An improved alphaviruses-specific RT-qPCR facilitates monitoring and prevention of alphaviruses.

Molecular surveillance is vital for monitoring arboviruses, often employing genus-specific quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Despite this, an overlooked chikungunya fever outbreak occurred in Yunnan province, China, in 2019 and false negatives are commonly encountered during alphaviruses screening practice, highlighting the need for improved detection methods. In this study, we developed an improved alphaviruses-specific RT-qPCR capable of detecting chikungunya virus, eastern equine encephalitis virus, western equine encephalitis virus, Venezuelan equine encephalitis virus, Sindbis virus, Mayaro virus, and Ross River virus with high sensitivity and specificity. The assay identified three chikungunya virus-positive cases out of 188 sera retrospectively. Later genetic characterization suggested that imported cases from neighboring countries may be responsible for the neglected chikungunya fever outbreak of 2019 in Yunnan. Our findings underscore the value of improved alphaviruses-specific RT-qPCR in bolstering alphaviruses surveillance and informing preventive strategies.

Development and application of a colloidal-gold immunochromatographic strip for detecting Getah virus antibodies.

Getah virus (GETV) is a re-emerging mosquito-borne alphavirus that is highly pathogenic, mainly to pigs and horses. There are no vaccines or treatments available for GETV in swine in China. Therefore, the development of a simple, rapid, specific, and sensitive serological assay for GETV antibodies is essential for the prevention and control of GETV. Current antibody monitoring methods are time-consuming, expensive, and dependent on specialized instrumentation, and these features are not conducive to rapid detection in clinical samples. To address these problem, we developed immunochromatographic test strips (ICTS) using eukaryotically expressed soluble recombinant p62-E1 protein of GETV as a labelled antigen, which has good detection sensitivity and no cross-reactivity with other common porcine virus-positive sera. The ICTS is highly compatible with IFA and ELISA and can be stored for 1 month at 37 °C and for at least 3 months at room temperature. Hence, p62-E1-based ICTS is a rapid, accurate, and convenient method for rapid on-site detection of GETV antibodies. KEY POINTS: • We established a rapid antibody detection method that can monitor GETV infection • We developed colloidal gold test strips with high sensitivity and specificity • The development of colloidal gold test strips will aid in the field serologic detection of GETV.

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.

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.

Ross River Virus Antibody Prevalence, Fiji Islands, 2013-2015.

A unique outbreak of Ross River virus (RRV) infection was reported in Fiji in 1979. In 2013, RRV seroprevalence among residents was 46.5% (362/778). Of the residents who were seronegative in 2013 and retested in 2015, 10.9% (21/192) had seroconverted to RRV, suggesting ongoing endemic circulation of RRV in Fiji.

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.

Madariaga Virus: Identification of a Lineage III Strain in a Venezuelan Child With Acute Undifferentiated Febrile Illness, in the Setting of a Possible Equine Epizootic.

We report identification of Madariaga virus (MADV) in plasma and urine samples from a child with acute undifferentiated febrile illness in Venezuela. Our data document the occurrence of milder MADV infections (ie, without encephalitis), with a symptom complex that resembles that seen with other arboviral infections, including dengue and zika.

Improved detection of genus-specific Alphavirus using a generic TaqMan® assay.

BACKGROUND: Alphaviruses are arthropod borne RNA viruses of medical importance. Geographical expansion of mosquitoes of the Aedes genus in the past decades has been associated with major Alphavirus-associated outbreaks. Climate changes and intensification of air travels have favored vector expansion and virus dissemination in new territories leading to virus emergence not only in tropical areas but also in temperate regions. The detection of emergence is based upon surveillance networks with epidemiological and laboratory investigation. METHOD: A specific, sensitive and rapid screening test for genus-specific Alphavirus is critically required. To address this issue, we developed a new molecular assay targeting nsP4 gene and using a TaqMan® real time RT-PCR method for the specific detection of all major Alphavirus genus members. RESULTS: This assay was tested for specificity using several Alphavirus species. We also tested successfully clinical sensitivity using patient's samples collected during the Chikungunya outbreak of 2005-2006 in the Indian Ocean. CONCLUSIONS: This new pan-Alphavirus molecular diagnostic tool offers great potential for exclusion diagnosis and emergence detection given its broad specificity restricted to Alphavirus genus.

Selective precipitation reaction: a novel diagnostic test for tissue pathology in Atlantic salmon, Salmo salar, infected with salmonid alphavirus (SAV3).

