Andrew E. Shaw

Identification and characterization of a novel non-structural protein of bluetongue virus.

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77–79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.

Clinical signs and pathology shown by British sheep and cattle infected with bluetongue virus serotype 8 derived from the 2006 outbreak in northern Europe

Four poll Dorset sheep and four Holstein-Friesian cattle were infected with the northern European strain of bluetongue virus (btv), btv-8, to assess its pathogenicity in uk breeds. The time course of infection was monitored in both species by using real-time reverse transcriptase-pcr (rt-pcr), conventional rt-pcr and serology. Two of the sheep developed severe clinical signs that would have been fatal in the field; the other two were moderately and mildly ill, respectively. The cattle were clinically unaffected, but had high levels of viral rna in their bloodstream. Real-time rt-pcr detected viral rna as early as one day after infection in the cattle and three days after infection in the sheep. Antibodies against btv were detected by six days after infection in the sheep and eight days after infection in the cattle. Postmortem examinations revealed pathology in the cattle that was more severe than suggested by the mild clinical signs, but the pathological and clinical findings in the sheep were more consistent.

Transmission Pathways of Foot-and-Mouth Disease Virus in the United Kingdom in 2007

Foot-and-mouth disease (FMD) virus causes an acute vesicular disease of domesticated and wild ruminants and pigs. Identifying sources of FMD outbreaks is often confounded by incomplete epidemiological evidence and the numerous routes by which virus can spread (movements of infected animals or their products, contaminated persons, objects, and aerosols). Here, we show that the outbreaks of FMD in the United Kingdom in August 2007 were caused by a derivative of FMDV O1 BFS 1860, a virus strain handled at two FMD laboratories located on a single site at Pirbright in Surrey. Genetic analysis of complete viral genomes generated in real-time reveals a probable chain of transmission events, predicting undisclosed infected premises, and connecting the second cluster of outbreaks in September to those in August. Complete genome sequence analysis of FMD viruses conducted in real-time have identified the initial and intermediate sources of these outbreaks and demonstrate the value of such techniques in providing information useful to contemporary disease control programmes.

Schmallenberg Virus Pathogenesis, Tropism and Interaction with the Innate Immune System of the Host

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV.

Detection of Foot-and-Mouth Disease Virus: Comparative Diagnostic Sensitivity of Two Independent Real-Time Reverse Transcription-Polymerase Chain Reaction Assays

Rapid and accurate diagnosis is central to the effective control of foot-and-mouth disease (FMD). It is now recognized that reverse-transcription polymerase chain reaction (RT-PCR) assays can play an important role in the routine detection of FMD virus (FMDV) in clinical samples. The aim of this study was to compare the ability of 2 independent real-time RT-PCR (rRT-PCR) assays targeting the 5′ untranslated region (5′UTR) and RNA polymerase (3D) to detect FMDV in clinical samples. There was concordance between the results generated by the 2 assays for 88.1% (347 of 394) of RNA samples extracted from suspensions of epithelial tissue obtained from suspect FMD cases. The comparison between the 2 tests highlighted 19 FMDV isolates (13 for the 5′UTR and 6 for the 3D assay), which failed to produce a signal in 1 assay but gave a positive signal in the other. The sequence of the genomic targets of selected isolates highlighted nucleotide substitutions in the primer or probe regions, thereby providing an explanation for negative results generated in the rRT-PCR assays. These data illustrate the importance of the continuous monitoring of circulating FMDV field strains to ensure the design of the rRT-PCR assay remains fit for purpose and suggest that the use of multiple diagnostic targets could further enhance the sensitivity of molecular methods for the detection of FMDV

Reassortment between Two Serologically Unrelated Bluetongue Virus Strains Is Flexible and Can Involve any Genome Segment

ABSTRACT Coinfection of a cell by two different strains of a segmented virus can give rise to a “reassortant” with phenotypic characteristics that might differ from those of the parental strains. Bluetongue virus (BTV) is a double-stranded RNA (dsRNA) segmented virus and the cause of bluetongue, a major infectious disease of livestock. BTV exists as at least 26 different serotypes (BTV-1 to BTV-26). Prompted by the isolation of a field reassortant between BTV-1 and BTV-8, we systematically characterized the process of BTV reassortment. Using a reverse genetics approach, our study clearly indicates that any BTV-1 or BTV-8 genome segment can be rescued in the heterologous “backbone.” To assess phenotypic variation as a result of reassortment, we examined viral growth kinetics and plaque sizes in in vitro experiments and virulence in an experimental mouse model of bluetongue disease. The monoreassortants generated had phenotypes that were very similar to those of the parental wild-type strains both in vitro and in vivo. Using a forward genetics approach in cells coinfected with BTV-1 and BTV-8, we have shown that reassortants between BTV-1 and BTV-8 are generated very readily. After only four passages in cell culture, we could not detect wild-type BTV-1 or BTV-8 in any of 140 isolated viral plaques. In addition, most of the isolated reassortants contained heterologous VP2 and VP5 structural proteins, while only 17% had homologous VP2 and VP5 proteins. Our study has shown that reassortment in BTV is very flexible, and there is no fundamental barrier to the reassortment of any genome segment. Given the propensity of BTV to reassort, it is increasingly important to have an alternative classification system for orbiviruses.

