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.

Deciphering the Nucleotide and RNA Binding Selectivity of the Mayaro Virus Macro Domain.

Mayaro virus (MAYV) is a member of Togaviridae family, which also includes Chikungunya virus as a notorious member. MAYV recently emerged in urban areas of the Americas, and this emergence emphasized the current paucity of knowledge about its replication cycle. The macro domain (MD) of MAYV belongs to the N-terminal region of its non-structural protein 3, part of the replication complex. Here, we report the first structural and dynamical characterization of a previously unexplored Alphavirus MD investigated through high-resolution NMR spectroscopy, along with data on its ligand selectivity and binding properties. The structural analysis of MAYV MD reveals a typical "macro" (ßßαßßαßαßα) fold for this polypeptide, while NMR-driven interaction studies provide in-depth insights into MAYV MD-ligand adducts. NMR data in concert with thermodynamics and biochemical studies provide convincing experimental evidence for preferential binding of adenosine diphosphate ribose (ADP-r) and adenine-rich RNAs to MAYV MD, thus shedding light on the structure-function relationship of a previously unexplored viral MD. The emerging differences with any other related MD are expected to enlighten distinct functions.

Efficacy and safety of an inactivated vaccine against Salmonid alphavirus (family Togaviridae).

Pancreas disease (PD) in salmonid fish is caused by an infection with Salmonid alphavirus (SAV) and remains as one of the major health problems in the European fish farming industry. Sequence studies have revealed a genetic diversity among viral strains. A subtype of SAV (SAV3) is causing an epizootic in farmed salmonids in Norway. Here we evaluate efficacy and safety of an inactivated virus vaccine based on ALV405, a strain of SAV3 that was isolated from Norwegian salmon. The vaccine provided an average relative percent survival (RPS) of 98.5 in an intraperitoneal challenge model, and induced nearly total protection against PD in a cohabitant challenge model. It provided significant protection against SAV-induced mortality also in a field trial under industrial conditions. Local reactions seen as melanization and adhesions in the visceral cavity were less severe than those induced by two commercial vaccines. Finally, we demonstrated that the protection is not impaired when the ALV405 antigen is combined with other viral or bacterial antigens in a polyvalent vaccine. The results confirm that efficient and safe protection against SAV infection and development of PD is possible using an inactivated virus vaccine, both alone and as a component in a polyvalent vaccine.

Seasonal variation and age-related correlates of Buggy Creek virus (Togaviridae) infection in nestling house sparrows.

Wild birds are rarely found with active arbovirus infections, and relatively little is known about the patterns of viremia they exhibit under field conditions or how infection varies with date, bird age, or other factors that potentially affect transmission dynamics. Buggy Creek virus (BCRV; Togaviridae, Alphavirus) is an arbovirus associated with colonially nesting Cliff Swallows (Petrochelidon pyrrhonota) and transmitted by its vector, the hematophagous swallow bug (Oeciacus vicarius), an ectoparasite of the Cliff Swallow. Introduced House Sparrows (Passer domesticus) that have occupied swallow nests at colony sites in peridomestic settings are also exposed to BCRV when fed upon by swallow bugs. We used data from 882 nestling House Sparrows in western Nebraska from 2006 to 2008 to examine seasonal variation and age-related correlates of virus infection in the field. Over 17% of nestling House Sparrows had active infections. Prevalence was higher in 2007 than in 2008 when birds from all colony sites were analyzed, but there was no significant difference between years for sites sampled in both seasons. Buggy Creek virus prevalence was similar in early and late summer, with a peak in midsummer, coinciding with the greatest swallow bug abundance. Nestlings 10 days of age and younger were most commonly infected, and the likelihood of BCRV infection declined for older nestlings. Average viremia titers also declined with age (but did not vary with date) and were high enough at all nestling ages to likely infect blood-feeding arthropods (swallow bugs). Length of viremia for nestlings in the field was ≥4 days, in agreement with an earlier study of BCRV. Nestling birds offer many advantages for field studies of arbovirus amplification and transmission.

