Investigating the Role of African Horse Sickness Virus VP7 Protein Crystalline Particles on Virus Replication and Release.

African horse sickness is a deadly and highly infectious disease of equids, caused by African horse sickness virus (AHSV). AHSV is one of the most economically important members of the Orbivirus genus. AHSV is transmitted by the biting midge, Culicoides, and therefore replicates in both insect and mammalian cell types. Structural protein VP7 is a highly conserved major core protein of orbiviruses. Unlike any other orbivirus VP7, AHSV VP7 is highly insoluble and forms flat hexagonal crystalline particles of unknown function in AHSV-infected cells and when expressed in mammalian or insect cells. To examine the role of AHSV VP7 in virus replication, a plasmid-based reverse genetics system was used to generate a recombinant AHSV that does not form crystalline particles. We characterised the role of VP7 crystalline particle formation in AHSV replication in vitro and found that soluble VP7 interacted with viral proteins VP2 and NS2 similarly to wild-type VP7 during infection. Interestingly, soluble VP7 was found to form uncharacteristic tubule-like structures in infected cells which were confirmed to be as a result of unique VP7-NS1 colocalisation. Furthermore, it was found that VP7 crystalline particles play a role in AHSV release and yield. This work provides insight into the role of VP7 aggregation in AHSV cellular pathogenesis and contributes toward the understanding of the possible effects of viral protein aggregation in other human virus-borne diseases.

Safety and efficacy of inactivated African horse sickness (AHS) vaccine formulated with different adjuvants.

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae causing African Horse Sickness (AHS) in equids with a mortality of about 95% in naïve horses. AHS causes serious losses in developing countries where horses play a central role in draft power and transportation. There are nine AHSV serotypes inducing no or low cross-neutralizing antibodies. AHSV is spread by biting Culicoides midges. AHS is endemic in sub-Saharan Africa, and a serious threat outside Africa, since Culicoides species in moderate climate conditions are spreading the closely related bluetongue virus. AHS outbreaks will be devastating for the equestrian industry in developed countries. Live-attenuated vaccines (LAVs) are licensed, marketed and in use in Africa. Their application is controversial with regard to safety issues. LAVs are not allowed in AHS-free countries. We here studied inactivated AHSV with different adjuvants in guinea pigs and horses. Subcutaneous and intramuscular vaccination were studied in horses. Local reactions were observed after prime and boost vaccination. In general, neutralizing antibodies (nAbs) titres were very low after prime vaccination, whereas boost vaccination resulted in high nAb titres for some adjuvants. Vaccinated horses were selected based on local reactions and nAb titres to study efficacy. Unfortunately, not all vaccinated horses survived virulent AHSV infection. Further, most survivors temporarily developed clinical signs and viremia. Further, the current prototype inactivated AHS vaccine is not suitable as emergency vaccine, because onset of protection is slow and requires boost vaccinations. On the other hand, inactivated AHS vaccine is completely safe with respect to virus spread, and incorporation of the DIVA principle based on NS3/NS3a serology and exploring a vaccine production platform for other serotypes is feasible. A superior adjuvant increasing the protective response without causing local reactions will be required to develop payable and acceptable inactivated AHS vaccines.

Molecular characterization of a new botybirnavirus that infects Botrytis cinerea.

Eight different double-stranded RNA (dsRNA) molecules were found in the wild-type fungal strain Botrytis cinerea CCg427. The electrophoretic profile displayed molecules with approximate sizes of 1, 1.3, 1.6, 1.8, 3.3, 4.1, 6.5, and 12 kbp. Sequences analysis of the molecules in the 6.5-kbp band revealed the presence of two different dsRNA molecules (dsRNA-1 and dsRNA-2) of 6192 and 5567 bp. Each molecule contained a unique ORF (5487 and 4836 nucleotides in dsRNA-1 and dsRNA-2, respectively). The ORF of dsRNA-1 encodes a 205-kDa polypeptide that shares 58% amino acid sequence identity with the RNA-dependent RNA polymerase (RdRp) encoded by dsRNA-1 of Alternaria sp. SCFS-3 botybirnavirus (ABRV1), whereas the ORF of dsRNA-2 encodes a 180-kDa polypeptide that shares 52% amino acid sequence identity with an unclassified protein encoded by dsRNA-2 of ABRV1. Genome organization and phylogenetic analysis based on the amino acid sequences of RdRps in members of different dsRNA virus families showed that the dsRNAs in the 6.5-kbp band correspond to the genome of a new botybirnavirus that we have named "Botrytis cinerea botybirnavirus 1".

