 strain- F27) Cx. quinquefasciatus (2 strains F0, F1) Dak84 (10^7.4 PFU/ml) PaRi_2015 (10^5.8 PFU/ml) MAS66 (10^6.9 PFU/ml) 26±1°C, 80% RH 12 h: 12 h L:D	For the two Cx. quinquefasciatus strain after challenge with all three virus strains, this species was found to be completely refractory, with no virus detected in bodies, heads, or saliva by plaque assay at either 14 or 21 dpi. For Ae. albopictus, one specimen out of 64 individuals from the location Sainte-Marie was able to be infected, to disseminate and to transmit MAS66, but was refractory to PaRi_2105. The Ae. aegypti strain also showed a low susceptibility to MAS66 as infectious viral particles were only detected in the body of 1/32 individuals at 14 dpi, but a limited number of specimens had disseminated PaRi_2015 strain. The highest VC parameters were observed for the African ZIKV strain Dak84 from Ae. albopictus and Ae. aegypti.	QAS: ≥ 90% (TS = 34, MS = 22) Strengths: Infectious assay to detect ZIKV. Field collected mosquitoes. Multiple virus strains. Weakness: Experimental metadata difficult to extract.
[75]	Li C-X et al. PloS NTD 2020; 14: e0008450. https://doi.org/10.1371/journal.pntd.0008450 Susceptibility of Armigeres subalbatus Coquillett (Diptera: Culicidae) to Zika virus through oral and urine infection. (Research Article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #SG+/#tested (CSGR) #transm./#tested (CTR) Transmission to neonatal mice and urine infection in larvae	Ar. subalbatus Coquillett (Colony) SZ01 (KU866423) 6.5 log₁₀ PFU/ml 26±1°C, 75±5% RH 14 h: 10 h L:D	Midgut, salivary gland, ovary, and saliva tested by PCR at 4, 7, 10 dpi. At 4 dpi, IR for midgut (53% [8/15]), salivary gland (13% [2/15}), ovary (7% [1/15]) and saliva (7% [1/15]. At 7 dpi, IR for midgut (32% [8/25]), salivary gland (16% [4/15]), ovary (12% [3/25]) and saliva (12% [3/25]. At 10 dpi IR for midgut (36% [9/25]), salivary gland (28% [7/25]), ovary (16% [4/25]) and saliva (8% [2/25]. 90% of the infant mice had viral RNA in their brain after being bitten by infectious mosquitoes. ZIKV RNA was detected in 100% of the 4^th instar larvae, but not confirmed by infectious assay. After the mosquitoes emerged as adults, the quantitative PCR results were all negative. Similarly, infectious virus was not detected from the saliva of any adult.	QAS: < 25% (TS = 24, MS = 14) Strengths: Transmission to neonatal mice is convincing. Weakness: Used infectious assays retrospectively indicating much of results may be residual RNA and not represent infectious virus. VC parameters not properly defined, and dpi range is short.
[13]	MacLeod HJ, Dimopoulos G. Mbio. 2020;11. Doi:10.1128/mBio.01765-20 Detailed analyses of Zika virus tropism in Culex quinquefasciatus reveal systemic refractoriness. (Research Article)	VC: #inf./#tested (IR) #transm./#tested (CTR) IT Retest HAI strain previously found to transmit ZIKV. Invitro studies	Ae. aegypti (Rockefeller) Cx. quinquefasciatus JHB (South Africa) HAI (China) FSS13025 (Cambodia, pre-epidemic) 6.9 log₁₀ and 9.3 log₁₀ PFU/ml (oral); 5.5 log₁₀ PFU/ml (IT) 27°C, 80% RH 12 h: 12 h L:D	Midguts tested at 7 dpi, salivary glands, and saliva at 14 dpi. No positive midgut samples at 7 dpi in Cx. quinquefasciatus JHB or HAI strains, but 11% of JHB and 23% of HAI strain midguts had indeterminant titers with ZIKV-Cambodia, which likely did not reflect infectious virus. No positive salivary glands for either of the Cx. quinquefasciatus strains, but 12% of JHB and 51% of HAI salivary gland samples were of indeterminate infectious status. Intrathoracically: 8% of JHB salivary glands were positive and no saliva samples were positive for infectious ZIKV by titer; 44% were negative, and 56% were of indeterminate status. Evidence suggest virus can enter midgut but not replicate but that other barriers exist in Cx. quinquefasciatus.	QAS: ≤ 50% (TS = 30, MS = 17) Strength: Study focused on specific mechanisms to identify the mechanisms resulting incompetence of Cx. quinquefasciatus. Focused on possible mechanisms for previous evidence for VC of species. Weakness: Not a natural system.
[76]	Uchida L et al. Pathogens 2021;10:938 https://doi.org/10.3390/pathogens10080938 Zika virus potential vectors among Aedes Mosquitoes from Hokkaido, Northern Japan: Implications for potential emergence of Zika disease (Research article)	VC: #inf./#tested (IR) #dissem/#inf. (SDR) Temperate species (Japan) Entomology survey	Ae. japonicus, Ae. punctor, Ae. galloisi PRVABC59 (KU501215) 5 log₁₀ FFU/ml 6 log₁₀ FFU/ml 26±2°C, 65–85% RH 12 h: 12 h L:D	No evidence of infection or dissemination of ZIKV was found for Ae. punctor. For Ae. galloisi, 71% (5/7) of tested abdomens contained ZIKV RNA at 5–10 dpi, but by FFA IR was 25% (1/4) at 10 dpi, the latter specimen’s head and thorax was also positive, however titers were low. For Ae. japonicus, ZIKV RNA was detected in 22% of abdomens at 0 and 10 dpi. IR at 10 dpi was 10% (1/10) but no additional evidence of dissemination or transmission was observed.	QAS: < 25% (TS = 21, MS = 11) Strengths: Field collected mosquitoes. Weakness: No transmission assessment. Low sample sizes and cutoffs for assay not clear. Data in supplementary tables inconsistent with text. Poor study design.
[77]	Zimler RA et al. J Med Entomol. 2021; 58: 1405–1411. Doi: 10.1093/jme/tjaa286 Transmission potential of Zika virus by Aedes aegypti (Diptera: Culicidae) and Ae. mediovittatus (Diptera: Culicidae) populations from Puerto Rico. (Research article)	VC: #inf./#tested (IR) #dissem/#tested (CDR) #transm./#tested (CTR)	Ae. aegypti (F2) Ae. mediovittatus (F1) PRVABC59 (KU501215.1) 7 log10 PFU/ml, 6 log10 PFU/ml 5 log10 PFU/ml 4 log10 PFU/ml 28±1°C, 80% RH 15 h: 9 h L:D	Mosquitoes were tested at 15 dpi. Aedes aegypti females were approximately twice as susceptible to Zika infection as Ae. mediovittatus and infection rates increased with viral dose. IRs ranged 70 to 85% at (7log₁₀ PFU/ml) compared to 0–10% for 4 log₁₀ PFU/ml). Aedes aegypti had 5-fold higher disseminated infection than Ae. mediovittatus. Significant differences were observed between Ae. aegypti and Ae. mediovittatus at 6 and 7 log₁₀ PFU/ml, with rates of disseminated infection being 5-fold to 22-fold higher for Ae. aegypti than Ae. mediovittatus. Only two saliva samples were available for Ae. mediovittatus, and not analyzed. Saliva infection was 14.81% at 7 log₁₀ PFU/ml. The remaining lower viral doses had 0% saliva infection for Aedes. aegypti.	QAS: ≥ 75% (TS = 33, MS = 20) Strengths: Field collected mosquitoes., multiple viral doses. Weakness: No infectious assay (PCR only). Only 15 dpi. Difficult to extract experimental metadata.

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Quality grading tool assessment

Our quality grading tool scores (QAS) showed a positively skewed distribution ranging from 20 to 35 out of a possible 38 maximum points. The mean (± SD) score was 29.0 + 4.2, with the lowest quartile representing scores from 20–25 and the top quartile ranging from 33–35 points out of a maximum of 38 points. Methodology scores (MS), a component representing 63% of the total score (TS) showed a similar distribution with a mean (± SD) score of 17.2 + 3.2, with the bottom quartile scores ranging from 11–14 and top quartile from 21–22, out of a possible 24 methodology points. It is notable that the article scoring lowest was also the only article published before 2015 which does not include an infectious virus assay or assess transmission. Four of five of the lowest scores were from papers where no infectious assay was used. Interestingly, of the seven publications in short format (letters, dispatches, short communications), five (71%) scored in the lowest 25%, in large part because important metadata was excluded.

Description of the included studies

Time and geographical clustering of studies

The included papers could be categorized in three-time periods representing the period prior to, during, and after the 2015–2018 ZIKV epidemic in the western hemisphere. Among our selected articles, one was published before 2015, 27 were published between 2015 and 2018, and 17 published after 2018 (Fig 2). The earliest study was on Ae. hensilli, the most abundant Aedes species found on the island of Yap, in the Federated States of Micronesia, the location of the only described ZIKV outbreak reported prior to 2015, prompting interest in the vector capacity of this species [37].

Fig 2 — Figure created in ARCGIS using the following map which the information in the links claim is open source. https://www.arcgis.com/home/item.html?id=30e5fe3149c34df1ba922e6f5bbf808f https://services.arcgisonline.com/ArcGIS/rest/services/World_Topo_Map/MapServer/0 https://www.arcgis.com/apps/mapviewer/index.html?layers=30e5fe3149c34df1ba922e6f5bbf808f.

Among the papers published during the 2015–2018 ZIKV epidemic, the majority (21 of 27) were laboratory studies principally from the USA and Europe that examined 1) laboratory colony mosquitoes that were rapidly available for VC studies; or 2) assessed local mosquito species in those areas that could represent risk of transmission to populations in those regions. For example, laboratory colonies of An. gambiae, An. stephensi, and Cx. quinquefasciatus were tested for competence for ZIKV [58]. Although the laboratory methodology was sound, they did not include an Ae. aegypti comparator, and this study is illustrative of the rush to publish this type of work at the time. There were also articles from Brazil [31] and China [30] that argued the role of Cx. quinquefasciatus as a possible secondary vector of Zika virus, especially in Brazil, where its role in the ongoing and devastating outbreak was of great public health concern.

Starting in 2019, the rational for published studies continued with the evaluation of local species [62,65,67,68,70,71,73,76,77] but also expanded to examining the role of sylvatic species [64], clarify the role of Cx. quinquefasciatus [13,63,66,72,74], and ask more specific questions about the mechanisms of ZIKV transmission within their respective vectors [69,71].

Of the total number of the articles included in this review, 38% (17 articles) were focused in North and Central America, and the Caribbean, (Panama, Puerto Rico, Mexico, Guadeloupe, Canada and USA), 10 papers focused on Europe (Switzerland-France, Netherlands, Spain, Germany, Italy, United Kingdom and Reunion Islands), five on Asia (China and Japan), four on Oceania (French Polynesia- New Caledonia—Samoa-Wallis and Futuna, Papua New Guinea, Australia and French Polynesia), four on South America (Brazil), two on Africa (Tunisia and Senegal), and one was not identified. Two papers tested mosquitoes from different continents: one from Oceania (New Caledonia) and South America (Brazil) and another from North America (USA) and Africa (Tunisia).

Mosquito species tested for vector competence

Table 2 summarizes the mosquito species undergoing VC assays for ZIKV. A total of 27 Aedes species other than Ae. aegypti and Ae. albopictus were studied. Seven, two, and one species from the genus Culex, Anopheles, and Coquillettidia were studied, respectively. Finally, Li et al. [75] examined the vector competence of Armigeres subalbatus as a mosquito species associated with human waste pools and potential infection through exposure to urine. Culex quinquefasciatus was the focus of almost half of VC examining species other than Ae. aegypti and Ae. albopictus (22 articles). Other species studies by multiple authors were Cx. pipiens (9 articles), Ae. japonicus (5 articles), and Ae. vexans, Ae. caspius, and Cx. tarsalis (3 articles each), and Ae. triseriatus, Ae. polynesiensis, and Ae. notoscriptus (2 articles each).

Table 2. Summary of species studied country of origin.

