Stephen R. Moss

Molecular epidemiology of Rabbit haemorrhagic disease virus

Millions of domestic and wild European rabbits (Oryctolagus cuniculus) have died in Europe, Asia, Australia and New Zealand during the past 17 years following infection by Rabbit haemorrhagic disease virus (RHDV). This highly contagious and deadly disease was first identified in China in 1984. Epidemics of RHDV then radiated across Europe until the virus apparently appeared in Britain in 1992. However, this concept of radiation of a new and virulent virus from China is not entirely consistent with serological and molecular evidence. This study shows, using RT-PCR and nucleotide sequencing of RNA obtained from the serum of healthy rabbits stored at 4 degrees C for nearly 50 years, that, contrary to previous opinions, RHDV circulated as an apparently avirulent virus throughout Britain more than 50 years ago and more than 30 years before the disease itself was identified. Based on molecular phylogenetic analysis of British and European RHDV sequences, it is concluded that RHDV has almost certainly circulated harmlessly in Britain and Europe for centuries rather than decades. Moreover, analysis of partial capsid sequences did not reveal significant differences between RHDV isolates that came from either healthy rabbits or animals that had died with typical haemorrhagic disease. The high stability of RHDV RNA is also demonstrated by showing that it can be amplified and sequenced from rabbit bone marrow samples collected at least 7 weeks after the animal has died.

Recombination in rabbit haemorrhagic disease virus: Possible impact on evolution and epidemiology ☆

Rabbit haemorrhagic disease (RHD) was first recognised in 1984 following the introduction of apparently healthy rabbits into China from Germany. The aetiological agent Rabbit haemorrhagic disease virus (RHDV) has subsequently killed hundreds of millions of domestic and wild rabbits particularly in Europe, China and Australia. Previously, using phylogenetic analysis we have attempted to understand the underlying factors that determine why this virus emerged, and why it has such an unpredictable epidemiology. Here we report the use of tree congruency supported by bootscanning analysis to detect recombination amongst both closely related, and widely divergent strains of RHDV. We show that recombination occurs commonly and in several different regions of the RHDV genome. Moreover, the first identified strain of RHDV, i.e. from China in 1984, showed evidence of recombination in the capsid gene, with a virus or viruses containing lineages in German strains. These observations imply that recombination may play a significant role in the evolution, epidemiology and diversity of RHDV.

Evolution and dispersal of encephalitic flaviviruses.

There are two major groups of encephalitic flaviviruses, those that infect and are transmitted by ticks, particularly Ixodes spp. and those that infect and are transmitted by mosquitoes, particularly Culex spp. The tick-borne encephalitic flaviviruses exhibit evolutionary characteristics that are largely determined by the protracted life cycle of the tick, its habitat and the prevailing climatic conditions. These viruses appear to have evolved gradually from non-encephalitic viruses that radiated eastwards and north eastwards out of Africa into Asia and the southern islands, then northwards to far east Asia and finally westwards across Eurasia to western Europe, during the past two to four thousand years. Only one of these recognized species has found its way to North America viz. Powassan virus. In contrast, the evolution of the recognized mosquito-borne encephalitic flaviviruses reflects the wide range of mosquito species that they infect. They emerged out of Africa relatively recently and at roughly the same time, i.e., probably during the past few centuries. Although many of these mosquito-borne viruses are geographically widely dispersed, with the exception of West Nile virus, they are found either in the Old World or the New World, never in both, and we are now beginning to understand the reasons. Phylogenetic trees will be used here to describe the evolution, epidemiology and dispersal characteristics of these viruses, taking into account the importance of virus persistence and recombination.

