Establishment and application of an indirect ELISA for Getah virus E2 antibody detection.

Getah virus (GETV) is a mosquito-transmitted disease that affects animals, causing fever, aseptic meningitis, and abortion. Its prevalence in China poses risks to both animal health and public well-being. Currently, there is a scarcity of seroepidemiological data on GETV due to the absence of commercial antibody detection kits for pigs. The aim of this study is to develop a rapid, accurate, and sensitive ELISA, providing a reliable tool for GETV seroepidemiology and laying the foundation for future commercial assay development. In this study, we removed specific hydrophobic domains and intracellular structures from E2 proteins and constructed the recombinant plasmid pCold-TF-E2. The recombinant protein was expressed using a prokaryotic expression system, and efficient purification of the rE2 protein was achieved using a nickel affinity column. The purified rE2 protein is suitable for the development of an indirect ELISA (rE2 ELISA). Following the optimization of reaction conditions for the rE2-ELISA, the cut-off value was 0.356. Additionally, the rE2-ELISA method showed a positive rate of 37.1% for IgG antibodies against GETV when testing 986 pig clinical serum samples collected from pigs in Sichuan between May 2022 and September 2022. The rE2-ELISA method displayed a 95.1% overall agreement with VNT, boasting a sensitivity of 98.2% and a specificity of 92.6%. These results indicate that IgG ELISA based on rE2 protein is an efficient and economical method for the detection of GETV antibodies in pigs, facilitating the diagnosis and prevention of GETV.

Seroconversion of anti-Getah virus antibody among Japanese native Noma horses around 2012.

Getah virus (GETV), an arthropod-borne virus transmitted by mosquitoes, has been isolated from several animals. GETV infection in horses shows clinical signs such as fever, rash, and edema in the leg. Noma horses are one of the eight Japanese native horses. The present study aimed to clarify the occurrence of GETV infection in Noma horses. Serum samples collected from Noma horses were analyzed using a virus neutralization test and enzyme-linked immunosorbent assay and showed that the anti-GETV antibody titers in the samples collected in 2017 were significantly higher than those collected in 2012. We concluded that a seroconversion of anti-GETV antibodies was occurred in the Noma horse population around 2012, providing evidence of the GETV epidemic in Japan circa 2012.

Epidemiological and Genomic Characterisation of Middelburg and Sindbis Alphaviruses Identified in Horses with Febrile and Neurological Infections, South Africa (2014-2018).

Although Old World alphaviruses, Middelburg- (MIDV) and Sindbis virus (SINV), have previously been detected in horses and wildlife with neurologic disease in South Africa, the pathogenesis and clinical presentation of MIDV and SINV infections in animals are not well documented. Clinical samples from horses across South Africa with acute or fatal neurologic and febrile infections submitted between 2014-2018 were investigated. In total, 69/1084 (6.36%) and 11/1084 (1.01%) horses tested positive for MIDV and SINV, respectively, by real-time reverse transcription (RT) PCR. Main signs/outcomes for MIDV (n = 69): 73.91% neurological, 75.36% fever, 28.99% icterus and anorexia, respectively, 8.70% fatalities; SINV (n = 11): 54.54% neurological, 72.73% fever, 36.36% anorexia and 18.18% fatalities. MIDV cases peaked in the late summer/autumn across most South African provinces while SINV cases did not show a clear seasonality and were detected in fewer South African provinces. MIDV could still be detected in blood samples via RT-PCR for up to 71,417 and 21 days after onset of signs in 4 horses respectively, suggesting prolonged replication relative to SINV which could only be detected in the initial sample. Phylogenetic analyses based on partial sequences of the nsP4 (MIDV n = 59 and SINV n = 7) and E1 (MIDV n = 45) genes, as well as full genome sequences (MIDV n = 6), clustered the MIDV and SINV strains from the present study with previously detected strains. MIDV infection appears to be more prevalent in horses than SINV infection based on RT-PCR results, however, prevalence estimates might be different when also considering serological surveillance data.

Sindbis virus outbreak and evidence for geographical expansion in Finland, 2021.

Sindbis virus (SINV) caused a large outbreak in Finland in 2021 with 566 laboratory-confirmed human cases and a notable geographical expansion. Compared with the last large outbreak in 2002, incidence was higher in several hospital districts but lower in traditionally endemic locations in eastern parts of the country. A high incidence is also expected in 2022. Awareness of SINV should be raised in Finland to increase recognition of the disease and prevent transmission through the promotion of control measures.