Double-Stranded RNA Analog Poly(I:C) Inhibits Human Immunodeficiency Virus Amplification in Dendritic Cells via Type I Interferon-Mediated Activation of APOBEC3G

ABSTRACT Human immunodeficiency virus (HIV) is taken up by and replicates in immature dendritic cells (imDCs), which can then transfer virus to T cells, amplifying the infection. Strategies known to boost DC function were tested for their ability to overcome this exploitation when added after HIV exposure. Poly(I:C), but not single-stranded RNA (ssRNA) or a standard DC maturation cocktail, elicited type I interferon (IFN) and interleukin-12 (IL-12) p70 production and the appearance of unique small (15- to 20-kDa) fragments of APOBEC3G (A3G) and impeded HIVBal replication in imDCs when added up to 60 h after virus exposure. Comparable effects were mediated by recombinant alpha/beta IFN (IFN-α/β). Neutralizing the anti-IFN-α/β receptor reversed poly(I:C)-induced inhibition of HIV replication and blocked the appearance of the small A3G proteins. The poly(I:C)-induced appearance of small A3G proteins was not accompanied by significant differences in A3G mRNA or A3G monomer expression. Small interfering RNA (siRNA) knockdown of A3G could not be used to reverse the poly(I:C)-induced protective effect, since siRNAs nonspecifically activated the DCs, inducing the appearance of the small A3G proteins and inhibiting HIV infection. Notably, the appearance of small A3G proteins coincided with the shift of high-molecular-mass inactive A3G complexes to the low-molecular-mass (LMM) active A3G complexes. The unique immune stimulation by poly(I:C) with its antiviral effects on imDCs marked by the expression of IFN-α/β and active LMM A3G renders poly(I:C) a promising novel strategy to combat early HIV infection in vivo.

RNA Interference Targets Arbovirus Replication in Culicoides Cells

ABSTRACT Arboviruses are transmitted to vertebrate hosts by biting arthropod vectors such as mosquitoes, ticks, and midges. These viruses replicate in both arthropods and vertebrates and are thus exposed to different antiviral responses in these organisms. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism that has been shown to play a major role in the antiviral response against arboviruses in mosquitoes. Culicoides midges are important vectors of arboviruses, known to transmit pathogens of humans and livestock such as bluetongue virus (BTV) (Reoviridae), Oropouche virus (Bunyaviridae), and likely the recently discovered Schmallenberg virus (Bunyaviridae). In this study, we investigated whether Culicoides cells possess an antiviral RNAi response and whether this is effective against arboviruses, including those with double-stranded RNA (dsRNA) genomes, such as BTV. Using reporter gene-based assays, we established the presence of a functional RNAi response in Culicoides sonorensis-derived KC cells which is effective in inhibiting BTV infection. Sequencing of small RNAs from KC and Aedes aegypti-derived Aag2 cells infected with BTV or the unrelated Schmallenberg virus resulted in the production of virus-derived small interfering RNAs (viRNAs) of 21 nucleotides, similar to the viRNAs produced during arbovirus infections of mosquitoes. In addition, viRNA profiles strongly suggest that the BTV dsRNA genome is accessible to a Dicer-type nuclease. Thus, we show for the first time that midge cells target arbovirus replication by mounting an antiviral RNAi response mainly resembling that of other insect vectors of arboviruses.

Inhibition of feline immunodeficiency virus infection by CD9 antibody operates after virus entry and is independent of virus tropism.

A monoclonal antibody which blocks infection with feline immunodeficiency virus (FIV) was found previously to react with the cell surface molecule CD9, implicating CD9 in the process of virus entry. We report here that inhibition by anti-CD9 antibody does not operate at the level of virus entry but at a subsequent stage in the virus life-cycle. Moreover, inhibition of infection is independent of the passage history of the virus or the virus subtype. Inhibition of FIV infection by anti-CD9 antibody does not operate in 3201 cells, which do not express this surface antigen. However, ectopic expression of CD9 on 3201 cells enhances infection with FIV, suggesting that the role of CD9 may be direct rather than via cellular signalling pathways. These results suggest a novel control point in the lentivirus life-cycle which might be susceptible to modulation by natural antagonists.

Virus and Host Factors Affecting the Clinical Outcome of Bluetongue Virus Infection

ABSTRACT Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype affect the clinical course of bluetongue. Results obtained indicate that in small ruminants, there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected toward the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low-frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants, and viral population diversity can be another factor influencing BTV virulence. IMPORTANCE Bluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected, while at the breed level differences were less evident. No differences were observed in the virulence of two different BTV serotypes (BTV-8 and BTV-2). In contrast, we show that the European BTV-8 strain isolated at the beginning of the bluetongue outbreak in 2006 was more virulent than a strain isolated toward the end of the outbreak. In addition, we show that there is a link between the variability of the BTV population as a whole and virulence, and our data also suggest that Culicoides cells might function as an “incubator” of viral variants.