Revisiting the clinal concept of evolution and dispersal for the tick-borne flaviviruses by using phylogenetic and biogeographic analyses.

Tick-borne flaviviruses (TBF) are widely dispersed across Africa, Europe, Asia, Oceania, and North America, and some present a significant threat to human health. Seminal studies on tick-borne encephalitis viruses (TBEV), based on partial envelope gene sequences, predicted a westward clinal pattern of evolution and dispersal across northern Eurasia, terminating in the British Isles. We tested this hypothesis using all available full-length open reading frame (ORF) TBF sequences. Phylogenetic analysis was consistent with current reports. However, linear and nonlinear regression analysis of genetic versus geographic distance combined with BEAST analysis identified two separate clines, suggesting that TBEV spread both east and west from a central point. In addition, BEAST analysis suggested that TBF emerged and dispersed more than 16,000 years ago, significantly earlier than previously predicted. Thus, climatic and ecological changes may have played a greater role in TBF dispersal than humans.

Genome-scale phylogeny of the alphavirus genus suggests a marine origin.

The genus Alphavirus comprises a diverse group of viruses, including some that cause severe disease. Using full-length sequences of all known alphaviruses, we produced a robust and comprehensive phylogeny of the Alphavirus genus, presenting a more complete evolutionary history of these viruses compared to previous studies based on partial sequences. Our phylogeny suggests the origin of the alphaviruses occurred in the southern oceans and spread equally through the Old and New World. Since lice appear to be involved in aquatic alphavirus transmission, it is possible that we are missing a louse-borne branch of the alphaviruses. Complete genome sequencing of all members of the genus also revealed conserved residues forming the structural basis of the E1 and E2 protein dimers.

Analysis of intrahost variation in Venezuelan equine encephalitis virus reveals repeated deletions in the 6-kilodalton protein gene.

RNA viruses exist as a spectrum of mutants that is generated and maintained during replication within the host. Consensus sequencing overlooks minority genotypes present in the viral sample that may impact the population's phenotype. In-depth sequencing of an original field isolate of subtype IE Venezuelan equine encephalitis virus (VEEV) demonstrated the presence of multiple deletions within the 6,000-molecular-weight (6K) protein gene. Using in vitro and in vivo experiments, similar deletions were generated in an additional VEEV strain originating from an infectious cDNA clone. Time course experiments demonstrated that the deletions are produced during acute infection although not until 24 h postinfection. Molecular clones containing some of these deletions were generated, and although the larger deletions appear to be noninfectious, viruses with the smaller deletions were viable and formed small plaques. Serial passages provided no evidence that these deletion mutants function as defective interfering particles. Furthermore, since wild-type infections generally occur at a low multiplicity of infection, it is unlikely that these deletions are propagated in natural transmission cycles. However, they could affect pathogenesis at later stages of infection. Because they are ubiquitously generated both in vivo and in vitro, further investigation is warranted to understand the generation of these deletions and their significance for disease.

Co-circulation of widely disparate strains of rabbit haemorrhagic disease virus could explain localised epidemicity in the United Kingdom.

Serum and liver samples collected monthly, during 2005, from healthy wild rabbits at a site in Pitroddie, Scotland, were analysed by ELISA and RT-PCR sequencing. Sera collected in January and February had high antibody titres against RHDV. However, during the rabbit breeding season average antibody titres were lower but increased again as the year progressed. Between March and August, RHDV-specific RNA was detected in healthy rabbits spanning a wide range of age and antibody titres. Importantly, two virus lineages were identified; a novel widely divergent strain, recovered between March and August, and a strain related to UK epidemic strains, was recovered between May and July from juvenile rabbits. We propose that a non-virulent widely divergent strain of RHDV circulated asymptomatically amongst the wild rabbits potentially inducing immunity against the introduced epidemic strain that predominantly causes high fatality rates in young immunologically naïve rabbits.

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.

Pseudocowpox virus: the etiological agent of contagious ecthyma (Auzdyk) in camels (Camelus dromedarius) in the Arabian peninsula.

We have determined the nucleotide sequence of DNA extracted from pustules, saliva, and blood of camels presenting with contagious ecthyma, in Bahrain and also from a sample (SACamel) of infected tissue from a camel that had presented with contagious ecthyma in 1998 in Saudi Arabia (1). Sequence homologies and phylogenetic analysis showed that this extracted DNA was more closely related to Pseudocowpox virus (PCPV) than Orf virus (ORFV), which infects sheep, goats, and other animal species. The phylogeny also demonstrated that PCPV in Arabian camels was phylogenetically distinct from, and circulates independently of, ruminant-associated PCPV from Europe.