Continent	Country	Species (no of reference)
Africa (n = 4)	Senegal	(Aedes unilineatus, Ae. vittatus, Ae. luteocephalus) [38]
	Tunisia	(Ae. caspius, Ae.detritus) [62]; (Culex quiquefaciatus, Cx. pipiens) [40]
	Reunion Islands^*	Cx. quinquefasciatus [74]
Asia (n = 5)	China	Cx. quinquefasciatus [13,30,54]; Armigeres subalbatus Coquillett [75]
Asia (n = 5)	Japan	(Ae. japonicus, Ae. punctor, Ae. galloisi) [76]
Europe (n = 9)	Italy	Cx. pipiens [41]
	Germany	(Cx. pipiens, Cx. torrentium) [52]; Ae. japonicus [59]
	Spain	Ae. caspius [65,67]
	Netherlands	Cx. pipiens [68,69]
	Switzerland/France	Ae. japonicus [73]
	UK	Ae. detritus [70] (reported as Ochlerotatus detritus)
North- Central America and the Caribbean (n = 17)	USA	Cx. quinquefasciatus [32,40,44,46,51,53,58,61]; Cx. pipiens [39,40,44,46,53]; Cx. tarsalis [46,61]; Ae. triseriatus [39,71]; Ae. vexans [50,56]; Ae. taeniorhynchus [51]; Ae. japonicus [71]; (Anopheles gambiae, An. stephensi) [58]
	Canada	(Ae. cinereus, Ae. euedes, Ae. fitchii, Ae. sticticus, Ae. vexans, Coquillettidia perturbans, Cx. restuans, Cx. tarsalis) [47]
	Mexico	Cx. quinquefasciatus [63]
	Panama	Sabethes cyaneus [60]
	Guadeloupe	Cx. quinquefasciatus [66]
	Puerto Rico	Ae. mediovittatus [77]
Oceania (n = 6)	Papua New Guinea	Ae. hensilli [37]
	French Polynesia	Ae. polynesiensis [45,57]
	Australia	Ae. vigilax [43]; Ae. procax [43]; Ae. notoscriptus [43,48]; Ae. camptorhynchus [48]; Cx. annulirostris [43,48]; Cx. sitiens [43]; Cx. quinquefasciatus [43,48]
	New Caledonia	Ae. polynesiensis [57]; Cx. quinquefasciatus [72]
	Samoa, Wallis, Futuna	Ae. polynesiensis [57]
South America (n = 5)	Brazil	Cx.. quinquefasciatus [31,42,56,72],; (Ae. terrens, Ae. scapulatus, Haemagogus leucocelaenus, Sa. identitus) [64]

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*Administratively belongs to France, and not part of WHO AFRO Member states but geographically in the Africa Region.

Mosquitoes used in vector competence experiments

Mosquitoes were infected orally with an artificial feeder in 41 of the 45 studies, whereas four articles used interferon deficient mice infected with ZIKV to infect Cx. pipiens and Ae. triseriatus [39], Cx. quinquefasciatus [51,61], Cx. tarsalis [61] and Sabethes cyaneus [60]. The dose used to infect mosquitoes ranged from 4 to 8 log₁₀ PFU/ml. Vector competence studies are best done with field derived material rather than strains that have been colonized for many generations. Twenty-four studies included mosquitoes captured from field populations recently from generations F₁-F₃ and three studies from F₄-F₉. Of these, 10 studies also tested some colonized strains. Eighteen studies only used mosquitoes colonized for more than 10 generations and up to 50 years. In most cases, readers were able to infer or distinguish between if colonized or field derived mosquitoes were used, but many articles failed to clearly indicate the generations the mosquitoes had been in the laboratory.

Virus strains used

Studies using well described viral strains, including strain names, source and passage history were considered of high scientific quality. Study design elements including testing >1 strain, use of low passage strains (≤ 20 passages), and inclusion of strains from epidemiologically relevant endemic regions of the world were all items in our quality grading tool. Among the 45 articles included in our systematic review, we identified 27 ZIKV strains used in experiments (Table 3). The most used strain was PRVABC59 (GenBank accession numbers KU501215 or KX601168) isolated in 2015 used in 14 of the studies, followed by MR766 the prototype strain originally isolated from a sentinel monkey during YF surveillance studies in 1947 was included in seven studies. Virus strains used by researchers appear driven mostly by access, but details provided by investigators was not standardized. For example, not all investigators included GenBank accession numbers or detailed information on passage histories. It was clear, however, in 30 of 45 articles (67%) if the virus used had a low or high passage history, and 26 of 45 studies (58%) included a low passage virus in their experiments. Of the articles providing passage numbers, for most specific values that were generally < 10 passages for “low” passage, apart from one research group in Senegal reporting the use of 20 passages for strain MR766 [38]. This latter prototype strain was reported to have between 146–150 passages for most authors who used it in their experiments [37,43,46,53,78]. No recombinant viruses were used for experiments in the studies reviewed. Additionally, 56% (25 of 45) included clear descriptions of both virus and mosquito strains used.

Table 3. Virus strains used in 45 vector competence studies examining secondary mosquito vectors for Zika virus.

GenBank accession numbers were not included unless specified in original article.

Virus Strain	GenBank No.^# (reference)	Country of origin	Year of isolation	Host	References
MR766 (prototype)	AY632535 [46]	Uganda	1947	Sentinel Monkey	[37,38,43,46,53,58,67]
MAS66 MYS/P6-740	KX694533 [66,74] KX601167 [61]	Malaysia	1966	Aedes aegypti	[61,66,72,74]
DAK41525	KU955591	Senegal	1984	Ae. africanus	[46,51]
DAK84	KU955592 [66,73]	Senegal	1984	Ae. taylori	[66,72–74]
ArD128000		Senegal	1997	Ae. luteocephalus	[38]
ArD132912		Senegal	1998	Ae. dalzieli	[38]
ArD157995		Senegal	2001	Ae. dalzieli	[38]
ArD165522		Senegal	2002	Ae. vittatus	[38]
HD78788		Senegal	1991	Human patient	[38]
Cambodia 2010 FSS13025	KU955593 [48] JN860885 [13,65]	Cambodia	2010	Human patient	[13,48,51,65] [48]
H/PF13 PF13/251013-18	KX369547 KJ776791 [41]	French Polynesia	2013	Human patient	[41,45]
PLCal_ZV	KF993678	Thailand^*	2013	Human patient	[47]
NC-2014-5132	SRR5309452 [57]	New Caledonia	2014	Human patient	[40,55,57,62]
MRS_OPY_Martinique_PaRi_2015	KU647676 [66,74]	Martinique	2015	Human patient	[66,72,74]
MEX1-7	KX247632 [60]	Mexico	2015	Ae. aegypti	[51,60]
MEX1-44		Mexico	2015	Ae. aegypti	[51]
Pariaba_01	KX280026	Pariaba, Brazil	2015	Human patient	[13]
PRVABC59	KU501215 [32,39,46,47,50,65,71,76,77] KX601168 [61]	Puerto Rico	2015	Human patient	[32,39,46,47,50,51,53,58] [44,61,65,71,76,77]
PE243	KX197192 [31]	Recife, Brazil	2015	Human patient	[31,49,70]
SPH2015	KU321639	Sao Paulo, Brazil	2015	Human patient	[13,49,61]
SZ01	KU866423	China	2016	Human patient^***	[30]
ZJO3	KU820899	China	2016	Human patient	[54]
FB-GWUH	KU870645	Guatemala^**	2016	Fetal brain	[52,59]
HND/R103451/2015	KX694534	Honduras	2016	Placenta^****	[53]
Unspecified Puerto Rico Strain		Puerto Rico	2016	Human patient	[56]
Rio-S1	KU926310	Rio de Janeiro, Brazil	2016	Human patient	[42,64]
Rio-U1	KU926309 [42,64]	Rio de Janeiro, Brazil	2016	Human patient	[42,49,64]
EVAg Ref-SKU 011V-01621	KU937936 [68,69]	Suriname	2016	Human patient	[67–69]

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^#GenBank Accession numbers were not reported by all authors, we include the references when reported.

*Individual with travel history to Thailand;

**Individual with travel history to Guatemala;

***Individual with travel history to American Samoa;

****Individual with travel to Honduras in 2015.

Vector competence parameters

The principal objective of the 45 studies evaluated was a straightforward evaluation of the ability of one or more mosquito species to become infected, disseminate, and transmit ZIKV at different viral titers and rearing temperatures. There were only a few exceptions where the authors objectives were focused on understanding the biology behind VC patterns observed [13,55]. Although, the main VC parameters (infection, dissemination, and transmission) were universally recognized, terminology used to describe these parameters was variable and more alarming, inconsistent; the terms dissemination rate (DR) and transmission rate (TR) had distinct definitions depending on the article. Fortunately, most authors provided definitions for the terms used, but unless readers are meticulous, mistakes in interpretation are inevitable. All 45 articles estimated infection rates either by testing mosquito bodies (usually the abdomen/thorax) or midguts for Zika virus or RNA. The denominator used was the total number mosquitoes tested which represented mosquitoes that were blood fed and held for 7–21 days. Dissemination rates were estimated in 37 of the 45 studies through testing heads, wings, legs, and in one case salivary glands and another ovaries/exoskeleton. Either the number of infected mosquitoes (stepwise approach), or alternatively, all mosquitoes tested (cumulative approach) were used in the denominator. The most relevant parameter, transmission was estimated in 39 of the articles. Two papers used salivary glands as a surrogate for virus transmission [31,54] whereas 37 articles tested saliva. In two articles, infected mosquitoes were observed to transmit Zika virus to neonatal mice [30,75]. As with dissemination, the denominator used to estimate transmission was the number of mosquitoes with disseminated infections (stepwise) or all the total number of mosquitoes tested (cumulative). Some authors use the term transmission efficiency and more recently dissemination efficiency [79] to make the distinction between stepwise and cumulative rates. Articles using the term “efficiency” distinguish between stepwise rates (e.g., dissemination rate [DR], transmission rate [TR]) from cumulative rates (e.g., dissemination efficiency [DE], transmission efficiency [TE]). The major cause of confusion is that among the 45 articles in our review, 22 used DR and TR to signify cumulative rates, whereas 17 articles used the same terminology, “DR” and “TR” to describe stepwise rates while sometimes also presenting cumulative rates using the terms “DE” and “TE”. Other authors avoided these terms and used their own well-defined definitions. Other terms used are dissemination infection rate (DIR) and transmission infection rate (TR[D]) to contrast these two different VC parameters.

Laboratory assays used to detect ZIKV or ZIKV RNA

Protocols employed to test mosquitoes for ZIKV or RNA varied across studies, but there is a clear distinction between studies that only used PCR to detect RNA as opposed to the more labor intensive and technical assay that directly measured infectious virus (Table 4). About 43% of the studies only used PCR assays to measure infection and dissemination, but for transmission 29% of the studies relied solely on PCR. For the identification of the transmission status of the tested mosquitoes a variation of laboratory assays was used. About 20% of the studies used a combined strategy of screening by either PCR or an infectious assay, followed by confirmation or titration with the other type of assay.

Table 4. Laboratory Assays used to detect Zika virus or RNA for infection, dissemination, and transmission assays.

Number of studies (%).

Assay	Description	Infection (n = 45)	Dissemination (n = 40)	Transmission (n = 41)
PCR	Both RT and qRT PCR	10 (22%)	17 (42.5%)	12 (29%)
Infectious virus	Plaque, TCID₅₀ or Fluorescent Focus assay using Vero or C6/36 cells,	16 (36%)	15 (37.5%)	20 (49%)
Combination	Varied protocols, some screening with infectious assay followed by titration with qPCR or PCR screen and confirmation of all or subset of samples by infection assay.	19 (42%)	8 (20.0%)	9 (22%)

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Studies using mice

In six studies, mice were utilized (either neonatal or genetically modified) to infect mosquitoes in a more natural and efficient manner or to confirm transmission of virus from the mosquito to a new host. Vector competence parameters were higher for mosquitoes fed on mice than those using artificial feeding strategies or direct testing of saliva, consistent with the observation that blood from viremic animals are typically more infectious for mosquitoes than artificial meals [80,81]. Four studies used interferon deficient mice infected with ZIKV to infect Cx. pipiens and Ae. triseriatus [39], Cx. quinquefasciatus [51], Cx. tarsalis [61] and Sa. cyaneus [60] mosquitoes experimentally, compared to Ae. aegypti. All species infected using mice were unable to transmit ZIKV, except for Sa. spp., where a single mosquito (11%) that fed on a mouse with 6.8 log₁₀ PFU/ml after being held for 21 days tested positive for ZIKV by plaque assay, compared to 70% of Ae. aegypti.

