Identification of Mayaro Virus Genotype D in Rondônia, Brazil

ABSTRACT.

The Mayaro virus (MAYV) is a significant reemerging arbovirus of public health concern, responsible for outbreaks in several countries including Brazil. In this study, 857 samples of patients with acute fever in the state of Rondônia, Brazil, were analyzed by reverse transcriptase qualitative polymerase chain reaction (RT-qPCR) to detect Zika, dengue, and chikungunya viruses. The mean age of the population was 38 years (SD = 17.46). Negative samples were subjected to duplex RT-qPCR to detect MAYV and Oropouche virus. One MAYV-positive sample with a negative result for all other viruses tested was identified and subsequently sequenced using the automated Sanger method and, through phylogenetic analysis, was characterized as belonging to genotype D, making it the first case of Mayaro in humans isolated in Rondônia. The symptoms reported by the positive patient were fever, vomiting, back pain, nausea, severe arthralgia, and retro-orbital pain. The study reinforces the need for differential diagnosis for Mayaro in the laboratory routine and the importance of genomic surveillance of this virus, mainly due to the similarity of symptoms with other arboviruses, which makes this screening difficult.

INTRODUCTION

Belonging to the Togaviridae family and the Alphavirus genus, the Mayaro virus (MAYV) consists of an enveloped viral particle that has a ssRNA+ genome of approximately 12 kb composed of five structural proteins and four nonstructural proteins.¹ First isolated in Trinidad and Tobago in 1954,² MAYV is one of the main arboviruses that cause infectious diseases of public health importance in tropical and subtropical countries.³ In Brazil, there are records of Mayaro fever outbreaks, and the reemergence of the virus in the Amazon region is the highest number of reported cases of MAYV infection in the country.^4–8 Mayaro fever has as its main clinical manifestations chills, fever, intestinal manifestations, retro-orbital pain, and myalgia, in addition to the polyarthralgia characteristic of MAYV infection, and is a significant public problem.⁹ Due to the similarity of symptoms with other arboviruses and the diagnosis of the disease being limited, epidemiological surveillance for MAYV is a challenge that makes it difficult to understand the mechanisms related to the spread of the virus in the population. In the present work, we report the first identification of MAYV infection in humans in Rondônia, Brazil, in an individual without a travel history.

MATERIALS AND METHODS

This study was approved by the Research Ethics Committee of the Centro de Pesquisa em Medicina Tropical de Rondônia-CEPEM/RO under protocol number 1.474.102. The collection of serum samples from individuals with up to 7 days of onset of febrile symptoms was carried out between January 2022 and July 2023 in Health Units in 28 municipalities in Rondônia and a border municipality in Amazonas. The individuals selected for the study were aged between 18 and 65 years with acute febrile symptoms for up to 7 days.

Viral RNA from the samples was isolated from 140 µL of serum using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions and eluted in 60 µL of Elution Buffer (AVE). Reverse transcriptase qualitative polymerase chain reaction (RT-qPCR) initially screened samples for zika, dengue (serotypes 1–4), and chikungunya viruses using the Biomol ZDC Kit (IBMP/PR, Curitiba, Brazil) following the manufacturer’s instructions. Negative samples were tested by duplex RT-qPCR assay to detect Mayaro and Oropouche viruses, where the chimeric plasmid containing the viral targets previously described by the authors was used as a positive control.¹⁰ Thus far, no samples have tested positive for the Oropouche virus.

The RNA extracted from the MAYV-positive sample was subjected to reverse transcription using the SuperScript™ IV RT (Thermo Fisher Scientific, Waltham, MA) enzyme. The cDNA was amplified by conventional PCR using primers 9368-F (5′-ACCAGTGGTGCAACCATAAAC-3′) and 10344-R (5′-TCGTGTTTRCACACGTCAGC-3′) to amplify a 976 bp product corresponding to a partial region of MAYV E1 and E2 surface glycoproteins¹¹ using Platinum™ SuperFi II PCR Master Mix (Thermo Fisher Scientific).

The automated Sanger method was used to sequence the conventional PCR product by SeqStudio Genetic Analyzer platform (Thermo Fisher Scientific). To perform the phylogenetic analysis, deposited sequences of MAYV genotypes and subgenotypes were collected through the Genbank available at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) and aligned together with the sequence obtained in the study through the online software MAFFT v.7. The phylogenetic tree was generated using the iQTREE software and edited using the FigTree v.1.4.4 program (http://tree.bio.ed.ac.uk/software/Figtree/). The support values of the obtained branches were estimated using bootstrap in 1,000 replicates for the reliability of the results.