Benign circulation of rabbit haemorrhagic disease virus on Lambay Island, Eire.

With the exception of virus strains Ashington and RCV, other recognised strains of Rabbit haemorrhagic disease virus (RHDV) share relatively close genetic homology. Using serology and phylogenetic analysis, we have identified a third disparate virus lineage in healthy rabbits on Lambay Island off the east coast of Eire, where disease due to RHDV has never been observed. ELISA tests revealed high titre RHDV-specific antibodies in 81% of the sera from 11 healthy rabbits captured on this island, indicating that the virus is actively circulating amongst these rabbits. Nevertheless, infectious virus has not been isolated from rabbits living on this island. The detection of antibodies and the disparate Lambay virus lineage in an apparently healthy and isolated wild rabbit population provides the most convincing evidence yet that at least some strains of RHDV can circulate harmlessly for long periods of time in wild rabbits possibly by producing persistent or latent infections.

Phylogenetic analysis of rabbit haemorrhagic disease virus strains from the Arabian Peninsula: did RHDV emerge simultaneously in Europe and Asia?

Rabbit haemorrhagic disease virus (RHDV) emerged in 1984 in China and subsequently a single strain apparently dispersed worldwide killing millions of rabbits. Two isolates that caused outbreaks in Saudi Arabia and Bahrain have been sequenced and analysed phylogenetically. The Saudi Arabian lineage is directly descended from the Chinese strain, but the Bahrain isolate occupies a distinct and more divergent lineage than the Chinese virus implying that epidemic RHDV strains have emerged at least twice during the past 20 years and are co-circulating in both domestic and wild rabbits.

Epidemiology of rabbit haemorrhagic disease virus in the United Kingdom: evidence for seasonal transmission by both virulent and avirulent modes of infection.

Rabbit haemorrhagic disease virus (RHDV) has killed many millions of wild rabbits in Europe and Australia, but has had little impact in the United Kingdom, despite outbreaks having occurred since 1994. High seroprevalence detected in the absence of associated mortality had suggested the presence of an endemic non-pathogenic strain which may be 'protecting' UK populations. Following the first detailed field study of RHDV epidemiology in the United Kingdom, using mark-recapture with serum sampling, we report that RHDV caused highly prevalent persistent infection in seropositive rabbits in the absence of associated mortality. Furthermore the virus strains responsible could not be distinguished phylogenetically from known pathogenic isolates, and were clearly very different from the only previously identified non-pathogenic strain of RHDV. These findings suggest that many--perhaps most--strains of RHDV may be propagated through both 'pathogenic' and 'non-pathogenic' modes of behaviour. Transmission occurred predominantly during and just after the breeding season.

Long-term survival of New Zealand rabbit haemorrhagic disease virus RNA in wild rabbits, revealed by RT-PCR and phylogenetic analysis.

Because Rabbit haemorrhagic disease virus (RHDV) is highly pathogenic for rabbits, farmers illegally introduced it as a bio-control agent onto New Zealand farms in 1997. The virus was dispersed rapidly, initially causing high fatality rates in rabbits. Nevertheless, many survived and these surviving rabbits have been investigated for evidence of infection by RHDV. Livers from healthy rabbits contained RHDV-specific RNA, as shown by nested RT-PCR sequencing. The sequences of the viral capsids were related closely to the released Czech strain of RHDV, although the sequence from one rabbit was related most closely to a Spanish strain of RHDV. Phylogenetic analysis of the capsid sequences of 38 samples implied that there have been at least two introductions of the Czech virus into New Zealand, probably corresponding firstly to the original illegal introduction by farmers and secondly to the introduction of the same virus under governmental control. Genomic length sequence of two samples was obtained, suggesting that they may have retained the potential to be infectious, although this has not yet been demonstrated. The detection of genomic-length RNA in the liver of healthy rabbits suggests that even though a highly virulent virus was introduced into New Zealand, it rapidly established persistent or latent infections in a proportion of rabbits. This might account for their ability to survive in the face of virulent released virus. Moreover, the co-circulation of other strains of RHDV in the same rabbit population, such as the Spanish strain, might also impact on their susceptibility to the bio-control agent.

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.