Serological Responses in Patients Infected with Mayaro Virus and Evaluation of Cross-Protective Responses against Chikungunya Virus.

Mayaro virus (MAYV) is an alphavirus endemic to both Latin America and the Caribbean. Recent reports have questioned the ability of MAYV and its close relative, Chikungunya virus (CHIKV), to generate cross-reactive, neutralizing antibodies to one another. Since CHIKV was introduced to South America in 2013, discerning whether individuals have cross-reactive antibodies or whether they have had exposures to both viruses previously has been difficult. Using samples obtained from people infected with MAYV prior to the introduction of CHIKV in the Americas, we performed neutralizing assays and observed no discernable neutralization of CHIKV by sera from patients previously infected with MAYV. These data suggest that a positive CHIKV neutralization test cannot be attributed to prior exposure to MAYV and that previous exposure to MAYV may not be protective against a subsequent CHIKV infection.

Development of a reverse transcription recombinase-aided amplification assay for detection of Getah virus.

GETV, an arbo-borne zoonotic virus of the genus Alphavirus, which causes diarrhea and reproduction disorders in swine, lead to serious economic losses to the swine industry in China. At present, the existing methods for GETV detection are time-consuming and low sensitivity, so, a rapid, accurate and sensitive GETV detection method is urgently needed. In this study, a fluorescent reverse transcription recombinase-assisted amplification method (RT-RAA) was successfully established for the rapid detection of GETV. The sensitivity of this method to GETV was 8 copies/reaction and 20 TCID50/reaction. No cross-reaction with other viruses. A total of 118 samples were prepared for GETV detection using fluorescent RT-RAA and SYBR Green I RT-qPCR, the coincidence rate of the two methods was 100%. The results suggest that the RT-RAA method is rapid, sensitive and specific for GETV detection and can be applied in the clinical.

Performance assessment of a multi-epitope chimeric antigen for the serological diagnosis of acute Mayaro fever.

Mayaro virus (MAYV), which causes mayaro fever, is endemic to limited regions of South America that may expand due to the possible involvement of Aedes spp. mosquitoes in its transmission. Its effective control will require the accurate identification of infected individuals, which has been restricted to nucleic acid-based tests due to similarities with other emerging members of the Alphavirus genus of the Togaviridae family; both in structure and clinical symptoms. Serological tests have a more significant potential to expand testing at a reasonable cost, and their performance primarily reflects that of the antigen utilized to capture pathogen-specific antibodies. Here, we describe the assembly of a synthetic gene encoding multiple copies of antigenic determinants mapped from the nsP1, nsP2, E1, and E2 proteins of MAYV that readily expressed as a stable chimeric protein in bacteria. Its serological performance as the target in ELISAs revealed a high accuracy for detecting anti-MAYV IgM antibodies. No cross-reactivity was observed with serum from seropositive individuals for dengue, chikungunya, yellow fever, Zika, and other infectious diseases as well as healthy individuals. Our data suggest that this bioengineered antigen could be used to develop high-performance serological tests for MAYV infections.

Development of a Sensitive Detection Method for Alphaviruses and Its Use as a Virus Neutralization Assay.

Alphaviruses have a single-stranded, positive-sense RNA genome that contains two open reading frames encoding either the non-structural or the structural genes. Upon infection, the genomic RNA is translated into the non-structural proteins (nsPs). NsPs are required for viral RNA replication and transcription driven from the subgenomic promoter (sgP). Transfection of an RNA encoding the luciferase gene under the control of the sgP into cells enabled the detection of replication-competent chikungunya virus (CHIKV) or Mayaro virus (MAYV) with high sensitivity as a function of the induced luciferase activity. This assay principle was additionally used to analyze virus-neutralizing antibodies in sera and might be an alternative to standard virus neutralization assays based on virus titration or the use of genetically modified tagged viruses.

Arthritogenic alphaviruses: epidemiological and clinical perspective on emerging arboviruses.

