Characterisation of an Australian fowlpox virus carrying a near-full-length provirus of reticuloendotheliosis virus.

Fowlpox virus (FWPV), which is the type member of the genus Avipoxvirus, subfamily Chordopoxvirinae, family Poxviridae, can lead to significant losses to the poultry industry. Although a large number of fowlpox virus genomes have been sequenced and characterised globally, there are no sequences available at the genomic level from Australian isolates. Here, we present the first complete genome sequence of a fowlpox virus vaccine strain (FWPV-S) containing an integrated near-full-length reticuloendotheliosis virus (REV) provirus. The genome of FWPV-S showed the highest sequence similarity to a fowlpox virus from the USA (97.74% identity). The FWPV-S genome contained 16 predicted unique genes, while a further two genes were fragmented compared to previously reported FWPV genome sequences. Subsequent phylogenetic analysis showed that FWPV-S was most closely related to other fowlpox viruses. This is the first reported genome sequence of FWPV from Australia.

Hayes Yard virus: a novel ephemerovirus isolated from a bull with severe clinical signs of bovine ephemeral fever is most closely related to Puchong virus.

Bovine ephemeral fever is a vector-borne disease of ruminants that occurs in tropical and sub-tropical regions of Africa, Asia and Australia. The disease is caused by a rhabdovirus, bovine ephemeral fever virus (BEFV), which occurs as a single serotype globally. Although several other closely related ephemeroviruses have been isolated from cattle and/or arthropods, only kotonkan virus from Nigeria and (tentatively) Mavingoni virus from Mayotte Island in the Indian Ocean have been previously associated with febrile disease. Here, we report the isolation of a novel virus (Hayes Yard virus; HYV) from blood collected in February 2000 from a bull (Bos indicus) in the Northern Territory of Australia. The animal was suffering from a severe ephemeral fever-like illness with neurological involvement, including recumbency and paralysis, and was euthanised. Histological examination of spinal cord and lung tissue identified extensive haemorrhage in the dura mata with moderate perineuronal oedema and extensive emphysema. HYV displayed cone-shaped morphology, typical of rhabdoviruses, and was found to be most closely related antigenically to Puchong virus (PUCV), isolated in 1965 from mosquitoes in Malaysia. Analysis of complete genome sequences of HYV (15 025 nt) and PUCV (14 932 nt) indicated that each has a complex organisation (3' N-P-M-G-GNS-α1-α2-ß-γ-L 5') and expression strategy, similar to that of BEFV. Based on an alignment of complete L protein sequences, HYV and PUCV cluster with other rhabdoviruses in the genus Ephemerovirus and appear to represent two new species. Neutralising antibody to HYV was also detected in a retrospective survey of cattle sera collected in the Northern Territory.

Genomic analysis of bluetongue virus episystems in Australia and Indonesia.

The distribution of bluetongue viruses (BTV) in Australia is represented by two distinct and interconnected epidemiological systems (episystems)-one distributed primarily in the north and one in the east. The northern episystem is characterised by substantially greater antigenic diversity than the eastern episystem; yet the forces that act to limit the diversity present in the east remain unclear. Previous work has indicated that the northern episystem is linked to that of island South East Asia and Melanesia, and that BTV present in Indonesia, Papua New Guinea and East Timor, may act as source populations for new serotypes and genotypes of BTV to enter Australia's north. In this study, the genomes of 49 bluetongue viruses from the eastern episystem and 13 from Indonesia were sequenced and analysed along with 27 previously published genome sequences from the northern Australian episystem. The results of this analysis confirm that the Australian BTV population has its origins in the South East Asian/Melanesian episystem, and that incursions into northern Australia occur with some regularity. In addition, the presence of limited genetic diversity in the eastern episystem relative to that found in the north supports the presence of substantial, but not complete, barriers to gene flow between the northern and eastern Australian episystems. Genetic bottlenecks between each successive episystem are evident, and appear to be responsible for the reduction in BTV genetic diversity observed in the north to south-east direction.

Evolution of bluetongue virus serotype 1 in northern Australia over 30 years.