Two studies evaluated transmission in parallel with saliva testing for Cx. quinquefasciatus [30] and Ar. subalbatus [75]. The former study continues to be cited as the only credible evidence for the competence of Cx. quinquefasciatus for ZIKV transmission. Ten days after being fed on by orally infected mosquitoes, the brains of eight of nine neonatal mice tested positive for ZIKV RNA. Similarly, 8-day post exposure 80% of 10 mosquitoes had ZIKV RNA positive saliva, but only 1 of 3 mosquitoes >12 d post exposure had RNA positive saliva. Biologically, we would expect that once saliva tests positive, it would remain so; low sample numbers made clear evaluation difficult, but the infection of the neonatal mice is difficult to interpret as anything other than evidence of virus transmission. Using almost identical methodology for Ar. subalbatus, saliva tested positive for ZIKV RNA in 7–12% of the mosquitoes exposed and nine of ten of the neonatal mouse brains tested positive for RNA. Although both studies presented virus titers observed in the saliva samples, these were generated from standard curves developed in the laboratory, not based on the mosquito samples themselves. In both studies, the failure to use and infectious virus assay to test for ZIKV prevented unequivocal conclusions about each species as a vector.

Review of evidence for individual vector species other than Ae. aegypti and Ae. albopictus

Existing evidence for VC of vector species other than Ae. aegypti and Ae. albopictus for ZIKV can be divided into the following relevant epidemiological contexts: 1) African [38] and South American [60,64] forests cycles; 2) local vector species from areas directly affected by ZIKV transmission generally associated with Micronesia/Polynesia in island settings [37,45,57]; 3) local vector species in normally temperate areas where risk of ZIKV transmission was evaluated for outbreak readiness which included species in Australia [43,48], Mediterranean region [62,65,67], Europe [40,41,52,68,69], Canada [47], USA [39,50,51,56,61,71,77] and the examination of one cosmopolitan species Ae. japonicus [59,76]; and 4) in settings similar to endemic DENV/CHIKV transmission where Cx. species are potentially involved. The latter category resulted a considerable focus on Cx. species in areas with and without ZIKV transmission. Table 5 summarizes the existing evidence, by species and the four categories above, of VC by mosquito species.

Table 5. Vector competence (VC) parameters of species investigated as possible vectors of Zika virus.

Infection Rate (IR, [#inf./#tested]) is defined as the percentage of mosquitoes contain virus in bodies or midguts (number positive/number tested). Terminology used for dissemination and transmission are expressed as cumulative (C) or stepwise (S) rates depending on the experimental design. For dissemination, we use cumulative dissemination rate (CDR, [#dissem./#tested]) or stepwise dissemination rate (SDR, [#dissem./#inf]), defined as the percentage of mosquitoes containing virus in head, legs+wings, or salivary glands/ovaries (number positive/number of engorged mosquitos tested for infection [CDR] or number positive /number of infected mosquitoes [SDR]). For transmission, we use cumulative transmission rate (CTR, [#transm./#tested]) or stepwise transmission rate (STR, [#transm./#dissem.]), defined as the number of mosquitos with virus in saliva or transmitting ZIKV to a mouse (number positive/number of engorged mosquitoes tested [CTR] or number positive/number disseminated infections [STR]). NT = not tested. Raw data presented in S1 Data.

Type	Species	IR	SDR	CDR	STR	CTR
Forest Africa	Ae. unilineatus (5 ZIKV strains) [38] Ae. vittatus (5 ZIKV strains) [38] Ae. luteocephalus (5 ZIKV strains) [38]	19% 14% 75%	5% 27% 42%	1% 4% 32%	0% 20% 50%	0% 1% ~17%^a
Forest South America	Ae. terrens (21 dpi) [64] Ae. scapularis [64] Sa. identicus [64] Sa. albiriprivus (2 strains) [64] Hg. leucocelaenus (7,14,21 dpi) [64] Sa. cyaneus (21 dpi) [60]	0% 2% 0% 1% 27% 1%	0% 0% 0% 0% 11% 100%	0% 0% 0% 0% 3% 1%	0% 0% 0% 0% 0% 100%	0% 0% 0% 0% 0% 1%
Local Polynesia	Ae. polynesiensis (2 studies) [45,57] Ae. henselli (4.9-5.9 log₁₀ PFU/ml) [37]	36;87% 69%	50;45% 69%	18;39% 13%	0;3% NT	0;1%^b NT
Puerto Rico	Ae. mediovittatus (4-7 log₁₀PFU/ml) [77]	29%^c	2%	1%^c	NT	NT
Australia	Ae. camptorhynchus [48] Ae. notoscriptus (2 studies) [43] [48] Ae. vigilax [43] Ae. procax [43]	28% 57% 34% 57% 33%	14% 21% 3% 47% 50%	NT^d 12% 1% 27% 17%	14% 0% 50% 0% 0%	<1%^d 0% <1%^d 0% 0%
China	Armigeres subalbatus (10 dpi) [75]	36%	78%	28%(SR)	29%	8%^e
Local	Refractory species Ae. cinereus [47], Ae. punctor [76], Ae. galloisi [76], Cx. annulirostris [43,48], Cx. sitiens [43], An. gambiae [58], An. stephensi [58]	0%	0%	0%	0%	0%
Widespread Temperate	Ae. vexans (3 studies) 10^5.8 PFU/ml, 25°C [47] 10^6.8-7.2 PFU/ml, 27°C [50] 10^5.3 PFU/ml, 28°C [56] Ae. triseriatus (2 studies) [39,71] Ae. taeniorhynchus (10^6.8 PFU/ml)^g [51] Ae. caspius (3 studies) (PCR only) [62, 65] (Plaque assay) [67] Ae. detritus (2 studies) 10^7.2 PFU/ml, 14 dpi [62] 10⁶ PFU/ml, 14-28 dpi, 19°C [70] 10⁶ PFU/ml, 14-28 dpi, 21°C [70] 10⁶ PFU/ml, 14-28 dpi, 24°C [70] 10⁶ PFU/ml, 14-28 dpi, 28°C [70] 10⁶ PFU/ml, 14 dpi, 31°C [70] Ae. japonicus (5 studies) [76] 10^6.5-7.7 PFU/ml, 24 °C [71] 10^7.2 TCID₅₀/ml, 28 °C [68] 14,21 dpi, constant 27°C [73] 14,21 dpi, fluctuating 21-27°C [73] 14 dpi, 21°C [59] 14 dpi, 24°C [59] 14 dpi, 27°C [59] Cx. pipiens [40, 41,44,46,71] 18-27°C, 14,21 dpi [52] 10^4-7.1 PFU/ml, 28°C [53] 10⁷ TCID₅₀/ml, 28°C [69] Cx. molestus [69] 18 and 27°C, 14,21 dpi [52] Cx.. tarsalis [46,47] 14,21 dpi, 26°C [61] Cx. torrentium 14,21dpi 18°,27°C [52]	13% 80% 28% 12;25% 0% 10;40% 0% 75% 14% 24% 17% 15% 15% 10% 20% 10% 87% 50% 10% 24% 67% 0% 16% 9% 47% 0% 24% 0% 12% 11%	47% 20% 11% 0% 0% 0% 0% 0% NT NT NT NT NT NT 47% 83% 22% 48% NT NT NT 0% NT 0% NT 0% NT 0% 38% NT	6% 16% 3% 0% 0% 0% 0% 0% NT NT NT NT NT NT 9% 8% 19% 24% 0% ~26% ~13% 0% NT 0% NT 0% NT 0% 5% NT	NT 29% 5%^f 0% 0% 0% 0% 0% NT NT NT NT NT NT 29% 40% 57% 41% 0% 0% 14% 0% 0% 0% NT 0% 0% 0% 0% 0%	NT 5% 1%^f 0% 0% 0% 0% 0% 3% 9% 10% 13% 15% NT 3% 3% 11% 10% 0% 0% 10% 0% 0% 0% 3% 0% 0% 0% 0% 0%
Widespread Temperate	Refractory species Ae. euedes [47], Ae. fitchii [47], Ae. strictus [47], Cx. restuans [47,71], Coquillettidia perturbans [47]	0%	0%	0%	0%	0%
Worldwide	Cx. quinquefasciatus	0-2%^h	0%^h	0%^h	0%^h	0%^h

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^aTR was reported as 50% representing denominator of 19, but text states that 27 saliva specimens were tested.

^bAt 21 dpi CTR = 3%.

^cEstimated IR from a logistical model at 4 titers (18%). The range for IR at lower titers was 0–75% increasing with dose. Dissemination was observed only at the highest titers between 4–5%.

^dDissemination measured as dissected positive carcasses including ovaries and exoskeleton over number mosquitoes exposed and transmission had distinct denominator from IR, dissemination, and transmission experiments. It is impossible to extract the number exposed and denominators are higher for dissemination and transmission experiments. Because denominators are independent, we present as SDR and STR.

^e90% of neonatal mice that were fed on by infected mosquitoes had ZIKV RNA detected in their brains.

^f34% transmission rate after IT inoculation, used to estimate TR/TE.

^gInfected from Ifnar-/- mice with at 4.7 log₁₀ and 6.8 log₁₀ PFU/ml, only infected at higher titer.

^h18/22 studies

Forest cycles

Laboratory incrimination studies for African forest vectors was limited to a single study conducted in Senegal [38]. The only forest species with laboratory evidence incriminating them as a ZIKV vectors were Ae. vittatus and Ae. luteocephalus, both able transmit virus with CDR of 1 and 17%, respectively. The same study did not find Ae. unilineatus to be competent, and the author’s pointed out the need for further studies on other important forest species that could be involved in transmission. For South American forest species, Ae. terrens, Ae. scapularis, Sa. identicus were completely refractory to infection, Sa. albiprivus had 0.5% infection rate, but Haemagogus leucocelaenus had infection rates of 14.8 to 40%, dissemination rates of 2.2 to 5% depending on day post infection but showed no evidence of transmission [64]. A laboratory study from a long-term colony of Sa. cyaneus had one mosquito with evidence of infection, dissemination, and transmission out of 69 tested.

Vectors associated with Micronesia/Polynesia in Island settings

Evidence for Ae. hensilli and Ae. polynesiensis as vectors for ZIKV was evaluated because of their high abundance at the time of ZIKV outbreaks. Infection of Ae. hensilli was demonstrated but no evaluation of transmission (saliva or salivary gland testing) was conducted [37]. Aedes polynesiensis showed disseminated infections with ZIKV under laboratory conditions but did not transmit the virus [45]. Dissemination was far less efficient than observed for Ae. aegypti. In contrast, a later study showed two populations of Ae. polynesiensis from French Polynesia and Wallis and Futuna had high infection rates (71%) that were less variable than Ae. aegypti populations from the same region and showed high dissemination rates (SDR = 45%, CDR = 39% and low transmission rates (STR = 3%, CDR = 1%) [57].

Local vectors not found to be competent to transmit Zika Virus

In Australia, there are many local Aedes species examined for their ability to transmit ZIKV [43]. Ae. vigilax, Ae. procax, and Ae. notoscriptus showed infection and dissemination rates consistent with Ae. aegypti, but all failed to transmit the virus. In contrast, a later study [48] found some transmission (<1%), but lower infection and dissemination rates than the previous study for both Ae. notoscriptus and Ae. camptorhynchus.

In Canada, of the three Culex species examined, Cx. sitiens and Cx. annulirostris were completely refractory to infection and only 2 out of 30 Cx. quinquefasciatus were infected but none developed disseminated infections. Similarly, in Europe, no evidence was found for transmission in Cx. pipiens pipiens or Cx. pipiens molestus [52,69], although both species did accumulate virus in saliva after intrathoracic injection. North American species Ae. triseriatus [39,71] and Ae. taeniorhynchus [51] were both found to be refractory to ZIKV infection. Ae. mediovittatus from Puerto Rico was studied [77] and found to be half less susceptible to oral infection than Ae. aegypti, indicating a more effective midgut infection barrier in Ae. mediovittatus. Dissemination rates were < 5% compared to 40–95% in Ae. aegypti. Insufficient saliva samples were obtained to evaluate transmission for this species. A single study [58] found laboratory colonies of An. gambiae and An. stephensi to be refractory to ZIKV infection.

