Expression and Purification of Recombinant Mayaro Virus Envelope Glycoproteins E1 and E2 to Develop a Mayaro Virus Detection System

Mayaro virus (MAYV) is a mosquito-transmitted alphavirus that produces an acute, usually non-fatal, febrile illness including Mayaro fever. Like other alphaviruses, the MAYV E1 and E2 envelope glycoproteins are major viral surface antigens that play a key role in host recognition and infection. Here, we report expression and purification methods for recombinant MAYV E1 (rE1) and rE2 using a baculovirus system. Enzyme-linked immunosorbent assays (ELISA) revealed that rE1 and rE2 were antigenic and reacted with human anti-MAYV IgG and IgM. Cross-reactivity was also confirmed with human anti-Chikungunya virus (CHIKV) IgG and IgM. Furthermore, we developed an immunochromatographic strip test (IST) with rE2 to diagnose MAYV infection. Thus, purified rE2 may be valuable tool for rapidly diagnosing MAYV infection.

Mayaro fever: A brief review on the immune profile

Mayaro virus is an emergent alphavirus that infects humans, leading to Mayaro fever. Approximately fifty percent of infected patients develop arthritis symptoms in the recovery phase, a phase that can last up to a year. The literature about Mayaro virus infection and its immune response is scarce, which may hamper the development of treatment strategies. We summarize changes in cytokines and chemokines in the acute and recovery phase in Mayaro virus infected patients, and relate this molecular characterization with the immune response. VEGF and IL-12/p70 show pronounced changes in patients in the acute phase, suggesting the development of cellular immunity and Th1 response. IL-6, IL-7, CXCL8/IL-8, IL-13, IL-17, and IFN-γ are elevated in patients with arthritis symptoms in the long-term recovery phase, which may be related to the continuous inflammatory process, a possible Th2 inhibiting and promoting Th17 process. Although few studies discuss the issue, with a small number of patients and different backgrounds, inflammatory and immune response and manifestations seem to be closely linked. This information may help to develop the appropriate treatment strategies in Mayaro virus infection. Therefore, we analyzed and summarized data available in literature.

Momento de considerar otras arbovirosis luego del virus mayaro / Time to Consider Other Arbovirus Diseases after mayaro Virus

Introducción: En los últimos años, debido a los movimientos migratorios, se ha desarrollado una expansión de nuevas enfermedades, como chikungunya, zika, oropuche y mayaro. Caso clínico: Paciente que manifestaba síntomas de fiebre, cefalea y artralgias persistente. Después de un arduo estudio y eliminación de otras patologías se llega al diagnóstico de virus mayaro. El paciente residía en una zona nororiental del Perú. Se brindó tratamiento de soporte junto con hidratación, paracetamol 500 mg cada 8 horas y se indicó cita diaria para evaluación. El paciente evolucionó favorablemente a los pocos días. Conclusiones: La vigilancia, las pruebas y el control vectorial siguen siendo claves para prevenir la propagación de este tipo de virus. La posibilidad de que el virus mayaro se urbanice aún más. Se debe tener siempre en cuenta el diagnóstico diferencial de virus mayaro(AU)

Introduction: In recent years, due to migratory movements, an expansion of new diseases has developed, such as chikungunya, zika, oropuche and mayaro. Clinical case: Patient with the following symptoms: fever, headache and persistent arthralgia. After an arduous study and ruling out other possible diseases, we diagnose mayaro virus. The patient resided in a northeastern part of Peru. Supportive treatment was provided along with hydration; paracetamol 500 mg every 8 hours and daily appointment for evaluation was indicated. The patient evolved favorably within a few days. Conclusions: Surveillance, testing and vector control are still key to monitoring and preventing the spread of this type of virus. The possibility of mayaro virus becoming more urbanized is worthy of attention. The differential diagnosis of mayaro virus should always be considered(AU)

Development of a triplex real-time RT-PCR assay to detect Zika, Chikungunya and Mayaro viruses / 中华实验和临床病毒学杂志