While investigating biomarkers for infection with salmonid alphavirus (SAV), the cause of pancreas disease (PD), a selective precipitation reaction (SPR) has been discovered in serum which could be an on-farm qualitative test and an in-laboratory quantitative assay for health assessments in aquaculture. Mixing serum from Atlantic salmon, Salmo salar, with SAV infection with a sodium acetate buffer caused a visible precipitation which does not occur with serum from healthy salmon. Proteomic examination of the precipitate has revealed that the components are a mix of muscle proteins, for example enolase and aldolase, along with serum protein such as serotransferrin and complement C9. The assay has been optimized for molarity, pH, temperature and wavelength so that the precipitation can be measured as the change in optical density at 340 nm (Δ340 ). Application of the SPR assay to serum samples from a cohabitation trial of SAV infection in salmon showed that the Δ340 in infected fish rose from undetectable to a maximum at 6 weeks post-infection correlating with histopathological score of pancreas, heart and muscle damage. This test may have a valuable role to play in the diagnostic evaluation of stock health in salmon.

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

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.

Arboviral diseases and malaria in Australia, 2013-14: Annual report of the National Arbovirus and Malaria Advisory Committee.

This report describes the epidemiology of mosquito-borne diseases of public health importance in Australia during the 2013-14 season (1 July 2013 to 30 June 2014) and includes data from human notifications, sentinel chicken, vector and virus surveillance programs. The National Notifiable Diseases Surveillance System received notifications for 8,898 cases of disease transmitted by mosquitoes during the 2013-14 season. The Australasian alphaviruses Barmah Forest virus and Ross River virus accounted for 6,372 (72%) total notifications. However, over-diagnosis and possible false positive diagnostic test results for these 2 infections mean that the true burden of infection is likely overestimated, and as a consequence, the case definitions have been amended. There were 94 notifications of imported chikungunya virus infection and 13 cases of imported Zika virus infection. There were 212 notifications of dengue virus infection acquired in Australia and 1,795 cases acquired overseas, with an additional 14 cases for which the place of acquisition was unknown. Imported cases of dengue were most frequently acquired in Indonesia (51%). No cases of locally-acquired malaria were notified during the 2013-14 season, though there were 373 notifications of overseas-acquired malaria. In 2013-14, arbovirus and mosquito surveillance programs were conducted in most jurisdictions. Surveillance for exotic mosquitoes at international ports of entry continues to be a vital part of preventing the spread of vectors of mosquito-borne diseases such as dengue to new areas of Australia, with 13 detections of exotic mosquitoes at the ports of entry in 2013-14.

Ross River virus infection in a Thuringian traveller returning from south-east Australia.

Ross River virus (RRV) is an arbovirus transmitted by Aedes and Culex mosquitos. It is endemic in Australia, New Zealand and south-east Asia. Clinical manifestation rates in adults range about 20-40%. Symptoms involve arthralgia, myalgia, lymphadenopathy, fever and rash. Here we report a case of RRV in a Thuringian traveller who visited the urban South-East of Australia.

Ross River virus disease in two Dutch travellers returning from Australia, February to April 2015.

We report two cases of Ross River virus (RRV) infection in Dutch travellers who visited Australia during February to April 2015. These cases coincided with the largest recorded outbreak of RRV disease in Australia since 1996. This report serves to create awareness among physicians to consider travel-related RRV disease in differential diagnosis of patients with fever, arthralgia and/or rash returning from the South Pacific area, and to promote awareness among professionals advising travellers to this region.

[DETECTION OF VENEZUELAN EQUINE ENCEPHALOMYELITIS VIRUS RNA IN BIOLOGICAL SAMPLES BY REVERSE-TRANSCRIPTION POLYMERASE CHAIN REACTION].

AIM: Detection-and identification of Venezuelan equine encephalomyelitis (VEE) virus RNA in biological samples by reverse-transcription polymerase chain reaction (RT-PCR) and RT-PCR in real time (rRT-PCR). MATERIALS AND METHODS: VEE, Sindbis, West Nile, Japanese and tick-borne encephalitis viruses were studied. Cell culture of chicken fibroblasts, outbred mice and rats, Javanese macaques were used in the experiments. Biological activity determination of the running culture of causative agents used in the experiments was carried out by negative colony method in monolayer cell culture under agar coating. and using intra-cerebral infection of mice. Reagent kits developed in the 48th Central Research Institute and Institute of Analytical Instrument Engineering were used during execution of experiments of VEE virus RNA detection by RT-PCR and rRT-PCR. RESULTS: VEE virus was detected in biological samples by various methods. Data from RT-PCR and rRT-PCR are in accordance with the results of virus detection in samples using sensitive animals. CONCLUSION: Use of molecular-diagnostics methods for detection in biological samples of a causative agent of a dangerous infectious disease is important for procuring biological safety of Russian Federation.