One study from China investigated Ar. subalbatus, a species of interest because it developed in human waste lagoons where ZIKV could be shed in urine. Average infection rate for this species was 43% (measured in midguts) and 90% of the infant mice that were bitten by infectious mosquitoes had viral RNA in their brain although was not detected in mosquito saliva. Furthermore, Ar. subalbatus larvae reared in water containing ZIKV and human urine did not result in any adult mosquitoes with detectable ZIKV RNA in saliva, providing no evidence of transmission via this route. Ae. galloisi and Ae. punctor were evaluated in Japan, showing very low rates of susceptibility to ZIKV infection but dissemination and transmission were not evaluated. These species provide a good example of the methodological difficulties associated with evaluation when blood feeding rates are low, and the numbers of infected mosquitoes are so low a true evaluation of dissemination and transmission is difficult.

Species with evidence of very low transmission potential

Species with evidence of transmission, albeit at very low levels, were Ae. vexans, Ae. detritus, and Ae. japonicus (Table 5). Three studies indicated that, Ae. vexans is a competent vector for ZIKV [47,50,56] and while infection rates were high in some of these studies, all of them reported low dissemination and transmission rates. In Canada, of the 4 (13%) Ae. vexans positive for ZIKV RNA, 2 (50%) had ZIKV RNA detected in their legs for an overall dissemination rate of 6%. Twenty-three percent of Ae. vexans infected by intrathoracic injection had ZIKV RNA detected in their saliva. Evidence for Ae. vexans transmission was also found in Colorado, USA, with relatively high infection rates ranging from 66–91%, dissemination rates from 3–25%, but low transmission rates from 2–7%. Consistent with these finding was another study [56] from the great plains region that had high transmission rates in Ae. vexans, after intrathoracic infection, suggesting that the primary barrier in this species is midgut escape barrier. In the United Kingdom Ae. detritus (reported as Ochlerotatus detritus in original article) became infected and showed infectious virus in saliva at rates lower than Ae. albopictus, but saliva positivity increased at higher temperatures [70]. In the Mediterranean Region neither Ae. detritus nor Ae. caspius showed the ability to disseminate or transmit ZIKV [62,65,67]. Additionally, Ae. caspius had low infection rates. Multiple studies have shown that Ae. japonicus is a competent vector for ZIKV. However, the infection, dissemination, and transmission rates of the virus were found to be temperature-dependent, especially at temperatures above 27°C [59,68,71,73].

Possible role of Culex species

A total of 24 of the 45 studies identified in our systematic review tested Culex species; of these 22 included Cx. quinquefasciatus, and 14 used mosquito strains recently brought from the field to laboratory. Thus, the overwhelming weight of evidence is that Culex species are unable to transmit ZIKV. Culex quinquefasciatus species were refractory in 10 laboratory studies using mosquitoes from colonies (n = 5) and field (n = 5). Of the remaining 11 studies infection rates were very low, dissemination rare, and one report on infectious saliva in one mosquito infected at very high virus titer. Three studies make claims that Cx. quinquefasciatus can transmit ZIKV. Guedes et al. [31] showed viral particles in salivary glands by electron microscopy, suggesting this was evidence of transmission, but was not able to demonstrate infectious virus in saliva. A study from China [30] showed transmission of ZIKV from laboratory infected mosquitoes to mice, however saliva from the same mosquitoes, tested by PCR only, showed presence of RNA at 8 dpi (80%) and 12 dpi (10%) but not 16 dpi, a result consistent with the observed detection of decreasing levels of noninfectious pieces of RNA rather than infectious virus. Finally, infectious virus was detected on FTA cards exposed to laboratory-infected field mosquitoes, evidence of transmission [32]. In summary, the studies evaluating Culex. laboratory competence indicated that Culex species has poor VC for ZIKV overall, but the possibility of geographically isolated strains that are competent must be considered. Macleod and Dimopoulos [13] provide a comprehensive review and interpretation of the data available for Cx. quinquefasciatus VC to date.

Role of temperature

Four of the reviewed articles conducted VC experiments that asked specific questions about the impact of rearing temperatures on European Culex species [52], Ae. detritus [70] and Ae. japonicus [59,73]. Transmission increased with rearing temperature for both Aedes species tested and when Ae. japonicus was reared at more realistic fluctuating temperature scheme (daily variation between 14°C and 27°C compared 27°C) there was no impact of fluctuating temperatures on VC (Table 5).

Discussion

As of March 2022, our systematic review confirms that, Ae. aegypti and Ae. albopictus are by far the most significant vectors of ZIKV worldwide, but that some temperate Aedes species, principally Ae. japonicus, Ae. vexans and to some degree Ae. detritus, are capable of transmitting ZIKV efficiently at higher temperatures. As climate change increases average temperatures in temperate regions of the world, these species could grow in importance as secondary vectors of ZIKV [82], if the behavioral characteristics of the vectors and humans favor contact between them. Although, Cx. quinquefasciatus received considerable interest and investigation (22 articles) as possible vector for ZIKV, the weight of the evidence suggests it is not important but recognizes the possibility that this species could be relevant for a limited number of localized genetic mosquito vector-virus strain combinations.

Our systematic review illustrates the inherent difficulties in the synthesis and interpretation of these kinds of studies because of the heterogeneities in experimental design and data presentation across them. A recent effort to address this issues published after the completion of our systematic review, provided guidance on minimum data reporting standards for VC studies highlighting this problem, stating “that the complexity of these experiments (vector competence), and the variety of conditions under which they are conducted, make it difficult to meticulously share (and synthesize) all relevant meta data, especially with consistent enough terminology to compare results across studies” [7,82].

Although, we developed and applied a quality assessment tool as part of our systematic review, its application proved cumbersome. First, not all the reviewed manuscripts provided data sets either posted online or in a data repository. New studies should be required to do so, and this requirement will help overcome problems associated with inconsistent reporting of study metadata. The purpose of our QAS tool was not to rank articles, but to clearly delineate minimum standards, and distinguish between different levels of evidence quality. As an analogy, for field trials, data from randomized controlled trials is seen as the gold standard, followed by non-randomized controlled trials, and finally observational studies [84]. For VC studies, those using infectious laboratory assays, with higher sample sizes (including replication and statistical analysis), a wider range of mosquito and virus strains, and clear measure of transmission represent higher quality data than those that only detect RNA, have smaller samples, and are limited by less natural conditions using colonized mosquito strains and heavily passaged viral strains, but these later studies can be informative is reporting is transparent and study limitations are understood. Our grading tool could not distinguish between well designed and executed studies where the reporting was limited (e.g., short format articles) and poorly designed studies. We encourage subject area experts to modify this tool if it is to be applied for future systematic reviews.

Research interest and study design often driven by outbreaks and financial considerations

Interest in mosquito vectors of ZIKV, in particular secondary vectors, has been driven by outbreak response with limited publications until 2016, about one year into the ZIKV outbreak in the Western Hemisphere, especially its association with fetal abnormalities.

In the following paragraphs we first describe the characteristics of ZIKV VC studies conducted on 1) forest species, 2) in response to the Yap Island outbreak in 2014, 3) rapid publications during 2015–2017 ZIKV outbreak, and 4) 2018 to present where study quality has improved, and research has been more systematic with the aim to better understand and predict ZIKV disease transmission [7].

Prior to 2007, sylvatic YF surveillance studies isolated ZIKV from wild-caught African forest species, but laboratory-based VC studies to properly assess the competence of these species was not a research priority and limited to a single study included in our systematic review [38]. These studies identified several mosquito species of interest for further study. Unfortunately, technical and logistical challenges for forest species that cannot survive or feed on blood in laboratory conditions in sufficient numbers required to conduct VC studies represented a significant barrier to VC evaluation. These limitations, extend to two studies conducted on forest species from the western hemisphere [60,64].

A second torrent of research on mosquito vectors occurred between 2007–2014 in response to ZIKV outbreaks in the South Pacific, but studies were quite limited overall, one conducted as part of the outbreak response did not include evaluation of transmission [37]. Only two studies on the potential secondary vector Ae. polynesiensis showed mixed transmission results [45,57]. This observation highlights the potential for large genetic and VC differences among vector populations of the same species. Calvez et al. [57] did evaluate three Ae. aegypti and two Ae. polynesiensis populations with a single virus strain from New Caledonia. Ideally, more strains from more locations with distinct virus strains could be evaluated, but these experiments are labor intensive and costly.

At the initiation of the South American ZIKV outbreak, VC studies appeared to be designed based on what mosquito colonies and virus strains could be obtained rapidly. Many of these initial publications, were short communications, and in many ways appeared rushed. There was considerable concern in Europe and the US, that ZIKV could potentially be transmitted by local species, leading to a higher proportion of studies from these countries. Although assessing the risk of potential vectors in these locations made sense, early studies used mosquito strains that had been in colony often for decades, transitioning into more studies that collected mosquitoes directly from the field that could be brought back to the laboratory. Also, reading between the lines were studies that appeared to retrospectively verify they were detecting infectious virus rather than relying exclusively on PCR to detect ZIKV RNA. Thus, these initial publications often lacked detailed metadata that are important for future meta-analyses or full assessments of a particular species’ vector potential. These studies did provide needed quick assessments for making Public Health decisions. ZIKV represents a prototype of what occurs during the initial response to emerging Public Health Emergency, in which an unprecedented amount of research was published very quickly [83].

Research priorities should include a more systematic and comprehensive approach to understanding forest species. ZIKV has been isolated from 15 Ae. species as well as a few species from Culex, Eretmapodites, Mansonia, and Anopheles genera [4,5,23] where no laboratory studies have been conducted. Virus isolation from field-collected mosquitoes does not constitute evidence for their role as a vector, only that the mosquito fed on an infected host, or contamination occurring within a trap. Laboratory VC studies are required for vector implication to justify further studies on the role of these species in potential spill-over events as well as species whose ecology has changed and could have important implications for ZIKV transmission. For example, Ae. africanus and Ae. furcifur became important vectors of YF in villages where these species moved freely between the forest canopy and villages nearby where Ae. aegypti was absent. Theoretically, the same is possible for ZIKV.

There was significant proliferation of studies on Cx. quinquefasciatus based on a premature press release [85] and data from a single study [30], despite significant evidence otherwise. It was not until 2020, that a clear effort was made to replicate the results of the original study from China and evaluate the existing evidence for competence of this species [13]. At the time we had completed our search in March 2022, many groups had evaluated this species as well as many other species within the Culex genera. Many of these studies were a part of coordinated research effort by the Zika Alliance. Obadia et al. [79], a study published a few months after concluding our search, provides an illustration of a coordinated research effort using a unified protocol across a wide geographic distribution using strains of Ae. albopictus and Ae. japonicus directly from the field involving multiple research groups. These kinds of efforts allow experimental studies that would be otherwise logistically impossible for a single research group. A collaborative approach where multiple research groups evaluate one or two local mosquito strains and if possible, an epidemiologically relevant virus strain (e.g., in sites with endemic transmission a strain from the same location), provides more credible and robust conclusions. We now have interesting candidates for future study: Ae. vexans, Ae. japonicus, and Ae. detritus for more comprehensive studies examining the role of ambient temperature.

Variability in laboratory methodologies

VC studies were generally of high quality methodologically, but have highly variable study designs, using different mosquito and ZIKV virus strains (appropriate), different blood feeding techniques (different artificial feeders, animal blood sources, murine models), virus preparations (dose, frozen versus fresh virus), methods for evaluating infection (testing mosquito bodies versus midguts), dissemination (testing mosquito heads, legs, wings, or salivary glands), and transmission (direct saliva testing, saliva amplified in cell culture, saliva amplified in mosquitoes by intrathoracic inoculations, with mice to confirm transmission). This variability makes comparison across studies difficult, although not impossible. Unfortunately, details on experimental designs were inconsistent across the articles reviewed.

Recently, a minimum data standard for VC experiments was proposed [7] which suggested metadata reporting for mosquito and virus strains used and experimental details and outcomes. Most articles provided most of the necessary experimental metadata, but experimental outcomes were less consistent overall. Mosquito and virus strain metadata varied across the publications, and for some articles the reader was left to infer the age of mosquito colonies and virus passage histories. Some authors provided this information clearly in tables with clear virus passage histories and the number of generations a mosquito strain had been in the laboratory, whereas other articles implied that a strain was local and “recent” but did not provide this information. The best example of this was the HAI strain of Cx. quinquefasciatus from China originally found to transmit ZIKV to neonatal mice [30] and later evaluated by MacLeod and Dimopoulous [13]. Neither publication provides precise information on when that colony was established other than the year 2014.