African horse sickness virus (AHSV) with a deletion of 77 amino acids in NS3/NS3a protein is not virulent and a safe promising AHS Disabled Infectious Single Animal (DISA) vaccine platform.

African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. Currently, nine serotypes have been defined showing limited cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African Horse Sickness (AHS) in equids with a mortality up to 95% in naïve domestic horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are competent vectors of closely related bluetongue virus. AHS outbreaks cause huge economic losses in developing countries. In the developed world, outbreaks will result in losses in the equestrian industry and will have an enormous emotional impact on owners of pet horses. Live-attenuated vaccine viruses (LAVs) have been developed, however, safety of these LAVs are questionable due to residual virulence, reversion to virulence, and risk on virulent variants by reassortment between LAVs or with field AHSV. Research aims vaccines with improved profiles. Reverse genetics has recently being developed for AHSV and has opened endless possibilities including development of AHS vaccine candidates, such as Disabled Infectious Single Animal (DISA) vaccine. Here, virulent AHSV5 was recovered and its high virulence was confirmed by experimental infection of ponies. 'Synthetically derived' virulent AHSV5 with an in-frame deletion of 77 amino acids codons in genome segment 10 encoding NS3/NS3a protein resulted in similar in vitro characteristics as published NS3/NS3a knockout mutants of LAV strain AHSV4LP. In contrast to its highly virulent ancestor virus, this deletion AHSV5 mutant (DISA5) was completely safe for ponies. Two vaccinations with DISA5 as well as two vaccinations with DISA vaccine based on LAV strain AHSV4LP showed protection against lethal homologous AHSV. More research is needed to further improve efficacy, to explore the AHS DISA vaccine platform for all nine serotypes, and to study the vaccine profile in more detail.

Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro.

The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family. IMPORTANCE: Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses.

Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus.

In an effort to simplify and expand the utility of African horse sickness virus (AHSV) reverse genetics, different plasmid-based reverse genetics systems were developed. Plasmids containing cDNAs corresponding to each of the full-length double-stranded RNA genome segments of AHSV-4 under control of a T7 RNA polymerase promoter were co-transfected in cells expressing T7 RNA polymerase, and infectious AHSV-4 was recovered. This reverse genetics system was improved by reducing the required plasmids from 10 to five and resulted in enhanced virus recovery. Subsequently, a T7 RNA polymerase expression cassette was incorporated into one of the AHSV-4 rescue plasmids. This modified 5-plasmid set enabled virus recovery in BSR or L929 cells, thus offering the possibility to generate AHSV-4 in any cell line. Moreover, mutant and cross-serotype reassortant viruses were recovered. These plasmid DNA-based reverse genetics systems thus offer new possibilities for investigating AHSV biology and development of designer AHSV vaccine strains.

Complete genome analyses of the first porcine rotavirus group H identified from a South African pig does not provide evidence for recent interspecies transmission events.