Objective@#To establish a method for the simultaneous identification of Zika, Chikungunya and Mayaro viruses.@*Methods@#The complete genome sequences of Zika, Chikungunya and Mayaro virus were retrieved from Global Shared Database for comparative analysis, estimate its conservative region and determine the target gene location, specific primers and probes were designed, then a triplex real-time RT-PCR assay was developed. The specificity, sensitivity and repeatability of the assay were assessed by viral nucleic acid of Zika virus, Chikungunya virus a, in vitro transcriptional RNA of Mayaro virus, normal human serum and related virus simulation sample.@*Results@#The result showed that the established method could detect Zika virus, Chikungunya virus, as well as simulated Mayaro virus samples, the limit of detection (LOD) of Zika and Chikungunya virus was 16.22 Copy/PCR and 12.02 Copy/PCR, respectively, the LOD for simulated Mayaro virus RNA was 2.82 Copy/PCR, no significant difference was detected between the triplex and monoplex assays. No cross reaction was found in the detection of dengue virus, Hantavirus, severe fever with thrombocytopenia syndrome (SFTS) virus, yellow fever virus and influenza virus, and 100 healthy adults blood samples, the specificity of the method was 100%. The repeatability result showed that the standard deviation of all three detections were blow 0.5 and the coefficient of variation was less than 2% by selecting viral nucleic acids or transcribed RNA with high, medium and low concentration gradients.@*Conclusions@#A triplex real-time RT-PCR assay for detection of Zika, Chikungunya and Mayaro virus has been established with an acceptable specificity, sensitivity and repeatability.

Prostaglandin A1 triggers Mayaro virus inhibition and heat shock protein 70 expression in an epithelial cell model

Abstract INTRODUCTION: The Mayaro virus (MAYV), which is an arbovirus closely related to the Chikungunya virus, causes a dengue-like acute illness that is endemic to Central and South America. We investigated the anti-MAYV activity of prostaglandin A1 (PGA1), a hormone which exhibits antiviral activity against both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) viruses. Further, we examined the effects of inducting the stress protein HSP70 following PGA1 treatment. METHODS: Hep-2 cells infected with MAYV were treated with PGA1 (0.1-6μg/ml) 12h before infection and for different periods post-infection. Inhibition of viral replication inhibition was analyzed via viral titer determination, whereas the effect of PGA1 on viral morphogenesis was examined via transmission electron microscopy (TEM). Autoradiography (with 35S methionine labeling) and western blotting were used to assess the effect of PGA1 treatment on viral and cellular protein synthesis, and on HSP70 induction, respectively. RESULTS: PGA1 strongly reduced viral replication in Hep-2 cells, particularly when added during the early stages of viral replication. Although PGA1 treatment inhibited viral replication by 95% at 24 hours post-infection (hpi), viral structural protein synthesis was inhibited only by 15%. TEM analysis suggested that PGA1 inhibited replication before viral morphogenesis. Western blot and densitometry analyses showed that PGA1 treatment increased HSP70 protein levels, although this was not detectable via autoradiography. CONCLUSIONS: PGA1 inhibits MAYV replication in Hep-2 cells at early stages of viral replication, prior to production of viral structural proteins, possibly via HSP70 induction.

Mayaro virus infection, the next epidemic wave after Zika? Evolutionary and structural analysis

Objective:To evaluate the evolution of the pathogen Mayaro virus, causing Mayaro fever (a mosquito-borne disease) and to perform selective pressure analysis and homology modelling.Methods:Nine different datasets were built, one for each protein (from protein C to non-structural protein 4) and the last one for the complete genome. Selective pressure and homology modelling analyses were applied.Results:Two main clades (A and B) were pointed in the maximum likelihood tree. The clade A included five Brazilian sequences sampled from 1955 to 2015. The Brazilian sequence sampled in 2014 significantly clustered with the Haitian sequence sampled in 2015. The clade B included the remaining 27 sequences sampled in the Central and Southern America from 1957 to 2013. Selective pressure analysis revealed several sites under episodic diversifying selection in envelope surface glycoprotein E1, non-structural protein 1 and non- structural protein 3 with a posterior probability P≤0.01. Homology modelling showed different sites modified by selective pressure and some protein-protein interaction sites at high interaction propensity.Conclusion:Maximum likelihood analysis confirmed the Mayaro virus previous circulation in Haiti and the successful spread to the Caribbean and USA. Selective pressure analysis revealed a strong presence of negatively selected sites, suggesting a probable purging of deleterious polymorphisms in functional genes. Homology model showed the position 31, under selective pressure, located in the edge of the ADP-ribose binding site predicting to possess a high potential of protein-protein interaction and suggesting the possible chance for a protective vaccine, thus preventing Mayaro virus urbanization as with Chikungunya virus.