The Great Island Subgroup of Tick-borne Orbiviruses Represents a Single Gene Pool

The geographical distribution of members of the Great Island (GI) subgroup in the Kemerovo serogroup of orbiviruses extends from the Arctic to the Sub-antarctic. To examine the gene pool size of this group, five topotypes whose origins ranged from Iceland in the northern hemisphere to Macquarie Island in the Southern Ocean were tested for their ability to reassort in vitro. All the isolates were distinguishable by plaque reduction neutralization tests, and their genome profile in polyacrylamide gels. They showed high frequency reassortment following dual infection of cell cultures with temperature-sensitive (ts) mutants and/or wild-type virus. Analysis of the dsRNA profile of the reassortants by PAGE confirmed the observation from reassortment assays that the Great Island subgroup constitutes a single gene pool. A seventh reassortment group was identified, distinct from the six groups previously described. The ts lesions for reassortment groups I, V and VII were considered to be in genome segments 9, 3 and 2, respectively. Segment 6 of GI virus (in contrast to segment 5 of Broadhaven and Wexford viruses) was shown to be the major genetic determinant of serotype specificity.

Comparison of the major structural core proteins of tick-borne and Culicoides-borne orbiviruses

Comparison of sequence data for Broadhaven (BRD) virus, a tick-borne orbivirus, and bluetongue virus (BTV), the type species of the genus, indicated that RNA segments 2 and 7 of BRD virus encode the two structural core proteins, VP2 and VP7, respectively. Segment 2 is 2792 nucleotides in length with a coding capacity for a protein (VP2) of 908 amino acids and a net charge of +8.5 at neutral pH. Segment 7 is 1174 nucleotides in length with a coding capacity for a protein (VP7) of 356 amino acids and a net charge of +11.5 at neutral pH. Comparison of the two sequences with BTV serotype 10 revealed amino acid identity of 35% between the product of segment 2 and BTV VP3, and 21% between the product of segment 7 and BTV VP7. The core proteins therefore show evidence of significant evolutionary divergence compared with that shown between different insect-borne orbiviruses. In particular, the amino terminus of BRD virus VP7 differed markedly from the equivalent region in VP7 of BTV and African horse sickness virus. This region is thought to interact with the outer capsid layer of insect-borne orbiviruses.

Subcore- and core-like particles of Broadhaven virus (BRDV), a tick-borne orbivirus, synthesized from baculovirus expressed VP2 and VP7, the major core proteins of BRDV

The genes encoding the two major core proteins (VP2 and VP7) of Broadhaven (BRD) virus, a tick-borne orbivirus, were inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) under the control of copies of the AcNPV polyhedrin promoter to produce two recombinant baculoviruses. Infection of Spodoptera frugiperda (Sf) cells with a recombinant AcNPV that synthesized BRDV VP2 produced large numbers of BRDV subcore-like particles. Co-infection of cells with the two recombinants that made either BRDV VP2 or VP7 produced core-like particles similar in appearance to authentic BRDV cores. No evidence was obtained for the formation of core-like particles between the major core proteins of BRDV and those of bluetongue virus (BTV) following the co-expression of BRDV VP2 and BTV VP7, or BRDV VP7 and BTV VP3, indicating that in this respect the proteins of these two orbiviruses are incompatible, unlike the situation previously described for epizootic haemorrhagic disease virus and BTV core proteins.

Cytotoxic T-cell activity is not detectable in Venezuelan equine encephalitis virus-infected mice

Previously published research has established that the immune response to the Venezuelan equine encephalitis virus (VEEV) vaccine strain TC-83 is Th 1-mediated, with local activation of both CD4+ and CD8+ T cells. This suggests that cytotoxic lymphocytes CTL may play a role in protection against virulent VEEV. Studies involving a variety of immunisation schedules with either TC-83 or strain CAAR 508 (serogroup 5) of VEEV, and six different haplotypes of mice, failed to reveal functional CTL activity against VEEV-infected targets in secondary antigen-stimulated lymphocyte cultures from either the draining lymph nodes (LN) or spleen. Nor were VEEV-specific CTL detected after immunisation of mice (three haplotypes) with recombinant vaccinia viruses (VV) expressing either the non-structural (nsP1-4) or the structural (C-E3-E2-6K-E1) genes of TC-83. Reciprocal experiments in which mice were immunised with TC-83, and their lymphocytes tested against VV recombinant-infected targets also failed to detect CTL activity. These data suggest that VEEV infection of mice does not elicit detectable CTL activity, and that CTL are unlikely to play a role in protection against virulent VEEV.