Mosquito-borne viruses, or arboviruses, have been part of the infectious disease landscape for centuries, and are often, but not exclusively, endemic to equatorial and subtropical regions of the world. The past two decades saw the re-emergence of arthritogenic alphaviruses, a genus of arboviruses that includes several members that cause severe arthritic disease. Recent outbreaks further highlight the substantial public health burden caused by these viruses. Arthritogenic alphaviruses are often reported in the context of focused outbreaks in specific regions (eg, Caribbean, southeast Asia, and Indian Ocean) and cause debilitating acute disease that can extend to chronic manifestations for years after infection. These viruses are classified among several antigenic complexes, span a range of hosts and mosquito vectors, and can be distributed along specific geographical locations. In this Review, we highlight key features of alphaviruses that are known to cause arthritic disease in humans and outline the present findings pertaining to classification, immunogenicity, pathogenesis, and experimental approaches aimed at limiting disease manifestations. Although the most prominent alphavirus outbreaks in the past 15 years featured chikungunya virus, and a large body of work has been dedicated to understanding chikungunya disease mechanisms, this Review will instead focus on other arthritogenic alphaviruses that have been identified globally and provide a comprehensive appraisal of present and future research directions.

Ross River Virus Infection of Horses: Appraisal of Ecological and Clinical Consequences.

Ross River virus (RRV) is a mosquito-borne arbovirus of the genus Alphavirus that causes disease in humans and horses in Australia. A temporal association of RRV infection in horses with clinical signs including pyrexia, malaise, and polyarthralgia has been reported, along with reduced athletic performance, often for extended periods. Despite these reports, disease due to RRV remains somewhat controversial as experimental infection of horses has resulted in obvious viraemia yet minimal signs of clinical disease. The relatively high viraemia demonstrated by horses infected with RRV has led to speculation that they could act as an important reservoir host of the virus, although this remains unclear. This review sought to appraise the existing literature relating to RRV infection of horses and to summarize the ecological and clinical consequences of RRV of relevance to the equine industry and to public health more broadly.

A micro-PRNT for the detection of Ross River virus antibodies in mosquito blood meals: A useful tool for inferring transmission pathways.

INTRODUCTION: Many arboviruses of public health significance are maintained in zoonotic cycles with complex transmission pathways. The presence of serum antibody against arboviruses in vertebrates provides evidence of their historical exposure but reveals nothing about the vector-reservoir relationship. Moreover, collecting blood or tissue samples from vertebrate hosts is ethically and logistically challenging. We developed a novel approach for screening the immune status of vertebrates against Ross River virus that allows us to implicate the vectors that form the transmission pathways for this commonly notified Australian arboviral disease. METHODS: A micro-plaque reduction neutralisation test (micro-PRNT) was developed and validated on koala (Phascolarctos cinereus) sera against a standard PRNT. The ability of the micro-PRNT to detect RRV antibodies in mosquito blood meals was then tested using two mosquito models. Laboratory-reared Aedes aegypti were fed, via a membrane, on sheep blood supplemented with RRV seropositive and seronegative human sera. Aedes notoscriptus were fed on RRV seropositive and seronegative human volunteers. Blood-fed mosquitoes were harvested at various time points after feeding and their blood meals analysed for the presence of RRV neutralising antibodies using the micro-PRNT. RESULTS: There was significant agreement of the plaque neutralisation resulting from the micro-PRNT and standard PRNT techniques (R2 = 0.65; P<0.0001) when applied to RRV antibody detection in koala sera. Sensitivity and specificity of the micro-PRNT assay were 88.2% and 96%, respectively, in comparison with the standard PRNT. Blood meals from mosquitoes fed on sheep blood supplemented with RRV antibodies, and on blood from RRV seropositive humans neutralised the virus by ≥50% until 48 hr post feeding. The vertebrate origin of the blood meal was also ascertained for the same samples, in parallel, using established molecular techniques. CONCLUSIONS: The small volumes of blood present in mosquito abdomens can be used to identify RRV antibodies and therefore host exposure to arbovirus infection. In tandem with the accurate identification of the mosquito, and diagnostics for the host origin of the blood meal, this technique has tremendous potential for exploring RRV transmission pathways. It can be adapted for similar studies on other mosquito borne zoonoses.

Arboviruses related with chronic musculoskeletal symptoms.

Arboviruses (ARthropods BOrne VIRUSES) are disease-causing viruses transmitted through the bite of hematophagous arthropods, such as mosquitoes and ticks. Among these, the alphavirus, genus of the Togaviridae family, is considered the most arthritogenic species, responsible for diseases such as chikungunya fever (CHIK), O'nyong-nyong virus fever, Ross River virus disease, Barmah Forest virus disease, Sindbis virus disease, and Mayaro fever. These arboviral diseases, especially CHIK, have impacted public health in recent decades, leading to devastating epidemics, particularly in developing countries, due to their high potential for chronicity, functional impairment, and great impact on the quality of life. In a similar way, chronic musculoskeletal symptoms have been described in all alphavirus infections. However, CHIK is the best studied. The purpose of this article is to review physiopathology, clinical manifestations, diagnosis, and treatment of alphaviruses, focusing on CHIK and chronic evolution of musculoskeletal symptoms.