Rotaviruses (RVs) are classified into eight species/groups (RVA-RVH) according to the migration patterns of their 11 genome segments, as well as by serological and molecular properties of Viral Protein 6 (VP6). In 1997 a new unclassified RV was reported infecting adults in Bangladesh and China. This virus was initially named novel adult diarrhoea rotavirus (ADRV-N), but later renamed as RVH. Since then, RVH has been detected in humans only very sporadically. However, RVH is increasingly being detected in pig populations in the USA, Brazil and Japan, but not yet in Africa. Unfortunately, whole genome sequence data of porcine RVH strains in GenBank is currently restricted to a single strain (SKA-1) from Japan. Porcine diarrhoeic samples were collected in South Africa and analysed for rotavirus using an RVA ELISA and electropherotyping by PAGE. One sample displayed a 4:2:1:1:1:1:1 migration pattern, typical for RVH. In order to further investigate this strain, sequence-independent amplification followed by random sequencing using the 454/Roche GS FLX Sequencer was performed, resulting in the second complete porcine RVH strain (MRC-DPRU1575) available in databases. Phylogenetically, all segments of MRC-DPRU1575 clustered closely with the SKA-1 strain and in some segments with known porcine RVH strains from Brazil and the USA. In contrast, the porcine RVH strains were only distantly related to human RVH strains from Asia and a partial RVH-like strain recently detected in bats from Cameroon. Overall, strain MRC-DPRU1575 is the first complete genome of a porcine RVH from Africa and allows for the development of improved RVH screening methods. Our analyses indicate that RVH strains cluster according to their host species, not suggesting any evidence of recent interspecies transmission events. However, more RVH genomes from a wider host range are needed to better understand their evolutionary pathways and zoonotic potential.

VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV.

UNLABELLED: African horse sickness virus (AHSV) is a virus species in the genus Orbivirus of the family Reoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species of Culicoides biting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate "synthetic" reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replication in vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes. IMPORTANCE: African horse sickness virus is transmitted by species of Culicoides biting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemic Culicoides species in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replication in vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since the in vitro release of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

Characterising Non-Structural Protein NS4 of African Horse Sickness Virus.

African horse sickness is a serious equid disease caused by the orbivirus African horse sickness virus (AHSV). The virus has ten double-stranded RNA genome segments encoding seven structural and three non-structural proteins. Recently, an additional protein was predicted to be encoded by genome segment 9 (Seg-9), which also encodes VP6, of most orbiviruses. This has since been confirmed in bluetongue virus and Great Island virus, and the non-structural protein was named NS4. In this study, in silico analysis of AHSV Seg-9 sequences revealed the existence of two main types of AHSV NS4, designated NS4-I and NS4-II, with different lengths and amino acid sequences. The AHSV NS4 coding sequences were in the +1 reading frame relative to that of VP6. Both types of AHSV NS4 were expressed in cultured mammalian cells, with sizes close to the predicted 17-20 kDa. Fluorescence microscopy of these cells revealed a dual cytoplasmic and nuclear, but not nucleolar, distribution that was very similar for NS4-I and NS4-II. Immunohistochemistry on heart, spleen, and lung tissues from AHSV-infected horses showed that NS4 occurs in microvascular endothelial cells and mononuclear phagocytes in all of these tissues, localising to the both the cytoplasm and the nucleus. Interestingly, NS4 was also detected in stellate-shaped dendritic macrophage-like cells with long cytoplasmic processes in the red pulp of the spleen. Finally, nucleic acid protection assays using bacterially expressed recombinant AHSV NS4 showed that both types of AHSV NS4 bind dsDNA, but not dsRNA. Further studies will be required to determine the exact function of AHSV NS4 during viral replication.

A wild-type Botrytis cinerea strain co-infected by double-stranded RNA mycoviruses presents hypovirulence-associated traits.

