Characterization of the virome associated with Haemagogus mosquitoes in Trinidad, West Indies.

Currently, there are increasing concerns about the possibility of a new epidemic due to emerging reports of Mayaro virus (MAYV) fever outbreaks in areas of South and Central America. Haemagogus mosquitoes, the primary sylvan vectors of MAYV are poorly characterized and a better understanding of the mosquito's viral transmission dynamics and interactions with MAYV and other microorganisms would be important in devising effective control strategies. In this study, a metatranscriptomic based approach was utilized to determine the prevalence of RNA viruses in field-caught mosquitoes morphologically identified as Haemagogus janthinomys from twelve (12) forest locations in Trinidad, West Indies. Known insect specific viruses including the Phasi Charoen-like and Humaiata-Tubiacanga virus dominated the virome of the mosquitoes throughout sampling locations while other viruses such as the avian leukosis virus, MAYV and several unclassified viruses had a narrower distribution. Additionally, assembled contigs from the Ecclesville location suggests the presence of a unique uncharacterized picorna-like virus. Mapping of RNA sequencing reads to reference mitochondrial sequences of potential feeding host animals showed hits against avian and rodent sequences, which putatively adds to the growing body of evidence of a potentially wide feeding host-range for the Haemagogus mosquito vector.

Pseudotyping Lentiviral Vectors: When the Clothes Make the Virus.

Delivering transgenes to human cells through transduction with viral vectors constitutes one of the most encouraging approaches in gene therapy. Lentivirus-derived vectors are among the most promising vectors for these approaches. When the genetic modification of the cell must be performed in vivo, efficient specific transduction of the cell targets of the therapy in the absence of off-targeting constitutes the Holy Grail of gene therapy. For viral therapy, this is largely determined by the characteristics of the surface proteins carried by the vector. In this regard, an important property of lentiviral vectors is the possibility of being pseudotyped by envelopes of other viruses, widening the panel of proteins with which they can be armed. Here, we discuss how this is achieved at the molecular level and what the properties and the potentialities of the different envelope proteins that can be used for pseudotyping these vectors are.

Viral RNA is a target for Wolbachia-mediated pathogen blocking.

The ability of the endosymbiont Wolbachia pipientis to restrict RNA viruses is presently being leveraged to curb global transmission of arbovirus-induced diseases. Past studies have shown that virus replication is limited early in arthropod cells colonized by the bacterium, although it is unclear if this phenomenon is replicated in mosquito cells that first encounter viruses obtained through a vertebrate blood meal. Furthermore, these cellular events neither explain how Wolbachia limits dissemination of viruses between mosquito tissues, nor how it prevents transmission of infectious viruses from mosquitoes to vertebrate host. In this study, we try to address these issues using an array of mosquito cell culture models, with an additional goal being to identify a common viral target for pathogen blocking. Our results establish the viral RNA as a cellular target for Wolbachia-mediated inhibition, with the incoming viral RNA experiencing rapid turnover following internalization in cells. This early block in replication in mosquito cells initially infected by the virus thus consequently reduces the production of progeny viruses from these same cells. However, this is not the only contributor to pathogen blocking. We show that the presence of Wolbachia reduces the per-particle infectivity of progeny viruses on naïve mosquito and vertebrate cells, consequently limiting virus dissemination and transmission, respectively. Importantly, we demonstrate that this aspect of pathogen blocking is independent of any particular Wolbachia-host association and affects viruses belonging to Togaviridae and Flaviviridae families of RNA viruses. Finally, consistent with the idea of the viral RNA as a target, we find that the encapsidated virion RNA is less infectious for viruses produced from Wolbachia-colonized cells. Collectively, our findings present a common mechanism of pathogen blocking in mosquitoes that establish a link between virus inhibition in the cell to virus dissemination and transmission.

Insect-specific virus evolution and potential effects on vector competence.

The advancement in high-throughput sequencing technology and bioinformatics tools has spurred a new age of viral discovery. Arthropods is the largest group of animals and has shown to be a major reservoir of different viruses, including a group known as insect-specific viruses (ISVs). The majority of known ISVs have been isolated from mosquitoes and shown to belong to viral families associated with animal arbovirus pathogens, such as Flaviviridae, Togaviridae and Phenuiviridae. These insect-specific viruses have a strict tropism and are unable to replicate in vertebrate cells, these properties are interesting for many reasons. One is that these viruses could potentially be utilised as biocontrol agents using a similar strategy as for Wolbachia. Mosquitoes infected with the viral agent could have inferior vectorial capacity of arboviruses resulting in a decrease of circulating arboviruses of public health importance. Moreover, insect-specific viruses are thought to be ancestral to arboviruses and could be used to study the evolution of the switch from single-host to dual-host. In this review, we discuss new discoveries and hypothesis in the field of arboviruses and insect-specific viruses.

Rio Negro virophage: Sequencing of the near complete genome and transmission electron microscopy of viral factories and particles.

Rio Negro virophage (RNV) was co-isolated with a strain of mimivirus named sambavirus, from Brazilian Amazon. We report the near complete genome sequence of RNV, the first virophage isolated in Brazil. We also present new microscopical data demonstrating that RNV particles have similar dimensions to that described to sputnik virophages.