Projeto Quantizika Humano ­ UFG / Human Quantizica Project ­ UFG

O Projeto Quantizika Humano, desenvolvido por docentes de várias unidades acadêmicas da Universidade Federal de Goiás (UFG), teve início em 2016, quando um grupo de pesquisadores composto por professores das áreas de biologia molecular e genética, epidemiologista e médico sanitarista, selecionaram pacientes que apresentavam sintomas semelhantes a dengue, inicialmente com a finalidade de rastrear aqueles que pudessem apresentar infecção pelo vírus da Zika. Durante o rastreamento molecular, e por meio de técnica própria desenvolvida na pesquisa, foram identificados também casos de infecção pelo vírus Mayaro (FERNANDES, M. 2019)

The Quantizica Humano Project, developed by professors from several academic units at the Federal University of Goiás (UFG), began in 2016, when a group of researchers composed of professors in the areas of molecular biology and genetics, an epidemiologist and a health doctor, selected patients who had symptoms similar to dengue, initially with the aim of tracking those who might have Zika virus infection. During the molecular screening, and by means of a proprietary technique developed in the research, cases of infection by the Mayaro virus were also identified (FERNANDES, M. 2019)

Projeto Quantizika Humano-UFG / Human Quantizika Project-UFG

O projeto QUANTIZIKA HUMANO, desenvolvido por docentes de várias unidades acadêmicas da Universidade Federal de Goiás (UFG), teve início em 2016, quando um grupo de pesquisadores composto por professores das áreas de biologia molecular e genética, epidemiologista e médico sanitarista, selecionaram pacientes que apresentavam sintomas semelhantes a dengue, inicialmente com a finalidade de rastrear aqueles que pudessem apresentar infecção pelo vírus da Zika. Durante o rastreamento molecular, e por meio de técnica própria desenvolvida na pesquisa, foram identificados também casos de infecção pelo vírus Mayaro (FERNANDES, M. 2019).

The QUANTIZIKA HUMANO project, developed by professors from several academic units of the Federal University of Goiás (UFG), began in 2016, when a group of researchers composed of professors from the fields of molecular biology and genetics, epidemiologist and sanitary doctor, selected patients with symptoms similar to dengue, initially with the purpose of tracking those who could present zika virus infection. During molecular screening, and through its own technique developed in the research, cases of Mayaro virus infection were also identified (FERNANDES, M. 2019).

High-Throughput Fluorescence-Based Screen Identifies the Neuronal MicroRNA miR-124 as a Positive Regulator of Alphavirus Infection.

MicroRNAs (miRNAs) are small regulatory RNAs which act by modulating the expression of target genes. In addition to their role in maintaining essential physiological functions in the cell, miRNAs can also regulate viral infections. They can do so directly by targeting RNAs of viral origin or indirectly by targeting host mRNAs, and this can result in a positive or negative outcome for the virus. Here, we performed a fluorescence-based miRNA genome-wide screen in order to identify cellular miRNAs involved in the regulation of arbovirus infection in human cells. We identified 16 miRNAs showing a positive effect on Sindbis virus (SINV) expressing green fluorescent protein (GFP), among which were a number of neuron-specific ones such as miR-124. We confirmed that overexpression of miR-124 increases both SINV structural protein translation and viral production and that this effect is mediated by its seed sequence. We further demonstrated that the SINV genome possesses a binding site for miR-124. Both inhibition of miR-124 and silent mutations to disrupt this binding site in the viral RNA abolished positive regulation. We also proved that miR-124 inhibition reduces SINV infection in human differentiated neuronal cells. Finally, we showed that the proviral effect of miR-124 is conserved in other alphaviruses, as its inhibition reduces chikungunya virus (CHIKV) production in human cells. Altogether, our work expands the panel of positive regulation of the viral cycle by direct binding of host miRNAs to the viral RNA and provides new insights into the role of cellular miRNAs as regulators of alphavirus infection.IMPORTANCE Arthropod-borne (arbo) viruses are part of a class of pathogens that are transmitted to their final hosts by insects. Because of climate change, the habitat of some of these insects, such as mosquitoes, is shifting, thereby facilitating the emergence of viral epidemics. Among the pathologies associated with arbovirus infection, neurological diseases such as meningitis and encephalitis represent a significant health burden. Using a genome-wide miRNA screen, we identified neuronal miR-124 as a positive regulator of the Sindbis and chikungunya alphaviruses. We also showed that this effect was in part direct, thereby opening novel avenues to treat alphavirus infections.