[Taxonomic structure of Orthomyxoviridae: current views and immediate prospects].

Analysis of taxonomic structure of Orthomyxoviridae was undertaken in view of its anticipated evolution. Four concepts of circulation of influenza A viruses in the biosphere are discussed, viz. anthrponose, zooanthroponose, metastrongilose, and protozoan. All of them may be considered in the framework of the general zooantroponose concept. Influenza B and C viruses can not be regarded as strictly anthroponose. Comparative molecular-genetic analysis of the genus Thogotovirus provides a basis for the designation of Thogoto and Batken-Dhori as independent geni. It is speculated that t he proof of transmission of Isaviruses by copepods Caligus elongates and Lepeophtheirus salmonis (Crustacea: Copepoda) may open up a new line of developments in arborvirology since crustacean vectors of viruses have never been described before.

[Virology of the family Togaviridae].

Many pathogens important for medicine, veterinary medicine or public health belong to the genera alphavirus and rubivirus within the family Togaviridae. 29 species of alphaviruses have been reported, and most of them are arboviruses. Chikungnya virus re-emerged in Kenya in 2004 and the epidemics spread to the Indian Ocean islands and many countries in South Asia, South-East Asia and Europe. On the other hand, rubella virus, a sole member of the genus rubivirus, is the causative agent of rubella and congenital rubella syndrome (CRS). Because human is only a natural host of the virus and effective live attenuated vaccines are available, immunization activities are strengthened globally to eliminate rubella and CRS, together with measles.

Mayaro fever virus, Brazilian Amazon.

In February 2008, a Mayaro fever virus (MAYV) outbreak occurred in a settlement in Santa Barbara municipality, northern Brazil. Patients had rash, fever, and severe arthralgia lasting up to 7 days. Immunoglobulin M against MAYV was detected by ELISA in 36 persons; 3 MAYV isolates sequenced were characterized as genotype D.

Development and evaluation of a 1-step duplex reverse transcription polymerase chain reaction for differential diagnosis of chikungunya and dengue infection.

Dengue (DEN) and chikungunya (CHIK) have emerged as the 2 most important arboviral infections of global significance. The similarities in clinical presentations, their circulation in the same geographic area, and the transmission through the same vector necessitate an urgent need for the differential diagnosis of these 2 infections. So far, no single assay is reported for differential diagnosis of these 2 infections. In this study, we report the development and evaluation of a 1-step single-tube duplex reverse transcription polymerase chain reaction (D-RT-PCR) assay by targeting E1 gene of CHIK and C-prM gene junction of DEN virus (DENV), respectively. The sensitivity of this assay was found to be better than conventional virus isolation and could detect as low as 100 copies of genomic RNA, which is equivalent to respective virus-specific RT-PCR. The evaluation was carried out with 360 clinical samples from recent CHIK and DEN outbreaks in India. This assay could also be able to detect dual infection of CHIK and DEN in 3 patients. The phylogenetic analysis based on the nucleotide sequencing of D-RT-PCR amplicon could precisely identify the genotypes of all the serotypes of DENV and CHIK viruses (CHIKV). These findings demonstrate the potential clinical and epidemiologic application of D-RT-PCR for rapid sensitive detection, differentiation, and genotyping of DENV and CHIKV in clinical samples.

Host and vector movement affects genetic diversity and spatial structure of Buggy Creek virus (Togaviridae).