ICTV Virus Taxonomy Profile: Togaviridae.

The Togaviridae is a family of small, enveloped viruses with single-stranded, positive-sense RNA genomes of 10-12 kb. Within the family, the genus Alphavirus includes a large number of diverse species, while the genus Rubivirus includes the single species Rubella virus. Most alphaviruses are mosquito-borne and are pathogenic in their vertebrate hosts. Many are important human and veterinary pathogens (e.g. chikungunya virus and eastern equine encephalitis virus). Rubella virus is transmitted by respiratory routes among humans. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Togaviridae, which is available at www.ictv.global/report/togaviridae.

Viral Macrodomains: Unique Mediators of Viral Replication and Pathogenesis.

Viruses from the Coronaviridae, Togaviridae, and Hepeviridae families ​all contain genes that encode a conserved protein domain, called a macrodomain; however, the role of this domain during infection has remained enigmatic. The recent discovery that mammalian macrodomain proteins enzymatically remove ADP-ribose, a common post-translation modification, from proteins has led to an outburst of studies describing both the enzymatic activity and function of viral macrodomains. These new studies have defined these domains as de-ADP-ribosylating enzymes, which indicates that these viruses have evolved to counteract antiviral ADP-ribosylation, likely mediated by poly-ADP-ribose polymerases (PARPs). Here, we comprehensively review this rapidly expanding field, describing the structures and enzymatic activities of viral macrodomains, and discussing their roles in viral replication and pathogenesis.

Rio Negro virophage: Sequencing of the near complete genome and transmission electron microscopy of viral factories and particles

ABSTRACT Rio Negro virophage (RNV) was co-isolated with a strain of mimivirus named sambavirus, from Brazilian Amazon. We report the near complete genome sequence of RNV, the first virophage isolated in Brazil. We also present new microscopical data demonstrating that RNV particles have similar dimensions to that described to sputnik virophages.

Viral RNAs are unusually compact.

A majority of viruses are composed of long single-stranded genomic RNA molecules encapsulated by protein shells with diameters of just a few tens of nanometers. We examine the extent to which these viral RNAs have evolved to be physically compact molecules to facilitate encapsulation. Measurements of equal-length viral, non-viral, coding and non-coding RNAs show viral RNAs to have among the smallest sizes in solution, i.e., the highest gel-electrophoretic mobilities and the smallest hydrodynamic radii. Using graph-theoretical analyses we demonstrate that their sizes correlate with the compactness of branching patterns in predicted secondary structure ensembles. The density of branching is determined by the number and relative positions of 3-helix junctions, and is highly sensitive to the presence of rare higher-order junctions with 4 or more helices. Compact branching arises from a preponderance of base pairing between nucleotides close to each other in the primary sequence. The density of branching represents a degree of freedom optimized by viral RNA genomes in response to the evolutionary pressure to be packaged reliably. Several families of viruses are analyzed to delineate the effects of capsid geometry, size and charge stabilization on the selective pressure for RNA compactness. Compact branching has important implications for RNA folding and viral assembly.

Comparison of complete polyprotein sequences of two isolates of salmon alphavirus (SAV) type I and their behaviour in a salmonid cell line.

Salmon pancreas disease virus is an alphavirus (family Togaviridae) affecting mainly Atlantic salmon (Salmo salar L.). Both polyprotein sequences of the Scottish isolate (SAV4640) were determined and compared with those of Irish isolate SAVF93-125. High amino acid sequence similarity (99.4 %) was found. Six amino acid deletions were found in the E2 gene of SAV4640. SAVF93-125 demonstrated a high viral load in culture despite high Mx expression. Approximately 50 % of cells infected with SAVF93-125 exhibited a cytopathic effect by day 8. SAV4640 successfully entered the cells, inducing 10,500-fold higher Mx expression at day 2 compared to SAVF93-25; however, no replication was observed based on results of the nsP1 qRT-PCR.

Probing Ca2+-binding capability of viral proteins with the EF-hand motif by grafting approach.

Ca(2+) is implicated in almost every step of the life cycle of viruses, including virus entry into host cells, virus replication, virion assembly, maturation, and release. However, due to the lack of prediction algorithms and rigorous validation methods, only limited cases of viral Ca(2+)-binding sites are reported. Here, we introduce a method to predict continuous EF-hand or EF-hand-like motifs in the viral genomes based on their primary sequences. We then introduce a grafting approach, and the use of luminescence resonance energy transfer and Ca(2+) competition assay for experimental verification of predicted Ca(2+)-binding sites. This protocol will be valuable for the prediction and identification of unknown Ca(2+)-binding sites in virus.

[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.

Phylogeographical structure and evolutionary history of two Buggy Creek virus lineages in the western Great Plains of North America.