Mayaro virus infection, the next epidemic wave after Zika? Evolutionary and structural analysis

Objective: To evaluate the evolution of the pathogen Mayaro virus, causing Mayaro fever (a mosquito-borne disease) and to perform selective pressure analysis and homology modelling. Methods: Nine different datasets were built, one for each protein (from protein C to non-structural protein 4) and the last one for the complete genome. Selective pressure and homology modelling analyses were applied. Results: Two main clades (A and B) were pointed in the maximum likelihood tree. The clade A included five Brazilian sequences sampled from 1955 to 2015. The Brazilian sequence sampled in 2014 significantly clustered with the Haitian sequence sampled in 2015. The clade B included the remaining 27 sequences sampled in the Central and Southern America from 1957 to 2013. Selective pressure analysis revealed several sites under episodic diversifying selection in envelope surface glycoprotein E1, non-structural protein 1 and non- structural protein 3 with a posterior probability P≤0.01. Homology modelling showed different sites modified by selective pressure and some protein-protein interaction sites at high interaction propensity. Conclusion: Maximum likelihood analysis confirmed the Mayaro virus previous circulation in Haiti and the successful spread to the Caribbean and USA. Selective pressure analysis revealed a strong presence of negatively selected sites, suggesting a probable purging of deleterious polymorphisms in functional genes. Homology model showed the position 31, under selective pressure, located in the edge of the ADP-ribose binding site predicting to possess a high potential of protein-protein interaction and suggesting the possible chance for a protective vaccine, thus preventing Mayaro virus urbanization as with Chikungunya virus.

Will Mayaro virus be responsible for the next outbreak of an arthropod-borne virus in Brazil?

Abstract Mayaro virus is an alphavirus from the Togaviridae family and is transmitted mainly by Hemagogus mosquitoes. This virus circulates in high-density tropical forests or rural areas of Central and South America causing a disease characterized by high-grade fever, maculopapular skin rash and marked arthralgia that, in some patients, can persist for long periods after infection and may be misinterpreted as chikungunya. Although only a few outbreaks involving this virus have been reported, in the last years the number of Mayaro virus infections has increased in the central and northern regions of Brazil. In this review, we describe the reported prevalence of this infection over the years and discuss the circumstances that can contribute to the establishment of an urban mayaro virus epidemic in Brazil and the problems encountered with the specific diagnosis, especially the antigenic cross-reactivity of this pathogen with other viruses of the same family.

Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses

ABSTRACT We describe a sensitive method for simultaneous detection of Oropouche and Oropouche-like viruses carrying the Oropouche S segment, as well as the Mayaro virus, using a multiplexed one-step reverse transcription real-time polymerase chain reaction (RT-qPCR). A chimeric plasmid containing both Mayaro and Oropouche targets was designed and evaluated for the in vitro production of transcribed RNA, which could be easily used as a non-infectious external control. To track false-negative results due to PCR inhibition or equipment malfunction, the MS2 bacteriophage was also included in the multiplex assay as an internal positive control. The specificity of the multiplex assay was evaluated by Primer-Blast analysis against the entire GenBank database, and further against a panel of 17 RNA arboviruses. The results indicated an accurate and highly sensitive assay with amplification efficiency greater than 98% for both targets, and a limit of detection between two and 20 copies per reaction. We believe that the assay described here will provide a tool for Mayaro and Oropouche virus detection, especially in areas where differential diagnosis of Dengue, Zika and Chikungunya viruses should be performed.