Determining the degree of genetic variability and spatial structure of arthropod-borne viruses (arboviruses) may help in identifying where strains that potentially cause epidemics or epizootics occur. Genetic diversity in arboviruses is assumed to reflect relative mobility of their vertebrate hosts (and invertebrate vectors), with highly mobile hosts such as birds leading to genetic similarity of viruses over large areas. There are no empirical studies that have directly related host or vector movement to virus genetic diversity and spatial structure. Using the entire E2 glycoprotein-coding region of 377 Buggy Creek virus isolates taken from cimicid swallow bugs (Oeciacus vicarius), the principal invertebrate vector for this virus, we show that genetic diversity between sampling sites could be predicted by the extent of movement by transient cliff swallows (Petrochelidon pyrrhonota) between nesting colonies where the virus and vectors occur. Pairwise F(ST) values between colony sites declined significantly with increasing likelihood of a swallow moving between those sites per 2-day interval during the summer nesting season. Sites with more bird movement between them had virus more similar genetically than did pairs of sites with limited or no bird movement. For one virus lineage, Buggy Creek virus showed greater haplotype and nucleotide diversity at sites that had high probabilities of birds moving into or through them during the summer; these sites likely accumulated haplotypes by virtue of frequent virus introductions by birds. Cliff swallows probably move Buggy Creek virus by transporting infected bugs on their feet. The results provide the first empirical demonstration that genetic structure of an arbovirus is strongly associated with host/vector movement, and suggest caution in assuming that bird-dispersed arboviruses always have low genetic differentiation across different sites.

A dual infection of infectious salmon anaemia (ISA) virus and a togavirus-like virus in ISA of Atlantic salmon Salmo salar in New Brunswick, Canada.

Two viruses, infectious salmon anaemia (ISA) virus and a novel togavirus-like virus, were isolated from ISA disease outbreaks that were first reported as a new syndrome, haemorrhagic kidney syndrome (HKS) affecting farmed Atlantic salmon Salmo salar L. on the East coast of Canada. Laboratory confirmation of ISA diagnosis was initially complicated by isolation of only the togavirus-like agent using the CHSE-214 cell line. Here we demonstrate that a clinical sample from a disease outbreak of ISA contained a mixture of ISA virus and togavirus-like virus. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the presence of both viruses during serial passage of cultures in SHK-1 and CHSE-214 cells. Virus harvested at passage level 3 in both cell lines caused high mortalities and severe gross pathology consistent with ISA virus infection in experimentally inoculated Atlantic salmon parr (approximately 35 g) in freshwater, beginning 12 d post inoculation. ISA virus was detected by virus isolation from kidney and liver tissues of all dead or moribund fish tested. A comparison of virus isolation, 1-step procedure RT-PCR and RNA dot-blot hybridization for detection of ISA virus (ISAV) in fish tissues showed virus isolation to have 100% sensitivity, followed by RT-PCR (66 and 28% sensitivity in kidney and liver, respectively), with RNA dot-blot hybridization as the least sensitive method (20 and 10% sensitivity in kidney and liver, respectively). No togavirus-like virus was detected in these samples by virus isolation. Moreover, another togavirus-like virus isolate grown in CHSE-214 cells in the absence of any other detectable pathogen was non-pathogenic in experimentally inoculated fish. This study confirms that the original ISA outbreaks in New Brunswick, Canada, were caused solely by ISAV.

Isolation and experimental oral transmission in pigs of a porcine reproductive and respiratory syndrome virus isolate.

A virus inducing a cytopathic effect on porcine alveolar macrophages was isolated from the lungs of a pig with respiratory problems and lesions of proliferative and necrotizing pneumonia. The isolate was found to react with porcine reproductive and respiratory syndrome virus (PRRSV) monoclonal antibodies by indirect immunofluorescence and was designated LHVA-93-3. The virus could also be propagated on the MARC-145 cell line. The LHVA-93-3 macrophage-passaged isolate was inoculated orally or intranasally in four-week-old specific pathogen-free pigs. Histologically, focal to multifocal lesions of proliferative, necrotizing and interstitial pneumonia could be observed in the lungs of pigs inoculated orally or intranasally, 6 and 10 days post-inoculation. Virus could be reisolated from essentially the same tissues including serum following both routes of infection. The distribution of PRRSV antigens in fixed tissues as determined by immunogold silver staining (IGSS) was similar in orally or intranasally inoculated pigs. The results of this experimental transmission study indicate that pigs may become infected by PRRSV following oral as well as intranasal exposure.