During the review, our team attempted to extract all the metadata mentioned and would recommend that authors include it in a tabular format in the main body of the publication, rather than placing these details in supplementary information or referring readers to a previous publication. Even if “none” or “not known” is included for mosquito colony generations or virus passage history, excluding this information leaves the perception of a lack of transparency. In the following sections, we highlight the more prominent issues associated with interpreting the reviewed articles, but that apply to the VC literature in general.

Definitions of Infection, Dissemination, Transmission, and other terms

When assessing VC, the following barriers have been described: midgut infection and escape barriers, and the salivary gland infection and escape barriers. Infection implies that virus has passed the midgut infection barrier, whereas dissemination -where virus is found throughout the body of the mosquito- suggests the virus has made it at a minimum through the midgut escape barrier [6,7,82]. Virus in saliva means virus has escaped from the salivary glands. Understanding where the barriers to VC exist for individual species is important to delineate biological mechanisms underlying the ability of virus to make it from the midgut to the saliva at titers sufficient to infect another host. These processes are essential for predicting the potential for emergence events where critical mutations in either the virus or vector would result in a virus-vector pair switch from incompetent to competent. Thus, presentation of stepwise rates has value, but in the context of a Public Health Emergency, like observed with ZIKV, the critical question is “can this species serve as a vector?”. To answer this later question, cumulative rates are the most informative since they are using the total number of mosquitoes exposed and tested in the denominator. There is no technical reason, that both stepwise and cumulative rates cannot be presented side by side, and many of the manuscripts did this, but the terms used to present these rates were not consistent. A major concern remains that the terms dissemination rate (DR) and transmission rate (TR) have different definitions depending on the article. One alternative has been the introduction of the terms dissemination efficiency (DE) and transmission efficiency (TE), terminology used in 11 of the reviewed articles but clearly distinguishes between stepwise and cumulative rates [79]. Moving forward, our recommendation is the universal adoption of the terminology for cumulative and stepwise rates as we have presented in Table 5.

Both definitions are presented clearly in Wu et al. [7], who make the additional recommendation that authors include raw data from their experiments in a supplementary appendix so that those calculations can be made by anyone reading the manuscript. They point out the “original raw data may never be reported and is often impossible to reconstruct from provided bar or line charts” and “derived quantities often follow different calculations, with (usually intentional but) very different biological meaning (e.g., the difference between ‘dissemination rate and ‘disseminated infection rate’ are often used interchangeably)” [7]. Although, most articles clearly defined their parameters, the choice on how to present data was often driven by the desire to tell a story. Interpretation of these parameters are also hindered by the inevitable problem of low sample sizes for dissemination and transmission assessments. When infection and dissemination rates are low, transmission rates presented as a percentage are often based on 1–2 mosquitoes.

Finally, in the reviewed articles the number of days after mosquitoes were fed blood infected with ZIKV was reported in days post-infection (dpi) for most articles and as days post-exposure (dpe) in a few. Although less problematic than the terms dissemination and transmission rate having distinct definitions, the term dpe is more appropriate and should be adopted to standardize terminology in the VC literature.

Methods that dissect out body parts versus testing bodies, legs, wings, and heads compared to murine models

Although, most published articles use the methodology of testing mosquito bodies (abdomens) to indicate infection, either wing, legs, heads, thoraces, and sometimes other organs to indicate dissemination, there is at the very least qualitative differences when comparing to papers testing dissected midguts and salivary glands. Experiments directly comparing these methodologies would be helpful to the field. The use of genetically modified murine models showed higher infection, dissemination, and transmission rates than experiments using artificial feeding methods. The observation that using a viremic animal to infect mosquitos represents a more realistic model than artificial blood feeding methods is not a new concept [80,81,86]. The ability to implement more realistic and appropriate study designs for VC studies in low- and middle-income countries where ZIKV and other arboviruses are endemic may be limited due to financial and logistical restraints. Thus, more detailed head-to-head comparisons of results from studies using artificial blood feeding with those using animal models or direct human feeds need to be conducted. The same is true for testing transmission from mosquitoes to a new host, that is—head-to-head comparisons of virus detection in saliva to infection of neonatal mice, especially under circumstances when mice become infected in the absence of a clear presence of infectious virus in saliva.

Minimum quality standards for vector competence studies and how to evaluate them

There are many challenges to the implementation of minimal quality standards for VC studies and there is significant recognition that their absence is a significant problem, but at the same time reluctance to impose undue burden on investigators, especially in locations with limited resources and facilities [7,82]. Our strongest recommendation is the transparent and clear reporting of metadata associated with VC experiments, as suggested by Wu et al. [7], and reflected by our quality grading scale. We strongly support the recommendation to provide access to raw data sets. There is strong consensus that presentation of experimental results needs to clearly report total counts of tested and positive mosquitoes at each stage evaluated (infection, dissemination, and transmission). Also critical is that transmission competence should only indicate the presence of virus in saliva, and not just positive salivary glands [83]. Reliance on PCR assays only, with no component to verify that there is infectious virus present is becoming unacceptable, with the minority of studies in the review not including some recognition of this.

At present, there have been efforts or suggestions for more standardized reporting of experimental data, but not for experimental design or preferred study laboratory assays. Vector competence studies require a balance between well controlled experimental conditions with approximation of natural conditions which are often at odds with each other [7,82]. We found the application of our quality grading tool difficult because much of the metadata especially on virus and mosquito strains, denominators used for competence rate calculations are incomplete or difficult to extract. Also apparent was the lack of experimental replication, which can only be accomplished if sufficient insectary space, mosquitoes, and labor are available to run replicates at the same time, all of which presents numerous technical challenges of rearing enough mosquitoes of the same generation to conduct these experiments. Laboratory conditions cannot be 100% duplicated, running multiple replicates for distinct species/virus or mosquito strain may be less of a priority that other components on the quality grading list. Related to this issue, however, is the observation that few manuscripts provided measures of variability or conducted statistical analyses, a more serious issue. Because our review focused on secondary vectors, the use of multiple virus and mosquito strains was limited, but either additional geographical isolates or studies that include a wide range of geographic mosquito strains together with multiple virus strains will provide convincing evidence of a species VC [79].

We agree with Azar and Weaver [83] that adherence to reporting guidelines such as those suggested by Wu et al. [7], would improve our ability to conduct meta-analyses and draw conclusions from reviews including systematic reviews, but also point out the need for establishing some experimental standards as well. Their recommendations including the use of viral doses that are consistent with viremias observed in human patients that may be easier to achieve using animal models which are not available for ZIKV except for knockout mice with their innate immunity altered for which many research teams may not have access. Insect specific viruses (ISVs) and vector microbiome also play a role in VC, but neither of these parameters were mentioned in the 45 articles we included in our review with one exception.

MacLeod and Dimopolous [13] hypothesized that the virus particles observed in Cx. quinquefasciatus mosquitoes challenged with ZIKV [31] represent ISVs rather than ZIKV particles which would be consistent with the absence of viral RNA found in mosquito saliva. We also argue that future systematic reviews potentially exclude articles where no infectious assays are included. We recognize there may be limited circumstances where publication of studies that rely on PCR assays to detect viral RNA are justified, the reliability of this evidence is greatly reduced. Another area that requires input from subject matter experts is minimum number of mosquitoes per stage the require evaluation to have confidence in rate estimates.

Our quality grading tool scored the title of each article, providing a point if the “vector competence” appeared in the title. We do not recommend this moving forward but the appropriate use of key parameters such as susceptible, refractory, transmit deserve discussion. Another item in our grading tool was the issue of sample size. We set 30 mosquitos per virus-mosquito pair evaluated in our quality grading tool as a minimum standard, but this is another experimental component where guidelines would be appropriate. This would be particularly important for reporting results for species deemed negative. We included publications reporting results for refractory species, but this may be an underestimate since there is often a bias against publishing negative results. We recognize, along with other authors [7,82] that complete standardization of VC studies is not possible, but the next logical extension toward this goal is developing quality guidelines, like our quality grading tool to help evaluate the relative quality of manuscripts for inclusion in future metanalyses which are evidently needed to clearly define vector competent species. We view our tool as a good start that needs additional revision by experts before widespread application.

Conclusions

As of March 2022, secondary vectors of ZIKV that show evidence of transmission, albeit at lower rates than primary vectors are Ae. japonicus, Ae. detritus and Ae. vexans, at higher temperatures, as well as local Australian species Ae. notoscriptus and Ae. camptorhynchus. There is ample evidence for Cx. quinquefasciatus not being an efficient vector of ZIKV. There is a strong need for future research that establishes what are significant differences between experimental approaches such as the use of dissected body parts (midguts, salivary glands) compared to whole body parts (heads, legs+wings, bodies) to evaluate infection and dissemination, and the impact of using murine models as well as other artificial feeding systems on VC parameters, and importantly, to develop large collaborative multi-country projects possibly taking advantage of research networks (eg. ZikaAlliance) [79], Centers for Research on Emerging Infectious Diseases [87] to conduct large scale simultaneous evaluations of specific mosquito species with common protocols to appropriately address the inherent geographic variation in both mosquito and virus strains.

Supporting information

S1 Table. Grading tool developed for quality assessment of VC studies using Reporting of Observational Studies in Epidemiology (STROBE) and Strengthening of the Reporting of Molecular Epidemiology for Infectious Diseases (STROME-ID) criteria.

(DOCX)

Click here for additional data file.^{(19.6KB, docx)}

S1 Data. Excel spreadsheet contain raw data extracted from manuscripts to calculate the infection rate (IR), stepwise dissemination rate (SDR), cumulative dissemination rate (CDR), stepwise transmission rate (STR) and cumulative transmission rate (CTR) presented in Table 5.

(XLSX)

Click here for additional data file.^{(17.8KB, xlsx)}

Acknowledgments

We thank Evangelia Zavitsanou for contribution to the development of Fig 2. Additionally, we are grateful to Mike Turell for technical advice and lively discussion about the issues highlighted in the manuscript.

Data Availability

All relevant data are within the paper and its Supporting information files.

Funding Statement

This study was a byproduct of original funding by the Special Programme for Research and Training in Tropical Diseases of the World Health Organization (WHO/TDR, contract number WCCPRD5295811 2017/700816 to OH, SRR, ACM, and PMS). MB, AK, and AM received salary support from the LIFE CONOPS (LIFE12 ENV/GR/000466 to AK) project co-funded by the European Commission and the participating Organizations in the framework of a LIFE + Environment Policy and Governance project (www.conops.gr). ACM received salary support from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (U01AI151814 to ACM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011591.r001

Decision Letter 0

Andrea Marzi, Andrea Morrison

13 Apr 2023

Dear Dr. Morrison,

Thank you very much for submitting your manuscript "Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Andrea Morrison, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: No, please see comments 9 and 17 in my Summary and General Comments

Reviewer #2: -Yes

-Yes

-N/A

-No

Reviewer #3: See the attached review report

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: I don't think that Figure 2 adds any value to the manuscript and should be deleted.

Reviewer #2: -Yes

-Yes

Reviewer #3: See the attached review report

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: No, again, please see comments 9 and 17 in my Summary and General Comments

Reviewer #2: -Yes

-Yes

Reviewer #3: See the attached review report

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: There are numerous minor editorial errors in capitalization, spelling, abbreviations, etc. Please see my Summary and General Comments

Reviewer #2: Minor Revision

Reviewer #3: See the attached review report

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: General Comment: Zika virus (ZIKV) created a major panic back in 2016-2018 when it caused millions of cases of disease in the Americas. As with the other anthroponotic arboviruses, e.g., yellow fever, dengue, and chikungunya viruses, Ae. aegypti is the principal vector. Yes, Ae. albopictus has been involved in a few outbreaks, but see comment 1 below. Because these anthroponotic viruses require the same mosquito to take two separate blood meals on a human, only anthropophilic mosquitoes that preferentially feed on humans are likely to be involved in the transmission of these viruses. This should be emphasized more in the paper. My biggest concern, as mentioned in comments 9 and 17, is that because authors use completely different definitions of a transmission rate, simply reporting the rate that these authors claimed is very misleading and may make an essentially incompetent mosquito appear highly efficient.

I understand that for most of the authors, English is a second language, but there were a lot of minor inconsistencies. Editors, reviewers, and readers can’t see how the study was done, but if the writing is careless (see numerous comments below), they are less likely to believe that the authors were careful in how they did their study.