RESULTS

From the cohort of 857 samples tested for the presence of the zika virus, serotypes 1, 2, 3, and 4 of dengue, chikungunya, Mayaro, and Oropouche in individuals with an acute febrile condition, one sample was positive for the MAYV with a cycle threshold value (Ct) of 20.2 for RT-qPCR, and had a negative result for all other viruses tested. In the study population, 49.12% (421/857) were male and 50.88% (436/857) female, the mean age of the population was 38 years (SD = 17.46), and none of the tested samples showed a positive result for Oropouche. The MAYV positive sample was submitted to genomic sequencing and phylogenetic analysis, where it was possible to confirm the identification of genotype D with characteristics similar to strains isolated in Peru (Figure 1).

Figure 1.

The phylogenetic tree containing 66 sequences retrieved from GenBank of representatives of the Mayaro virus (MAYV) genotypes (MAYV-D, MAY-N, and MAYV-L) and one isolate generated in the present study identified by the filled triangle. The support values (bootstrap) are contained in the main branches.

The positive patient is a 33-year-old man living in a rural area of Rondônia capital (Porto Velho, Brazil) who had symptoms for 2 days before the collection date. During medical care, reported symptoms were fever, vomiting, back pain, nausea, severe arthralgia, and retro-orbital pain. There were no reports of headaches, arthritis, rash, tourniquet test, myalgia, rash, conjunctivitis, or leukopenia. The patient also claimed not to have traveled to another region in the previous 15 days, which suggests that the infection occurred in the area close to his residence, thus ruling out the possibility of being imported from another region. In Figure 2, the MAYV detection localities and genotypes sequenced in Brazil between 2007 and 2022 are listed, with emphasis on the location where this study was developed.

Figure 2.

The distribution of Mayaro virus (MAYV) cases in Brazil, 2007–2022. The states highlighted in black have identified MAYV isolates; the symbols have been included to indicate the Brazilian federal units where viral genotyping was carried out.

DISCUSSION

This study describes the first report of infection in humans with the Mayaro virus in Rondônia, Brazil, in an individual with suspected initial infection for other arboviruses. The state of Rondônia is located within the Brazilian western Amazon, listed among the states with a high-risk level for the occurrence of zoonotic diseases, such as arboviruses, due to the natural characteristics of the Amazon biome.

We found that the diagnosis of Mayaro is an important public health problem in the west of the western Amazon, mainly because of the similarity of the clinical spectrum, initially characterized by acute fever, with other circulating arboviruses, such as dengue, zika and chikungunya.¹² More than 50% of individuals in the region who present with this symptom have a negative diagnosis for the main pathogens identified in the laboratory routine, which may suggest infection by other arboviruses, such as Mayaro.¹³

The description of this case in Rondônia also raises speculation related to its possible origin of infection, which may be related to the Haemagogus janthinomys vector already described as transmitting the Mayaro virus¹⁴ and potential urban vectors such as Aedes aegypti and Aedes albopictus¹⁵; and, more recently, a published study described the first report of natural infection by the virus in insects of the species Mansonia humeralis, collected from 2018 to 2020 in chicken coops in rural communities in Jaci Paraná, Porto Velho, Rondônia,¹⁶ indicating that these vectors can also be potential transmission agents of this arbovirus. Thus, considering the great biodiversity of the Amazon region, specifically in Rondônia, studies that address other possible vectors for MAYV are paramount to elucidate the region’s transmission cycle of this arbovirus.

A published study surveyed Brazil’s MAYV virus infection rate between 1955 and 2018. Of the total of 1,304 reported cases, 88% (1,142 of 1,304) were detected in the northern region, with the highest number of cases concentrated in Amazonas and Pará,¹⁷ which demonstrates its high prevalence in the area and highlights the need for epidemiological and genomic monitoring of this virus.

Although the D genotype of MAYV isolated in this study has been identified in other regions of the country in previous years, such as the midwest and north of Brazil,^7,18–20 the phylogenetic reconstruction observed in the isolate in this study shows a closer relationship with Peruvian strains, indicating that the D genotype described here possibly diverged from strains phylogenetically related to these strains. However, with the lack of publications of recent Brazilian MAYV sequences in public databases, we were unable to infer a direct link with Brazilian sequences. Given its high endemicity, this finding reinforces the importance of evaluating other arboviruses, such as MAYV, to gain a understanding of these diseases in the Amazon region.

CONCLUSION

The case reported in this study reinforces the need for a differential diagnosis for Mayaro in the laboratory routine due to the similarity of the symptoms with other arboviruses, mainly dengue, and the importance of genomic and epidemiological surveillance for tracking neglected arboviruses in the country. Furthermore, additional studies are needed to estimate the prevalence of Mayaro cases in Rondônia, Brazil.