BACKGROUND: Botrytis cinerea CCg378 is a wild-type strain infected with two types of double-stranded RNA (dsRNA) mycoviruses and which presents hypovirulence-associated traits. The objectives of the present study were to characterize the mycoviruses and investigate their relationship with the low virulence degree of the fungal host. RESULTS: B. cinerea CCg378 contains five dsRNA molecules that are associated with two different types of isometric viral particles of 32 and 23 nm in diameter, formed by structural polypeptides of 70-kDa and 48-kDa, respectively. The transfection of spheroplasts of a virus-free strain, B. cinerea CKg54, with viral particles purified from the CCg378 strain revealed that the 2.2-kbp dsRNAs have no dependency on the smaller molecules for its stable maintenance in the fungal cytoplasm, because a fungal clone that only contains the 2.2-kbp dsRNAs associated with the 32-nm particles was obtained, which we named B. cinerea CKg54vi378. One of the 2.2 kbpdsRNA segments (2219 bp) was sequenced and corresponds to the gene encoding the capsid protein of B. cinerea CCg378 virus 1 (Bc378V1), a putative new member of the Partitiviridae family. Furthermore, physiological parameters related to the degree of virulence of the fungus, such as the sporulation rate and laccase activity, were lower in B. cinerea CCg378 and B. cinerea CKg54vi378 than in B. cinerea CKg54. Additionally, bioassays performed on grapevine leaves showed that the CCg378 and CKg54vi378 strains presented a lower degree of invasiveness on the plant tissue than the CKg54 strain. CONCLUSIONS: The results show that B. cinerea CCg378 is coinfected by two mycoviruses and that the 2.2-kbp dsRNAs correspond to the 32-nm mycovirus genome, which would be a new member of the Partitiviridae family as it has the typical pattern of partitiviruses. On the other hand, the results suggest that the hypovirulence of B. cinerea CCg378 could be conferred by both mycoviruses, since the fungal clone B. cinerea CKg54vi378 presents an intermediate virulence between the CKg54 and CCg378 strains. Therefore, the putative partitivirus would be partially contributing to the hypovirulence phenotype of the CCg378 strain.

Structural insight into African horsesickness virus infection.

African horsesickness (AHS) is a devastating disease of horses. The disease is caused by the double-stranded RNA-containing African horsesickness virus (AHSV). Using electron cryomicroscopy and three-dimensional image reconstruction, we determined the architecture of an AHSV serotype 4 (AHSV-4) reference strain. The structure revealed triple-layered AHS virions enclosing the segmented genome and transcriptase complex. The innermost protein layer contains 120 copies of VP3, with the viral polymerase, capping enzyme, and helicase attached to the inner surface of the VP3 layer on the 5-fold axis, surrounded by double-stranded RNA. VP7 trimers form a second, T=13 layer on top of VP3. Comparative analyses of the structures of bluetongue virus and AHSV-4 confirmed that VP5 trimers form globular domains and VP2 trimers form triskelions, on the virion surface. We also identified an AHSV-7 strain with a truncated VP2 protein (AHSV-7 tVP2) which outgrows AHSV-4 in culture. Comparison of AHSV-7 tVP2 to bluetongue virus and AHSV-4 allowed mapping of two domains in AHSV-4 VP2, and one in bluetongue virus VP2, that are important in infection. We also revealed a protein plugging the 5-fold vertices in AHSV-4. These results shed light on virus-host interactions in an economically important orbivirus to help the informed design of new vaccines.

Rescue of recent virulent and avirulent field strains of bluetongue virus by reverse genetics.

Since 1998, Bluetongue virus (BTV)-serotypes 1, 2, 4, 9, and 16 have invaded European countries around the Mediterranean Basin. In 2006, a huge BT-outbreak started after incursion of BTV-serotype 8 (BTV8) in North-Western Europe. More recently, BTV6 and BTV11 were reported in North-Western Europe in 2008. These latter strains are closely related to live-attenuated vaccine, whereas BTV8 is virulent and can induce severe disease in ruminants, including cattle. In addition, Toggenburg orbivirus (TOV) was detected in 2008 in Swiss goats, which was recognized as a new serotype of BTV (BTV25). The (re-)emergency of known and unknown BTV-serotypes needs a rapid response to supply effective vaccines, and research to study this phenomenon. Recently, orbivirus research achieved an important breakthrough by the establishment of reverse genetics for BTV1. Here, reverse genetics for two recent BTV strains representing virulent BTV8 and avirulent BTV6 was developed. For this purpose, extensive sequencing of full-genomes was performed, resulting in the consensus sequences of BTV8/net07 and BTV6/net08. The recovery of 'synthetic BTV', respectively rgBTV8 and rgBTV6, completely from T7-derived RNA transcripts was confirmed by silent mutations by which these 'synthetic BTVs' could be genetically distinguished from wild type BTV, respectively wtBTV6 and wtBTV8. The in vitro and in vivo properties of rgBTV6 or rgBTV8 were comparable to the properties of their parent strains. The asymptomatic or avirulent properties of rgBTV6 and the virulence of rgBTV8 were confirmed by experimental infection of sheep. Reverse genetics of the vaccine-related BTV6 provides a perfect start to develop new generations of BT-vaccines. Reverse genetics of the virulent BTV8 will accelerate research on the special features of BTV8, like transmission by species of Culicoides in a moderate climate, transplacental transmission, and pathogenesis in cattle.