Comparison of the nonstructural protein, NS3, of tick-borne and insect-borne orbiviruses.

The complete nucleotide sequence of the smallest RNA segment (segment 10) of Broadhaven (BRD) virus, a tick-borne orbivirus, was determined from a full-length cDNA clone. The genome segment is 702 nucleotides in length and has a coding capacity for two proteins of either 205 or 199 amino acids, having net charges of +16.5 and +17.5, respectively, at neutral pH. Comparison of the sequence of RNA segment 10 of BRD, bluetongue, African horse sickness, and Palyam viruses revealed amino acid homology of 20 to 30% between the four orbiviruses, with one conserved region of 40 to 50% homology which, in segment 10 of BRD virus, is found between residues 26 and 71.

Enhanced neurovirulence of tick-borne orbiviruses resulting from genetic modulation.

The genome of orbiviruses (Reoviridae family) comprises 10 segments of double-stranded RNA. The fourth largest segment of the tick-borne Kemerovo (KEM) group orbiviruses is the genetic determinant of neurovirulence in experimentally infected mice, and segment 6 determines serotype. Reassortant viruses derived from a cross between two KEM-related viruses, Great Island (GI) and Wexford (WEX), that had the heterotypic gene combination W4G6 (segment 4 of WEX virus and segment 6 from GI virus) were nonpathogenic in mice. This apparent genetic modulation of neurovirulence may have resulted from steric interaction between the two outer capsid proteins of nonpathogenic reassortants. Further data are consistent with this hypothesis. Reassortants generated from additional KEM group viruses showed various degrees of enhanced neurovirulence in terms of their PFU/LD50 (ratio of infectivity in cell culture and in mice) and ASTmax (the average survival time at the highest virus dilution resulting in 100% mortality). Some reassortants were more pathogenic than either of their parental viruses. The results indicate that the gene determining neurovirulence dictates ASTmax, and the PFU/LD50 is a measure of the interaction between the products of the gene determining neurovirulence and that determining serotype. The nonpathogenic phenotype of a low passage isolate (St. Abbs 84-34 virus), derived from a single tick, generated neurovirulent reassortants. This result indicates that genetic modulation of KEM group viruses may occur in nature.

Genetic determinants modulating the pathogenic phenotype of tick-borne orbiviruses.

Genetic studies have been carried out on orbiviruses in the Great Island (GI) antigenic subgroup of the Kemerovo serogroup (Orbivrus, Reoviridae) to elucidate the functions of the 10 genomic double-stranded RNA segments. Such studies have shown that segment 4 is the major genetic determinant of neurovirulence (P.A. Nuttall, S.R. Moss, L.D. Jones, and D. Carey, 1989, Virology 172, 428-434), whereas segment 5 of Wexford (WEX) virus and segment 6 of GI virus are the major determinants of serotype specificity (S.R. Moss, C.M. Ayres, and P.A. Nuttall, 1987, Virology 157, 137-144; S.R. Moss, C.M. Ayres, and P.A. Nuttall, 1988, J. Gen. Virol. 69, 2721-2727). In studies with reassortants isolated following dual infection of cell cultures with WEX and GI viruses, the gene combination W4G6 (i.e., viruses deriving segment 4 from WEX virus and segment 6 from GI virus) resulted in nonpathogenic reassortants. Unlike the parental viruses, the avirulent reassortants did not produce clinical evidence of infection in inoculated 2-day-old mice although, suprisingly, they replicated in the brains of the mice. The alternate heterotypic gene combination, G4W5, resulted in typical neurovirulent reassortants. The results indicate that segment 6 of GI virus is able to modulate the phenotypic expression of segment 4 of WEX virus, but not vice versa. Modulation probably results from interactions between the products of these two genomic segments, possibly at the level of virion structure.