Specific Comments:

1. Line 46 and numerous comments throughout the entire manuscript. Yes, Ae, albopictus can readily transmit ZIKV and yes, Ae. albopictus readily feeds on humans. However, how important is Ae. albopictus in the transmission of ZIKV, chikungunya virus, or dengue virus? After their introduction into the Americas beginning in 2013, about 10,000 cases of chikungunya and Zika were diagnosed and reported in the U.S. (given the inefficiency in diagnosis and reporting, this was a severe underreporting of cases). Despite many, many reported imported cases of chikungunya and Zika in the southeastern U.S., where Ae. albopictus is one of the principal pest mosquitoes, not one case of locally transmitted chikungunya or Zika was reported from any location that did not also have a significant Ae. aegypti population. Because they are not as anthropophilic as Ae. aegypti, a single Ae. albopictus is not likely to take multiple blood meals from a human, and Ae. albopictus are not as likely to be involved in significant transmission of anthroponotic pathogens such as dengue, chikungunya, Zika, and yellow fever as Ae. aegypti. Note, in the outbreak on the islands around the Indian Ocean, there were very few other animals present to feed on, so the Ae. albopictus did take multiple blood meals from humans, and the same thing happened in a few locations in Italy.

2. Lines 64-66: Why are the use of murine models and infectious virus assays critical gaps? To me, it is critical that the studies use infectious virus assays as qRT-PCR may detect some lingering, non-infectious pieces of RNA remaining from the blood meal in an uninfected mosquito resulting in it inappropriately being called positive. Similarly, the use of artificial blood meals often produce lower infection rates than when mosquitoes ingest the same virus titer from a viremic host. Therefore, it is not immunocompromised murine models that are a critical gap, but rather the lack of use of these models.

3. Lines 86-87: I found this confusing. Testing mosquito bodies for infection is MUCH more efficient than testing midguts. Similarly, testing salivary glands for virus dissemination is very inefficient compared to testing either legs or heads. Note, I have seen papers that test heads to test the presence of virus in salivary glands, but in a mosquito, the salivary glands are in the thorax, not the head.

4. Line 110-120: Very well said!

5. Line 295: Yes, “Days post infection (DPI)” is commonly used, but shouldn’t it be “Days post exposure (DPE)” as many of the mosquitoes did NOT become infected and in some cases, none became infected, so how can it be days post INFECTION?

6. Line 296: What is a Transmission rate versus a Transmission efficiency? I have seen these terms misused numerous times in recent years, and they need to be defined in this paper. Because people define these terms differently, it is important that the authors first define how they are being used in this paper and then correct the citations so that the cited paper are using the same definition, i.e., if the paper states that it is using transmission efficiency, but it is using the definition of a transmission rate (as defined in this review), then the paper should be cited as using a transmission rate despite claiming to have used a transmission efficiency.

7. Line 297 (Table 1): I found this table to be very confusing. Obviously, the various “Results” were written by different people with different formats, i.e., summary first or methods first. These all need to be rewritten to be in a similar format. Either is fine, but they need to be consistent. There are also a bunch of minor errors which I will include here, rather than in the section below on Minor Comments. It is important that the writing be consistent so that the reader can easily go from one paper to the other and compare the results. Someone needs to go through ALL of these and make sure that they are consistently presented.

a. Reference 68 and 69: What is USUV? It is not established in the paper. I know that it is Usutu virus, but will the readers know this? Also, ZIKV is used in many of the papers. However, ZIKV was not established in the heading for this table and ALL abbreviations need to be established in figures and tables.

b. The titers of the infectious blood meal are listed in numerous different ways. For example, it is reported as “log10 PFU/ml” (most of the papers) or as “logs” (see paper 39 and others), or “3x10(5) PFU (See paper 30), or 105.4 PFU (note, the 5.4 was in a subscript) (see paper 47), or 2,530 PFU/ML (see paper 40), or merely as particles (see reference 40). Again, the authors need to be consistent. All of these “PFU” titers should be in the same format, either X.X log10 PFU/ml (note, the “10” should be a subscript) or 10x.x PFU/ml (note, the x.x should be a superscript). Minor, but reporting a titer to two decimal places is reporting data beyond the level of accuracy and should be rounded to the nearest tenth of a log (see reference 44).

c. Some of the English usage is a bit confusing. For example, in paper 45, it states, ‘Aedes aegypti and Ae. polynesiensis in ZIKV spread was also suspected in addition of Ae. aegypti, supported by its ability to transmit laboratory colonies (F16 to F18) were orally infected with a French Polynesian strain) at a 7 log10 TCID50/ml and evaluated at 2, 6, 9, 14, 21 d DPI.” This, and many of the others, needs to be completely rewritten. Note, why was it “…, 21 d DPI.?”

d. Why is “Hemotek” listed under “Mosquitoes/Virus” for reference 37? The method of virus exposure is not list for others under this column, but rather under results for some of the references.

e. Check spacing. There were numerous places where a space was left out. For example, in paper 57, “at 9dpi,”should be at “9 dpi,” (note, see above and this should be at 9 dpe). Likewise, in paper 58, “log10PFU/ml” should be “log10 PFU/ml,…”

f. Why are the infectious titers given in the “Mosquito/Virus” column for some papers, but in the “Results” column for other papers. You need to be consistent. Putting these critical elements in different places makes it much more difficult for the reader.

g. Also, is it “dpi” as in paper 57 and others or “DPI” as in paper 54 and others? Likewise, is it “ml” as used in most papers, “ML” as used in paper 37, or “mL” as used in paper 77?

h. What is a “PFUe” in paper 77?

i. Why in paper 77 does it have titers expressed as “log10” with only the “0” as a subscript?

8. Line 310 (Figure 2): I’m not sure what this adds to the paper. Yes, according to the criteria selected by some of the authors, some of the papers were stronger than others, but which papers received high scores and which received low scores, so that we know which papers’ results/conclusions are more believable, is already indicated in Table 1.

9. The difference in how the studies defined these rates is CRITICAL! For example if study A defined the Transmission Rate (TR) as the number of mosquitoes transmitting virus divided by the number of mosquitoes with a disseminated infection tested and study B defined the TR as the number of mosquitoes transmitting virus divided by the number tested (this is what is said on lines 418-420), and the actual numbers were 100 tested, 50 infected, 3 with a disseminated infection, and 2 transmitting, then in study A, the TR was 67%, yet in study B, it was 2%. How will the reader know which it really was? Again, you need to state one definition and adjust the rates so that the studies are comparing the same thing. As written, the species tested in study A would appear to be a highly effective vector, while those in study B would appear to be very poor vectors. This can be VERY misleading as if in study A, the numbers were above for mosquito species X, but in study B, the numbers were 100 tested, 50 infected, 20 with a disseminated infection, and 20 transmitting for mosquito species Y, so if study B used the definition for study B, it would report a TR of 20% for species Y, so in your paper, you would report that species X has a TR of 67%, while species Y has a TR of only 20%, despite the fact that the actual TR of species Y was 10-fold higher than species X. If you report the rates from the various studies, which this paper needs to do, you NEED to state and use a consistent definition of that rate, so if the TR is the percentage of all mosquitoes tested (which is the classical definition), then the rate for study A needs to be changed to 2%, even if they reported 67%. That allows the reader to compare the results of the various studies. If this is not done, then this paper is completely misleading and should not be published!

10. Line 433 (Table 4): How can 19 studies in the “Infection” column be only “20%?”

11. Line 478 (Table 5): See comment 9 above. It can be extremely misleading if the data presented here are using different definitions of transmission. These rates need to be adjusted to a single definition. Note, using the percentage of mosquitoes with a disseminated infection is itself very misleading as mosquitoes with a very low dissemination rate may appear to have a very high transmission rate. Look at the table, how can a mosquito have a 43% infection rate and a 90% transmission rate?

12. Lines 508-509: Again, how can you have “significant transmission of ZIKV” with only a 42% infection rate?

13: Lines 529, 531: First of all, it is Ochlerotatus, not Ochelerotatus. More importantly, is it Ochlerotatus detritus or Ae. detritus? The genus Ochlerotatus was briefly establish for some of the Aedes species a number of years ago, but nearly everyone has gone back to using Aedes. Note, if you use Ochlerotatus, then it should also be Oc. taeniorhynchus, which I do not recommend.

14. Line 535: This is the second time that the authors mention that one of the reasons that Ar. subalbatus was tested was because they develop in human waste lagoons where ZIKV could be shed in urine. I believe that the study found no risk of the larvae becoming infected despite exposure to very high titers of ZIKV in a water/urine suspension in the laboratory. Repeating the statement above, without clarification that they didn’t find that risk, is misleading.

15. Lines 575-579: Will warmer temperatures really affect ZIKV transmission? Remember, yellow fever and dengue were once common diseases in the United States, including Philadelphia, New York, and Boston. It was not warmer temperatures, but social economic status that allowed for the transmission of viruses by Ae. aegypti. However, warmer temperatures will allow for the greater and more efficient transmission of zoonotic viruses.

16. Lines 642-644: Yes, the mere isolation of a virus from a field-collected species does not mean that it is involved in the transmission of that virus. Even if a species is completely incompetent for a particular virus, if it had recently fed on a viremic host, it would contain virus. Similarly, when mosquitoes are sorted for virus detection, legs of numerous specimens break off and may adhere to the body of a mosquito of a different species. If that leg came from an infected mosquito, then whatever species it adhered to would appear to be positive. You might want to add a little about why the mere detection of virus from a species does not make it a vector.

17. Lines 703-708: Despite what was said in this paragraph, the use of the number of disseminated mosquitoes as the denominator is a relatively new use, and as I explained in comment 9, may be a very misleading number. If only 1/100 mosquitoes has a disseminated infection and that one transmits virus, then if only the disseminated mosquitoes are counted, the transmission rate for that species is 100%, making it appear that the species is very important, while if in another species, in which 100% develop a disseminated infection and only 20% transmit, that species would have a 20% transmission rate despite have 20-fold higher vector competence. This new definition came out of the Institute Pasteur, and I believe that they introduced it to make their work look more important, i.e., in the example above, a 100% transmission rate is much more publishable than a 1% one. From a public health standpoint, we need to know how efficiently a particular mosquito species is as a vector, saying that the species above has a 100% transmission rate might lead to the control of the wrong species. You sort of indicate this on line 717-718.

18. Line 733: What do you mean by “traditional methods?” the use of animal models and checking for infectious virus are the traditional methods.

19. Lines 734-736: Various studies have shown that the amount of virus injected when feeding on a vertebrate host is significantly higher as compared to the amount of virus injected during artificial methods to collect saliva. Similarly, transmission rates are usually higher to a vertebrate than when collecting saliva and testing it. Many laboratories don’t want to recognize this as it is difficult, or even impossible, for them to maintain the necessary animal colonies. Therefore, they use the less scientifically efficient artificial techniques.

20. Lines 769: Unfortunately, using viral doses that are consistent with viremias observed in humans in an artificial blood meal will result in significantly lower infection rates in mosquitoes than if they ingested the same dose of virus from a viremic animal (i.e., human). Therefore, studies using those doses would greatly underestimate the importance of that mosquito species.

21. Lines 794-796: Yes, if the study only tested 15 mosquitoes and did not find transmission, that really doesn’t mean that the mosquito tested was incompetent. However, if they tested 10 specimens and eight of them transmitted virus by bite, then I think that they have clearly shown that this species may be very important.

22. Line 812: I would be very surprised is there is any difference in infection rates determined by testing bodies or midguts, but it is a lot more labor intensive to test midguts.

23. Lines 812-813: Heads and salivary glands are testing two different things. It is possible to have a disseminated infection, i.e., positive head and the salivary glands be negative. Likewise, a positive salivary gland does not mean that there will be virus in the saliva.

24. References: These need to be formatted properly.

a. Only the first word and proper nouns in a reference title should be capitalized. See references 13, 36, 39, and others.

b. References should not be in all caps. See reference 20.

c. Shouldn’t the journal for reference 28 be “Trans R Soc Trop Med Hyg” instead of “Transactions of The Royal Society of Tropical Medicine and Hygiene?”

Minor Comments:

25. Lines 49-50: Should “…identify knowledge key gaps” be “…identify key knowledge gaps?”

26. Line 59: Why is there a “comma” after “quinquefasciatus?”

27. Line 98: “Aedes” needs to be written out the first time it is used in the body of the manuscript.

28. Line 103: Traditionally, numbers less than 10 are written out, so this should be “eight genera…” Note, it appears that there is an extra space after “genera.” In contrast, all numbers used in measurement are given as numerals. Therefore, on line 149, “five years…” should be “5 years…” Please check the rest of the manuscript and correct these.