Establishment of an enzyme-linked immunosorbent assay for Getah virus infection in horses using a 20-mer synthetic peptide for the E2 glycoprotein as an antigen.

An enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide for the E2 glycoprotein was developed for the serodiagnosis of Getah virus infection in horses. To identify an immunogenic epitope, a series of 20-mer peptides (n = 22) for the E2 protein was screened with pooled sera from horses infected with Getah virus. Peptide P11 (PTEEEIDMHTPPDIPDITLL) showed the strongest reaction. ELISA using P11 (E2-P11-ELISA) detected increased antibody levels in all seven experimentally infected horses and in five out of nine vaccinated horses. Out of 28 naturally infected horses, 25 were seronegative in their acute sera but turned seropositive in their convalescent sera. For the remaining three horses whose acute sera were seropositive, an endpoint method with serial dilutions detected a ≥ 4-fold increase in titer between paired sera. The concordance between E2-P11-ELISA and a virus-neutralization test in terms of seropositivity was assessed using a series of 220 horse sera, resulting in almost perfect agreement, with a kappa coefficient value of 0.865. E2-P11-ELISA had a sensitivity of 93.3% (95% CI 86.6-97.1%) and a specificity of 95.0% (95% CI 92.5-96.4%). This highly sensitive and specific E2-P11-ELISA should be useful for serodiagnosis of Getah virus infection in horses.

Divergent Barmah Forest Virus from Papua New Guinea.

We report a case of Barmah Forest virus infection in a child from Central Province, Papua New Guinea, who had no previous travel history. Genomic characterization of the virus showed divergent origin compared with viruses previously detected, supporting the hypothesis that the range of Barmah Forest virus extends beyond Australia.

Isolation of Mayaro Virus from a Venezuelan Patient with Febrile Illness, Arthralgias, and Rash: Further Evidence of Regional Strain Circulation and Possible Long-Term Endemicity.

Fifty-two febrile patients living in Barquisimeto, Venezuela, were screened for arbovirus infection by virus culture during an outbreak of what was thought to be Zika virus infection. We report identification of Mayaro virus (MAYV) on culture of plasma from one patient, an 18-year-old woman with acute febrile illness, arthralgias, and psoriasiform rash. The strain was sequenced and was found to be most closely related to a 1999 strain from French Guiana, which, in turn, was related to two 2014 strains from Haiti. By contrast, previously reported outbreak-related MAYV strains from a sylvatic area approximately 80 miles from where the case patient lived were most closely related to Peruvian isolates. The two strain groups show evidence of having diverged genetically approximately 100 years ago.

GloPID-R report on chikungunya, o'nyong-nyong and Mayaro virus, part 2: Epidemiological distribution of o'nyong-nyong virus.

The GloPID-R (Global Research Collaboration for Infectious Disease Preparedness) chikungunya (CHIKV), o'nyong-nyong (ONNV) and Mayaro virus (MAYV) Working Group has been established to identify gaps of knowledge about the natural history, epidemiology and medical management of infection by these viruses, and to provide adapted recommendations for future investigations. Here, we present a report dedicated to ONNV epidemiological distribution. Two large-scale ONNV outbreaks have been identified in Africa in the last 60 years, interspersed with sporadic serosurveys and case reports of returning travelers. The assessment of the real scale of ONNV circulation in Africa remains a difficult task and surveillance studies are necessary to fill this gap. The identification of ONNV etiology is made complicated by the absence of multiplex tools in co-circulation areas and that of reference standards, as well as the high cross-reactivity with related pathogens observed in serological tests, in particular with CHIKV. This is a specific obstacle for seroprevalence studies, that necessitate an improvement of serological tools to provide robust results. The scarcity of existent genetic data currently limits molecular epidemiology studies. ONNV epidemiology would also benefit from reinforced entomological and environmental surveillance. Finally, the natural history of the disease deserves to be further investigated, with a specific attention paid to long-term complications. Considering our incomplete knowledge on ONNV distribution, GloPID-R CHIKV, ONNV and MAYV experts recommend that a major effort should be done to fill existing gaps.