Mayaro virus and dengue virus 1 and 4 natural infection in culicids from Cuiabá, state of Mato Grosso, Brazil

This study aimed to verify the diversity of Culicidae species and their frequency of infection with flaviviruses and alphaviruses in Cuiabá, state of Mato Grosso, Brazil. Mosquitoes were captured with Nasci aspirators and hand net in 200 census tracts, identified alive at species level and pooled in one-20 (11,090 mosquitoes, 14 species). Female pools (n = 610) were subjected to multiplex seminested-reverse transcription-polymerase chain reaction (RT-PCR) for 11 flavivirus and five alphavirus. Positive pools were tested by single RT-PCR followed by nucleotide sequencing, by RT-PCR for E1 gene [Mayaro virus (MAYV)] and by inoculation in Vero cells (MAYV) or C6/36 cells (flaviviruses). One/171 Aedes aegypti was positive for dengue virus (DENV)-1, 12/403 Culex quinquefasciatus, and four/171Ae. aegypti for MAYV, which was isolated from two pools containing two nonengorged females of Ae. aegypti and two ofCx. quinquefasciatus. DENV-4 was detected in 58/171 pools of Ae. aegytpi, 105/403 Cx. quinquefasciatus, two/five Psorophora sp., two/11 Psorophora varipes/Psorophora albigenu, one/one Sabethes chloropterus, two/five Culex bidens/Culex interfor, and one/one Aedes sp. DENV-4 was isolated from two pools containing three and 16 nonengorged Cx. quinquefasciatus females. Phylogenetic analysis revealed MAYV belongs to genotype L, clustering with human samples of the virus previously identified in the city. Cuiabá has biodiversity and ecosystem favourable for vector proliferation, representing a risk for arbovirus outbreaks.

Prevalence of antibodies to equine alphaviruses in the State of Pará, Brazil / Prevalência de anticorpos para alphavirus em equinos do estado do Pará, Brasil

The State of Pará comprises 26% of Brazilian Amazon region, where a large diversity of arboviruses has been described. This study sought to assess the prevalence and distribution of hemagglutination inhibition (HI) antibodies against antigens of four alphaviruses (Togaviridae: Alphavirus ) from the species: Eastern equine encephalitis (EEEV), Western equine encephalitis (WEEV), Mayaro virus (MAYV), and Mucambo virus (MUCV) in 753 serum samples of horses in Pará State, Brazil. All investigated arboviruses were detected and indicate that horses are susceptible to these alphaviruses, and show evidences of their active circulation in farm animals in the Brazilian Amazon.(AU)

O estado do Pará corresponde a 26% da Amazônia brasileira, onde uma grande diversidade de arbovírus foi descrita. Este estudo procurou avaliar a prevalência e a distribuição de anticorpos inibidores da hemaglutinação (IH) contra antígenos de quatro alfavirus (Togaviridae: Alphavirus ), das espécies: Vírus da encefalite equina do leste (EEEV), Vírus da encefalite equina do oeste (WEEV), Vírus mayaro (MAYV) e Vírus mucambo (MUCV), de 753 amostras de soro de equinos no estado do Pará, Brasil. Todos os arbovirus pesquisados foram detectados, indicando que os equinos são suscetíveis a esses Alphavirus e mostrando evidências de sua circulação ativa em animais de fazenda na Amazônia brasileira.(AU)

Neutralising antibodies for Mayaro virus in Pantanal, Brazil

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

Molecular detection of Mayaro virus during a dengue outbreak in the state of Mato Grosso, Central-West Brazil

Mayaro virus (MAYV) is frequently reported in Pan-Amazonia. The aim of this study was to investigate the circulation of alphaviruses during a dengue outbreak in the state of Mato Grosso, Brazil. Serum samples from dengue-suspected patients were subjected to multiplex semi-nested reverse transcriptase polymerase chain reaction for 11 flaviviruses and five alphaviruses, to nucleotide sequencing and to viral isolation. MAYV was detected in 15 (2.5%) of 604 patients. Twelve were co-infected with dengue virus 4, which was isolated from 10 patients. The molecular detection of MAYV in dengue-suspected patients suggests that other arboviruses may be silently circulating during dengue outbreaks in Brazil.