29. Line 165: Why is there a space after the “)” and before the “comma?”

30. Line 169: Should “WHOLIS” be “(WHOLIS)?”

31. Line 191: Should “wild mosquitoes” be “field-collected mosquitoes?”

32. Line 203: “n= 8.986)…” should be “(n= 8,986)…” Note that a comma was correctly used elsewhere.

33. Line 291: “Medical Entomology [3] and” should be “Medical Entomology [3]), and” with both a close parenthesis and a comma.

34. Line 330 and many others: Why write out “Zika virus” when “ZIKV” was established and used many times earlier in the manuscript?

35. Line 371: Why are Aedes, Culex, Anopheles, etc. written out each time they are used in this table? They should only be written out the first time that they are used.

36. Line 377: Why is the “10” in “log(10)” in parentheses?

37. Line 380: “…ten also…” should be “…10 also…”

38. Line 393: “7 studies” should be “seven studies.” See also lines 452, 475, and others.

39. Lines 440-441: Why is “(Coffey et al. 2018)” listed here? Should this be assigned a reference number? Note, all of the reference numbers may need to be adjusted.

40. Line 441: Why is “Sabethes” written out throughout the manuscript? You don’t write out Aedes or Culex? These should all be “Sa. species” as Sabethes was established on line 376. The same is true for Armigeres.

41. Line 443: Again, reporting a titer to two decimal places is reporting data to a level that is not accurate. Yes, I know that some papers do this, but that does not make it right, and you should not make the same error. On this line, simply report the titer as “6.8 log10 PFU/ml.: Note, elsewhere in the paper, PFU is in all caps. Please be consistent.

42. Line 484: Change “vector were…” to “vectors were…”

43. Line 501: What does “more consistent infection rates” mean? Dose that mean that the rates were consistent, i.e., 5%, 6%, 4% in three separate trials or that they were consistently higher than those for Ae. aegypti tested from that area?

44. Line 515: Why is there a “comma” after “injection,?”

45. Line 516: “Colorado USA…” should be “Colorado, USA…”

46. Line 520: “Ae. triaseriatus” should be “Ae. triseriatus”

47. Line 521: Why is “(Hart et al. 2017)” listed as a reference by name rather than by reference number 51?

48. Line 531: Change “or Ae.” to “nor Ae.”

49. Line 624: Why “(2018)” rather than “(57)?”

50. Line 766: Why “(2019)” rather than “(79)?”

Reviewer #2: In the review “Secondary vectors of Zika Virus, a systematic review of laboratory vector competence

studies” Bisia and colleagues have conducted a thorough and transparent review of the existing Zika virus vector competence literature with respect to laboratory studies conducted on potential secondary vectors. As a laboratory construct, vector competence represents the research community’s best method to examine a complex interplay of factors that contribute to a given mosquito species or populations ability to serve as vector for a given agent. That said, the methodology/discipline suffers from a pervasive lack of unity with respect to methodologies, definitions and other critical parameters. In conducting these analyses, Bisia et al are able to illustrate this issue alongside their primary objective of identifying potential secondary vectors for ZIKV. Given the increased incidence in the emergence of zoonotic diseases, including arboviruses, increasing the rigor and reproducibility in this discipline is paramount. This manuscript illustrates this in the context of the ZIKV outbreak in the Americas from 2015-2018.

In total the manuscript is well-written, articulates its methodologies clearly and transparently, identifies promising avenues for further development, and draws appropriate and scientifically sound conclusions. Publication of this manuscript is therefore highly recommended albeit with the following suggestions and comments put forward for consideration.

Major Points

1. Lines 141-142 and throughout: Something to note; the connection between Culex quinquefasciatus and ZIKV was NOT initially facilitated by peer-reviewed research. A group that was affiliated with public health agency FioCruz in Recife issued a press release (e.g. https://www.theguardian.com/world/2016/mar/03/zika-virus-carried-more-common-mosquito-scientists-say) in the absence of public data a full year prior to the publication of their manuscript.

2. In the text and in the grading tool (Supplemental Table 1), the authors refer to the use of “low passage virus” as a parameter. While considering colonization of mosquito populations, authors define the association between the point value and the number of generations in the laboratory. I feel that a defined passage number should be indicated in the table, as “low passage” in the absence of a defined number is ambiguous.

a. Related; do the authors distinguish between passaged primary isolates or recombinant viruses in these experiments? Can this be noted in the text either way?

3. Lines 760-761: The authors note the lack of experimental replication in the vector competence literature. Although the authors note that this is understandable due to the labor cost of these experiments, it also worth noting that from a strict standpoint such replication is not technically possible. Exposure of a given laboratory population is associated with a given generation of colonization (e.g. F3, F4, etc). In the case of mosquitoes that do not lay eggs that can undergo desiccation and storage such as Culex spp, it is logistically impossible to perform an exact replica of an experiment with the same generation of mosquito from the same population. While such experimental replicates ARE technically feasible in the case of some mosquito species, it also requires substantial up-front effort to rear large quantities of mosquitoes and store substantial cohorts of eggs to allow for generation matching.

Minor Points

1. Line 138: This may be pedantic; however, I believe this sentence requires clarification. Specifically, the phrase “much of our knowledge comes from…” seems inappropriate as our current knowledge base far surpasses what was known solely based on the YFV surveillance.

2. Line 483: The word “both” on this line appears to be misplaced as there is no secondary factor after “laboratory evidence”.

3. Line 491: The correct spelling is Sabethes cyaneus

4. Lines 495 and 497: The authors here refer to Aedes polynensis, which is a misspelling of the species name Aedes polynesiensis.

5. In line 576, the authors identify Ae. japonicus, Ae. vexans, “and to some degree” Ae. detritus as potential secondary vectors given higher temperatures. In lines 659-661 the authors note that Ae. vexans, Ae. japonicus, and Ae. polynesiensis as possible candidates for follow up studies. Please correct for consistency.

Reviewer #3: See the attached review report

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Thomas Obadia

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PLoS Negl Trop Dis. 2023 Aug 31;17(8):e0011591. doi: 10.1371/journal.pntd.0011591.r002

Author response to Decision Letter 0

9 Jun 2023

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011591.r003

Decision Letter 1

Andrea Marzi, Andrea Morrison

10 Jul 2023

Dear Dr. Morrison,

Thank you very much for submitting your manuscript "Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Thank you for your resubmission! I would highly recommend reviewing the manuscript for remaining grammatical issues as indicated by the reviewers. Additionally, please pay close attention to Reviewer 1's comments regarding transmission rate.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Thank you again for your submission to our journal. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Andrea Morrison, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: See general comments.

Reviewer #2: Accept

Reviewer #3: Yes

Yes

N/A

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: See general comments.

Reviewer #2: Accept

Reviewer #3: Yes

Yes

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: There are some issues. Please see general comments.

Reviewer #2: Accept

Reviewer #3: Yes

Yes

--------------------

Editorial and Data Presentation Modifications?

Reviewer #1: There were numerous minor grammatical issues. I pointed many of these out in the general comments.

Reviewer #2: Accept

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Reviewer #1: General Comment: This version is much improved. However, I am still a little concerned about how transmission rate is used in this paper. The use in Table 5 is fine but its use in the text can be misleading (see comment 8 below). Also, there were numerous minor editorial errors (multiple uses of a numeral, at random, for numbers less than 10). To me, this illustrates some carelessness. More importantly, many of the rates given in Table 5 are wrong (see comment 5 below).

Specific Comments:

1. Line 124: Vector competence studies generally do NOT include “inoculation” or

‘intrathoracic inoculation,” but may use that technique to examine either midgut escape or salivary gland barriers. Inoculated mosquitoes cannot be used to determine a transmission rate as this technique bypasses two critical barriers, midgut escape and salivary gland barriers.

2. Table 1, Guo reference: As they did not check for actual, infectious virus, isn’t it much more likely that they were detecting lingering small pieces of noninfectious RNA, rather than a transient infection, which as you indicate would be totally new and unexplained paradigm?

3. Lines375-383: I added up the numbers for the various locations, 16, 10, 5, 4, 4, 2, 1 (not identified), and 2 (multiple continents), and the total was 44.Where was the missing study done? Based on Table 2, shouldn’t the 16 for the Americas be 17?

4. Lines 388-389: Why is Armigeres not listed on line 388? Li et al. did test the vector competence of this species for ZIKV.

5. Table 5: Much better, but there are still some problems.

a. For Ae. luteocephalus, how is it possible for the CDR to be 42% if the SDR was only 37%? By definition, the SDR has to be greater than or equal to the CDR.

b. For Ae. vigilax, how is it possible for the CDR to be 27% if the SDR was only 10%?

c. For Aedes vitalis, how can the CDR be 17% if only 14% of the mosquitoes were infected?

d. I am a little confused about the calculations for Ae. japonicus. If the IR was 87%, and the CDR was 19%, should the SDR have been 22%, not 27%. Alternatively, if the SDR was 27%, shouldn’t the CDR have been 22%. There is the same problem with the TRs. If the SDR was 27 and the STR was 57%, then the CTR should have been 15%, not 11%. However, if the SDR was 22% (see above) and the STR was 57%, then the CTR should have been 12.5% (note, the 1% difference might be due to the decimal points not shown in the original data.

e. As illustrated by the above four examples, there are some errors in the calculations of these rates. Please go back and confirm all of the rates as I found several others that are also wrong.

f. The importance of clarifying the transmission rate definitions is illustrated by Sa. cyaneus. If the paper defined TR as the number transmitting/number tested, then the TR was 1%. However, if they used the inappropriate definition of number transmitting/number disseminated, it was 100%. In the abstract of the paper, it would have said, the TR for Sa. cyaneus was 1% (or 100%) depending on the definition used in the paper. Unfortunately, most people only read the abstract to get this information.

6. Lines 549-551: As illustrated by the example of Sa. cyaneus in 5(f) above. The definition of transmission rate is critical. If you used the STR, the transmission rate would be 100%, i.e., more efficient that Ae. aegypti, but if you used the CTR, then it would be 1% and extremely inefficient. You need to explain which you are using, and if using the STR, then some extremely inefficient vectors would be classified as extremely efficient, and this is not only misleading, but public health people might focus their control on an extremely inefficient species and ignore more important vectors, resulting n more disease.

7. Lines 568: Again, you need to be clear as to which definition of dissemination you are using for the various studies. Species with a relatively low infection rate may appear to have a high dissemination rate, making them look much more efficient.

8. Lines 574-576: This really illustrates the problem. If only 34% of the Ae. notoscriptus became infected (and only 3% had a disseminated infection), how could they have a 42% transmission rate when only about 1% of the virus-exposed mosquitoes actually transmitted ZIKV? Obviously, this species is a very inefficient vector of ZIKV. However, someone reading this sentence would think that it was an efficient vector. This is very misleading.

9. Lines 632-634: You might want to add that noninfectious pieces of RNA have been shown to persist for long periods of time in a mosquito, and thus, this study detected these noninfectious pieces of RNA, but the amount of these noninfectious pieces would decline with time providing the results reported.

10. Lines 810-814: This is important.

11. Lines 817-820: Yes. If someone reads the paper, they can understand how the authors defined a TR. However, many of these papers simply state in the abstract that the TR for species X was 50% and most readers do not read the paper. However, if they had used a stepwise rate and only two of the 100 mosquitoes tested had a disseminated infection, and one of these transmitted, then instead of being an efficient vector, as indicated in the abstract, it would be a very inefficient vector. As indicated in the paragraph, this could have serious public health liabilities. We need to be consistent in our definition of a transmission rate, and if the purpose is to identify potential vectors, then it needs to be the number transmitting/number tested.

12. Lines 835-839: Thanks for pointing this out.

13. Lines 855-858: I’ve played with this quite a bit. I have often found that a single mosquito bite on the tip of a mouse’s tail usually contains >1,000 PFU of virus, but a saliva sample rarely contained mor than 100 PFU, and a mosquito that had transmitted to a rodent often contained no virus in its saliva sample. While it is easier and less costly to simply collect saliva and test it, it tends to underestimate the actual TR. This is consistent with what you are reporting.