Creation of a nonspreading Rift Valley fever virus.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic bunyavirus of the genus Phlebovirus and a serious human and veterinary pathogen. RVFV contains a three-segmented RNA genome, which is comprised of the large (L), medium (M), and small (S) segments. The proteins that are essential for genome replication are encoded by the L and S segments, whereas the structural glycoproteins are encoded by the M segment. We have produced BHK replicon cell lines (BHK-Rep) that maintain replicating L and S genome segments. Transfection of BHK-Rep cells with a plasmid encoding the structural glycoproteins results in the efficient production of RVFV replicon particles (RRPs). To facilitate monitoring of infection, the NSs gene was replaced with an enhanced green fluorescent protein gene. RRPs are infectious for both mammalian and insect cells but are incapable of autonomous spreading, rendering their application outside biosafety containment completely safe. We demonstrate that a single intramuscular vaccination with RRPs protects mice from a lethal dose of RVFV and show that RRPs can be used for rapid virus neutralization tests that do not require biocontainment facilities. The methods reported here will greatly facilitate vaccine and drug development as well as fundamental studies on RVFV biology. Moreover, it may be possible to develop similar systems for other members of the bunyavirus family as well.

Unusual assortment of segments in 2 rare human rotavirus genomes.

Using full-length genome sequence analysis, we investigated 2 rare G3P[9] human rotavirus strains isolated from children with diarrhea. The genomes were recognized as assortments of genes closely related to rotaviruses originating from cats, ruminants, and humans. Results suggest multiple transmissions of genes from animal to human strains of rotaviruses.

Are human P[14] rotavirus strains the result of interspecies transmissions from sheep or other ungulates that belong to the mammalian order Artiodactyla?

A limited number of human G6P[14] rotavirus strains that cause gastroenteritis in humans have been isolated in Europe and Australia. The complete genome sequences were determined for five of these human strains--B10925-97 (isolated in Belgium in 1997), 111/05-27 (Italy, 2005), PA169 (Italy, 1987), MG6 (Australia, 1993), and Hun5 (Hungary, 1997)--and their genetic relatedness to animal rotavirus strains was evaluated by sequencing the complete genome of the sheep rotavirus OVR762 (G8P[14]; Spain, 2002), the guanaco (Lama guanicoe) rotavirus strains Arg/Chubut/99 and Arg/Río Negro/98 (G8P[14] and G8P[1], respectively; Argentina, 1999 and 1998), the sable antelope strain RC-18/08 (G6P[14]; South Africa, 2008), and the bovine rotavirus strain Arg/B383/98 (G15P[11]; Argentina, 1998). These analyses revealed an overall consensus genomic constellation (G6/G8)-P[14]-I2-(R2/R5)-C2-M2-(A3/A11)-N2-T6-(E2/E12)-H3, together with a few gene reassortments, and the phylogenetic analyses confirmed that the P[14] human strains evaluated in this study were closely related to rotavirus strains isolated from sheep, cattle, goats, guanacos, and antelopes and to rabbits (albeit to a lesser extent), suggesting that one (or more) of these animal species might be the source of the human G6P[14] strains. The main feature of the genotype and phylogenetic analyses was the close overall genomic relatedness between the five human G6P[14] rotavirus strains and the ovine and antelope rotavirus strains. Taken together, these data strongly suggest a common origin for the human P[14] strains and those of the even-toed ungulates belonging to the mammalian order Artiodactyla, with sheep probably playing a key role in the interspecies transmission responsible for the introduction of P[14] rotavirus strains into the human population.