Buggy Creek virus (BCRV) is an unusual arbovirus within the western equine encephalitis complex of alphaviruses. Associated with cimicid swallow bugs (Oeciacus vicarius) as its vector and the cliff swallow (Petrochelidon pyrrhonota) and house sparrow (Passer domesticus) as its amplifying hosts, this virus is found primarily in the western Great Plains of North America at spatially discrete swallow nesting colonies. For 342 isolates collected in Oklahoma, Nebraska, Colorado and North Dakota, from 1974 to 2007, we sequenced a 2076 bp region of the 26S subgenomic RNA structural glycoprotein coding region, and analysed phylogenetic relationships, rates of evolution, demographical histories and temporal genetic structure of the two BCRV lineages found in the Great Plains. The two lineages showed distinct phylogeographical structure: one lineage was found in the southern Great Plains and the other in the northern Great Plains, and both occurred in Nebraska and Colorado. Within each lineage, there was additional latitudinal division into three distinct sublineages. One lineage is showing a long-term population decline. In comparing sequences taken from the same sites 8-30 years apart, in one case one lineage had been replaced by the other, and in the other cases there was little evidence of the same haplotypes persisting over time. The evolutionary rate of BCRV is in the order of 1.6-3.6x10(-4) substitutions per site per year, similar to that estimated for other temperate-latitude alphaviruses. The phylogeography and evolution of BCRV could be better understood once we determine the nature of the ecological differences between the lineages.

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.

Retinoblastoma protein purification and transduction of retina and retinoblastoma cells using improved alphavirus vectors.

PURPOSE: To develop and use improved Semliki Forest vectors (SFVs) for functional and structural analyses of the retinoblastoma protein (RB) in developing retina and retinoblastoma cells. METHODS: Virus was harvested from cells transfected with replicon and helper plasmids. Combinations of producer and target cells were tested for optimal virus production and protein expression. The replicon was improved by adding an expanded multiple cloning site, translational enhancer, and FLAG and HIS10 epitope and affinity tags. Affinity chromatography was used to purify beta-galactosidase or RB. RB function was assessed through interaction with E2F1. The efficacy of SFV as a retinal delivery system was tested on mouse explants and cultured human retinoblastoma cells. RESULTS: The optimal producer and target cell lines were an HEK-293 derivative (Phoenix Eco) and BHK-21, respectively. Stable expression of structural proteins in the BHK-21 helper line simplified virus production and amplified virus yield 100-fold. The translational enhancer improved expression three- to eightfold. Full-length, functional RB was produced without the truncated products characteristic of bacterially produced RB and was purified using the affinity tags. SFVs efficiently transduced mouse retinal explants and multiple hard-to-transduce retinoblastoma tumor lines. CONCLUSIONS: This study describes a simple, rapid, SFV vector system to produce recombinant proteins, such as RB, in mammalian cells. These SFV vectors represent an efficient approach to purification of proteins and protein complexes and transduction of retinal or retinoblastoma cells for the purpose of in vivo analysis of protein functions.

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.

Determination of natural versus laboratory human infection with Mayaro virus by molecular analysis.

A laboratory worker developed clinical signs of infection with Mayaro virus (Togaviridae), an arbovirus of South and Central America, 6 days after preparation of Mayaro viral antigen and 10 days after a trip to a rain forest. There was no evidence of skin lesions during the antigen preparation, and level 3 containment safety measures were followed. Therefore, molecular characterization of the virus was undertaken to identify the source of infection. RT-PCR and DNA sequence comparisons proved the infection was with the laboratory strain. Airborne Mayaro virus contamination is thus a hazard to laboratory personnel.

Universal diagnostic RT-PCR protocol for arboviruses.

A selected number of PCR protocols were evaluated to determine if they could serve as a universal protocol for detecting and identifying all arboviruses. In this study, four parameters that affect the efficacy of RT-PCR (RNA extraction method, choice of reverse transcriptase, choice of DNA polymerase and thermocycling program) were evaluated in combination. The most optimal combination of those parameters employed use of silica gel membrane spin column, RAV-2 reverse transcriptase, Tth DNA polymerase, and a simple modification of a published thermocycling program. By this modified protocol, viral RNA could be amplified satisfactorily with more than 50 pairs of primers designed for diagnosis of arboviruses representing five families. The sensitivity and specificity obtained by this universal protocol were comparable to those obtained by the original protocol for each primer pair tested; and for some primers, improved sensitivity was observed. It was also found that a simple modification of a suggested protocol of a commercial RT-PCR kit could produce nearly identical results and serve as another universal protocol. With the use of a universal diagnostic reverse transcriptase-polymerase chain reaction (RT-PCR) protocol, simultaneous screening of clinical or biological specimens against a large number of RNA viruses belonging to many families can be performed more efficiently for etiologic determination in the situations complicated by the difficulty of differential diagnosis. Furthermore, such a universal protocol facilitates reducing the cost of PCR-based diagnostic operation and standardizing the qualities of PCR-based diagnosis within an institution or among collaborating institutions. A logical strategy is to conduct diagnosis in two stages by using broadly group-reactive primers in the first stage to narrow the range of possible etiologic agents and using virus-specific primers in the second stage for identification. Before such a strategy is employed, however, more group-reactive primers for a large number of arboviruses, for which no such primers currently exist, must be made available. Furthermore, the best pair or pairs of primers need to be selected for each virus for the second stage of the strategy.