Analysis of antibody response (IgM, IgG, IgG3) to Chikungunya virus using panel of peptides derived from envelope protein for serodiagnosis.

BACKGROUND: Many epidemic outbreaks of Chikungunya fever (CHIKF) have been reported throughout the world including India after its reemergence in 2005. The immuno protective role of envelope proteins during Chikungunya virus (CHIKV) infection has been reported. With the aim of identifying the immunodominant epitopes within the envelope protein we investigated the detailed analysis of fine specificity of antibody response in different individuals during CHIKV infection. METHODS: The peptides corresponding to the full length of E1, E2 and E3 proteins of S27 strain of CHIKV were synthesized and their seroreactivity with CHIKV positive patients' sera collected from different epidemic regions of India was determined using indirect ELISA. RESULTS: The data analysis reveals many potent epitopes throughout the length of envelope E2 protein thus displaying it as the most promising antigen for diagnostic purpose. We found that the main IgG isotype response to envelope protein was predominantly of subclass IgG3. Interestingly, most of the epitopes were found to be conserved for detecting IgM, IgG and IgG3 antibody response. CONCLUSIONS: Peptides E2P3, E2P7, E2P16 and E2P17 were revealed as the most immunodominant peptides that together can form the basis for designing an accurate, economical and easy to synthesize a peptide-based immunodiagnostic for CHIKV. This study provides new and important insight into the humoral response generated by CHIKV S27 strain during the early phase of infection.

Cases of chikungunya virus infection in travellers returning to Spain from Haiti or Dominican Republic, April-June 2014.

Ten cases of chikungunya were diagnosed in Spanish travellers returning from Haiti (n=2), the Dominican Republic (n=7) or from both countries (n=1) between April and June 2014. These cases remind clinicians to consider chikungunya in European travellers presenting with febrile illness and arthralgia, who are returning from the Caribbean region and Central America, particularly from Haiti and the Dominican Republic. The presence of Aedes albopictus together with viraemic patients could potentially lead to autochthonous transmission of chikungunya virus in southern Europe.

Local and regional spread of chikungunya fever in the Americas.

Chikungunya fever (CHIKV), a viral disease transmitted by mosquitoes, is currently affecting several areas in the Caribbean. The vector is found in the Americas from southern Florida to Brazil, and the Caribbean is a highly connected region in terms of population movements. There is therefore a significant risk for the epidemic to quickly expand to a wide area in the Americas. Here, we describe the spread of CHIKV in the first three areas to report cases and between areas in the region. Local transmission of CHIKV in the Caribbean is very effective, the mean number of cases generated by a human case ranging from two to four. There is a strong spatial signature in the regional epidemic, with the risk of transmission between areas estimated to be inversely proportional to the distance rather than driven by air transportation. So far, this simple distance-based model has successfully predicted observed patterns of spread. The spatial structure allows ranking areas according to their risk of invasion. This characterisation may help national and international agencies to optimise resource allocation for monitoring and control and encourage areas with elevated risks to act.

Dengue virus serotype 4 and chikungunya virus coinfection in a traveller returning from Luanda, Angola, January 2014.

A concurrent dengue virus serotype 4 and chikungunya virus infection was detected in a woman in her early 50s returning to Portugal from Luanda, Angola, in January 2014. The clinical, laboratory and molecular findings, involving phylogenetic analyses of partial viral genomic sequences amplified by RT-PCR, are described. Although the circulation of both dengue and chikungunya viruses in Angola has been previously reported, to our knowledge this is the first time coinfection with both viruses has been detected there.

Ross River virus infection surveillance in the Greater Perth Metropolitan area--has there been an increase in cases in the winter months?

An increase in off-season (June to September) Ross River virus (RRV) notifications from the greater Perth metropolitan area was observed from 2006 to 2009. We investigated the increase to determine whether it is likely to have reflected a true increase in off-season cases. A single positive RRV IgM test result is sufficient for RRV notification but where follow-up testing was performed, the positive predictive value of an IgM test where IgG was negative was very low in the off-season and also in the season when using the only commercially available test kit. The increase in off-season notifications was not associated with an increase in off-season testing. Some Perth laboratories use more stringent notification criteria than the nationally agreed RRV case definition, and the geographical distribution of samples tested varies between laboratories. Our findings make a strong case to change the nationally agreed case definition for RRV to not accept a single IgM positive test result as laboratory definitive evidence where the IgG is negative. Our study also identified a range of challenges in interpreting changes in seasonal patterns and geographical distribution of RRV. Any such observed changes should be investigated through further data analysis and/or mosquito trapping and testing in order to assess validity.