Minor Comments:

14. Line 98: “VC” needs to be established in the main body text.

15. Line 119: “vector capacity…” should be “vectorial capacity…”

16. Line 148: What is “transmission efficiency?”

17. Line 163: This does not read well. Should “where” be added so that it is now “…and a period where VC studies…?”

18. Line 319: Why is “Days” post exposure capitalized, while “days” post infection is not?

19. Line 319: “contain…” should be “containing…”

20. Line 334: Why establish “MS” if it is not used again?

21. Line 335: Why establish “TS” if it is not used again?

22. Line 388: I don’t think that you need both “genus” and “genera” in this sentence. That is repetitive.

23. Line 400: “4 articles…” should be “four articles …”

24. Line 413: “using well described the viral strains…” should be “using well described viral strains…”

25. Line 469: Why establish “DIR” or “YR(D)” if they are not used again?

26. Line 493: Why report a titer to a hundredth of a log? This is not accurate. I don’t care if that was reported in the paper cited, it should be 6.8 logs.

27. Line 501: “ten mosquitoes…” should be “10 mosquitoes…”

28. Line 663: “these kind studies…” should be “these kinds of studies…”

29. Line 702: Why establish “YF” if it is not used again?

30. Line 760: “1 or 2 local…” should be “one or two local…”

31. Line 929: Shouldn’t “rates primary vectors…” be “rates than primary vectors…?

32. References

a #16 (Cao-Lormeau): Shouldn’t “Polynesia” be capitalized?

b. #18: Why is “Transactions of The Royal Society of Tropical Medicine and Hygiene” written out. Check others.

c. #37: Why is “chikungunya” capitalized?

Reviewer #2: In the revision of the review “Secondary vectors of Zika Virus, a systematic review of laboratory vector competence

studies” Bisia and colleagues have addressed and/or justified their rationale for comments and concerns expressed by myself and the other reviewers. As such, I support the publication of the manuscript in its current form.

Reviewer #3: Following my initial review and that of the other two anonymous reviewers, the authors have substantially improved their manuscript and I can only commend them for going through what must have been some very tedious modifications and proof-reading.

My main criticism at first submission was the long delay between the end date for including papers in this systematic review and timing for submission (more than a year). This resulted, among other things, in reviewing that paper after I had myself published what the authors were calling for as “future efforts” in the discussion. In an effort for transparency, I decided it was only fair that I disclosed my name during the review process, and that hopefully it did not make me sound like an author wanting his word credited. I appreciate that for any systematic review, and end date must be set and respected. It is unfortunate that publication of this paper will happen more than a year and a half later, though we can all agree that “publishing science takes time” and sometimes delays out of authors’ control contribute to lengthy publication process.

Among the strongly positive points now made by the authors is the call for harmonized reporting standards (e.g. clearly defining stepwise/cumulative rates), a major point raised by anonymous Reviewer #1. I can only hope that future VC work will build upon that terminology. The QAS, though remaining of high relevance, also feels better now that it is introduced as “guidelines” towards standardization rather than a rewarding metric.

I now only have minor comments that, once addressed, will make that paper acceptable for publication. Upon reading that R1 revision, I consider my major comments addressed and can recommend this article for publication PLoS Neglected Tropical Diseases pending corrections for remaining typos and few syntactical errors.

Minor comments

L155: yellow fever is first mentioned here, then again at L420, then at L702 (where it is abbreviated YF) and L745-746 (“yellow fever” again). The abbreviation may come at L155 and YF be used later for clarity and consistency.

L295: “considers” should probably not have an “s” here.

L539+: consider mentioning somewhere that the now harmonized “NT” stands for “Not tested”.

L725: “accessing” should probably be “assessing”. This was actually a minor suggestion from my first review and marked as “done” by the authors, could you double-check that other small typoes highlighted earlier were not corrected then wrongly rejected/reverted ?

L762: Missing an “s” at “provides”.

L801: accessing/assessing, again

L851-853: the sentence does not make sense as is though I think understand what the authors meant. Consider inverting the beginning so that it’s clear that these constraints dictate what can and can’t be reasonably expected from low/middle-income countries in terms of lab capacity and experiments.

L889: Shouldn’t “geographically” be “geographical”?

--------------------

PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Thomas Obadia

Figure Files:

Data Requirements:

Reproducibility:

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

PLoS Negl Trop Dis. 2023 Aug 31;17(8):e0011591. doi: 10.1371/journal.pntd.0011591.r004

Author response to Decision Letter 1

20 Jul 2023

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011591.r005

Decision Letter 2

Andrea Marzi, Andrea Morrison

10 Aug 2023

Dear Dr. Morrison,

Thank you very much for submitting your manuscript "Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

We appreciate the edits you have made in response to the reviewers' comments and think that this would be a valuable publication. We request a few additional edits be made in response to reviewer #1's comments as we believe they will even further improve upon the manuscript and provide the final details needed.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the manuscript.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Sincerely,

Andrea Morrison, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

***********************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Methods are fine and no concerns

Reviewer #2: Meets all review criteria and no ethical/regulatory issues noted

Reviewer #3: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: please see General comments

Reviewer #2: The results meet all the aforementioned criteria

Reviewer #3: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: please see General comments

Reviewer #2: The conclusions meet all the aforementioned criteria

Reviewer #3: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Reviewer #1: please see General comments

Reviewer #2: Accept

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Reviewer #1: General Comment: This version is much improved. However, the are still a few issues that need to be corrected. Please see my comments below.

Specific Comments:

1. Line 406: “ten generations…” should be “10 generations…”

2. Table 5

a. What does “Raw data presented in” at the end of the table heading mean?

b. For Ae. notoscriptus, you report IR- 29%, SDR-21%, CDR-10%. However, if 29% are infected, and 21% of those have a disseminated infection, then only 6% of the total would be infected. On the other hand, if the CDR was 10% and 29% were infected, the SDR would be 34%

c. The results for Ae. vigilax IR- 34%, SDR-47%, CDR-27% ref43 and Ae. procax IR- 57%, SDR-50%, CDR-17% ref 43 seem to be partially crossed. The SDR-50% and CDR-17% reported for Ae. procax work for Ae. vigilax, and the SDR-47% and CDR-27% reported for Ae. vigilax work for Ae. procax. Alternatively, the two infection rates were switched.

3. Line 550: What does, “with both laboratory evidence” mean?

4. Line 558: Yes, it was only one mosquito that became infected and transmitted ZIKV. However, the sentence gives no indication of the sample size, so if they had only tested that one Sa. cyaneus, it would appear to be a very efficient vector. The authors might want to modify the sentence to, “…had one mosquito out of 60 tested with…

5. Line 583: Why use the word “Important” for those two species, particularly as they were refractory. Delete “Important.”

6. Line 674: I agree that that data sets should be provided. However, how are the authors going to guarantee that they “will be required.” Shouldn’t this be, “New studies should be required to do so…”

7. Line 824: Shouldn’t “Both these definitions…” be “Both of these definitions…?

8. Lines 852-858: You might also want to cite Turell MJ. Reduced Rift Valley fever virus infection rates in mosquitoes associated with pledget feedings. Am J Trop Med Hyg. 1988 Dec;39(6):597-602 that clearly showed that when feeding mosquitoes (both Culex and Aedes) on blood from a viremic hamster or on the hamster itself, the infection rate in the mosquitoes was significantly higher if they had fed on the hamster although the amount of virus ingested was identical.

9. Line 949: Thanks

Reviewer #2: In the revision of the review “Secondary vectors of Zika Virus, a systematic review of laboratory vector competence

studies” Bisia and colleagues have further addressed concerns, in particular in regards to Table 5, and the definitional consideration between stepwise vs cumulative dissemination/transmission rate. In revising, the authors have strengthened an already phenomenal manuscript. Given that, I would wholeheartedly endorse the acceptance and publication of this work.

Reviewer #3: My minor concerns mentioned after Revision 1 have been addressed.

The authors now provide Table 5, which raised numerous concerns (most of these detected by the very thorough anonymous Reviewer #2), has now been derived from an Excel spreadsheet with automated calculations. This makes sure that IR >= CDR >= CTR. The formulas have not been applied to all rows, presumably to avoir '#ERROR' values due to some divisions by zero, and instead value was forced to zero in these cases. I am honestly not sure which is best since 0/0 is also not equal to 0, though readership will eventually be using "0" for any dissemination/transmission rate if infection was completely unsuccessful. So, this is fine by me.

There are rows where formulas do not reference existing cells:

- Some with hard-coded values that were derived based on extrapoliting percentages in original articles; this is fine by me

- Two have hard-coded mathematical operations, this is slightly worse because one has to make sure the numbers do match those from columns B, C, D and E. This applies to row 20 (I20 and J20)

Since this supplementary file is used as a basis for generating Table 5, it would be good that at least these two hard-coded mathematical operations are corrected to reference the corresponding denominators from columns B and D. The results are, nonetheless, valid.

I am fine with proceeding for publication (or asking the authors for that very minor modification to Table S2 and then only undergo editorial validation).

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Thomas Obadia

Figure Files:

Data Requirements:

Reproducibility:

References

PLoS Negl Trop Dis. 2023 Aug 31;17(8):e0011591. doi: 10.1371/journal.pntd.0011591.r006

Author response to Decision Letter 2

11 Aug 2023

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PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011591.r007

Decision Letter 3

Andrea Marzi, Andrea Morrison

14 Aug 2023

Dear Dr. Morrison,

We are pleased to inform you that your manuscript 'Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

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Andrea Marzi

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PLOS Neglected Tropical Diseases

***********************************************************

PLoS Negl Trop Dis. doi: 10.1371/journal.pntd.0011591.r008

Acceptance letter

Andrea Marzi, Andrea Morrison

21 Aug 2023

Dear Dr. Morrison,

We are delighted to inform you that your manuscript, "Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

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Data Availability Statement

All relevant data are within the paper and its Supporting information files.

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PERMALINK

Secondary vectors of Zika Virus, a systematic review of laboratory vector competence studies

Marina Bisia

Carlos Alberto Montenegro-Quinoñez

Peter Dambach

Andreas Deckert

Olaf Horstick

Antonios Kolimenakis

Valérie R Louis

Pablo Manrique-Saide

Antonios Michaelakis

Silvia Runge-Ranzinger

Amy C Morrison

Roles

Abstract

Background

Methods

Findings

Conclusion

Author summary

Introduction

Methods

Search strategy, databases, and search terms

Fig 1. PRISMA flow diagram (adapted from Page et al. [33]) describing identification of articles examining vector competence of mosquito species other than Aedes aegypti and Aedes albopictus for Zika virus transmission.

Quality assessment

Results

Descriptive results

Table 1. Evidence table, in chronological order from 2014 to 2021.

Quality grading tool assessment

Description of the included studies

Time and geographical clustering of studies

Fig 2. Geographic and temporal distribution of 45 vector competence studies of mosquito species other than Aedes aegypti and Aedes albopictus for Zika virus transmission included from an electronic search finalized on 15 March 2022 [78].

Mosquito species tested for vector competence

Table 2. Summary of species studied country of origin.

Mosquitoes used in vector competence experiments

Virus strains used

Table 3. Virus strains used in 45 vector competence studies examining secondary mosquito vectors for Zika virus.

Vector competence parameters

Laboratory assays used to detect ZIKV or ZIKV RNA

Table 4. Laboratory Assays used to detect Zika virus or RNA for infection, dissemination, and transmission assays.

Studies using mice

Review of evidence for individual vector species other than Ae. aegypti and Ae. albopictus

Table 5. Vector competence (VC) parameters of species investigated as possible vectors of Zika virus.

Forest cycles

Vectors associated with Micronesia/Polynesia in Island settings

Local vectors not found to be competent to transmit Zika Virus

Species with evidence of very low transmission potential

Possible role of Culex species

Role of temperature

Discussion

Research interest and study design often driven by outbreaks and financial considerations

Variability in laboratory methodologies

Definitions of Infection, Dissemination, Transmission, and other terms

Methods that dissect out body parts versus testing bodies, legs, wings, and heads compared to murine models

Minimum quality standards for vector competence studies and how to evaluate them

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Andrea Marzi

Andrea Morrison

Roles

Author response to Decision Letter 0

Decision Letter 1

Andrea Marzi

Andrea Morrison

Roles

Author response to Decision Letter 1

Decision Letter 2

Andrea Marzi

Andrea Morrison

Roles

Author response to Decision Letter 2

Decision Letter 3

Andrea Marzi

Andrea Morrison

Roles