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

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.