UNLABELLED: Bluetongue virus serotype 1 (BTV 1) was first isolated in Australia from cattle blood collected in 1979 at Beatrice Hill Farm (BHF), Northern Territory (NT). From long-term surveillance programs (1977 to 2011), 2,487 isolations of 10 BTV serotypes were made. The most frequently isolated serotype was BTV 1 (41%, 1,019) followed by BTV 16 (17.5%, 436) and BTV 20 (14%, 348). In 3 years, no BTVs were isolated, and in 12 years, no BTV 1 was isolated. Seventeen BTV 1 isolates were sequenced and analyzed in comparison with 10 Australian prototype serotypes. BTV 1 showed an episodic pattern of evolutionary change characterized by four distinct periods. Each period consisted primarily of slow genetic drift which was punctuated from time to time by genetic shifts generated by segment reassortment and the introduction of new genome segments. Evidence was found for coevolution of BTV genome segments. Evolutionary dynamics and selection pressure estimates showed strong temporal and clock-like molecular evolutionary dynamics of six Australian BTV genome segments. Bayesian coalescent estimates of mean substitution rates clustered in the range of 3.5 × 10(-4) to 5.3 × 10(-4) substitutions per site per year. All BTV genome segments evolved under strong purifying (negative) selection, with only three sites identified as under pervasive diversifying (positive) selection. The obligate replication in alternate hosts (insect vector and vertebrate hosts) imposed strong evolutionary constraints. The dominant mechanism generating genetic diversity of BTV 1 at BHF was through the introduction of new viruses and reassortment of genome segments with existing viruses. IMPORTANCE: Bluetongue virus (BTV) is the causative agent of bluetongue disease in ruminants. It is a disease of concern globally and is transmitted by biting midges (Culicoides species). Analysis of the evolutionary and selection pressures on BTV 1 at a single surveillance site in northern Australia showed strong temporal and clock-like dynamics. Obligate replication in alternate hosts of insect and vertebrate imposed strong evolutionary constraints, with all BTV genome segments evolving under strong purifying (negative) selection. Generation of genetic diversity of BTV 1 in northern Australia is through genome segment reassortment and the introduction of new serotypes.

Genomic sequences of Australian bluetongue virus prototype serotypes reveal global relationships and possible routes of entry into Australia.

Bluetongue virus (BTV) is transmitted by biting midges (Culicoides spp.). It causes disease mainly in sheep and occasionally in cattle and other species. BTV has spread into northern Europe, causing disease in sheep and cattle. The introduction of new serotypes, changes in vector species, and climate change have contributed to these changes. Ten BTV serotypes have been isolated in Australia without apparent associated disease. Simplified methods for preferential isolation of double-stranded RNA (dsRNA) and template preparation enabled high-throughput sequencing of the 10 genome segments of all Australian BTV prototype serotypes. Phylogenetic analysis reinforced the Western and Eastern topotypes previously characterized but revealed unique features of several Australian BTVs. Many of the Australian BTV genome segments (Seg-) were closely related, clustering together within the Eastern topotypes. A novel Australian topotype for Seg-5 (NS1) was identified, with taxa spread across several serotypes and over time. Seg-1, -2, -3, -4, -6, -7, -9, and -10 of BTV_2_AUS_2008 were most closely related to the cognate segments of viruses from Taiwan and Asia and not other Australian viruses, supporting the conclusion that BTV_2 entered Australia recently. The Australian BTV_15_AUS_1982 prototype was revealed to be unusual among the Australian BTV isolates, with Seg-3 and -8 distantly related to other BTV sequences from all serotypes.

Menangle virus, a pteropid bat paramyxovirus infectious for pigs and humans, exhibits tropism for secondary lymphoid organs and intestinal epithelium in weaned pigs.

This study is the first report of experimental infection and transmission of Menangle virus (MenPV) in pigs. Isolated in 1997 from piglets that were stillborn at a large commercial piggery in New South Wales, Australia, MenPV is a recently identified paramyxovirus of bat origin that causes severe reproductive disease in pigs and an influenza-like illness, with a rash, in humans. Although successfully eradicated from the infected piggery, the virus was only isolated from affected fetuses and stillborn piglets during the period of reproductive disease, and thus the mode of transmission between pigs was not established. To investigate the pathogenesis of MenPV, we undertook time-course studies in 6-week-old pigs following intranasal administration of a low-passage, non-plaque-purified isolate from the lung of an infected stillborn piglet. Viraemia was of short duration and low titre, as determined by real-time RT-PCR and virus isolation. Following an incubation period of 2-3 days, virus was shed in nasal and oral secretions, faeces and urine, typically for less than 1 week. Cessation of shedding correlated with the development of neutralizing antibodies in sera. Secondary lymphoid organs and intestine were identified, using quantitative real-time RT-PCR, as major sites of viral replication and dissemination, and this was confirmed by positive immunolabelling of viral antigen within various lymphoid tissues and intestinal epithelium. These data provide new insights into the pathogenesis of MenPV in weaned pigs, and will facilitate future control and eradication programmes should it ever re-emerge in the pig population.

Evidence of a Possible Viral Host Switch Event in an Avipoxvirus Isolated from an Endangered Northern Royal Albatross (Diomedea sanfordi).

Avipoxviruses have been characterized from many avian species. Two recent studies have reported avipoxvirus-like viruses with varying pathogenicity in reptiles. Avipoxviruses are considered to be restricted to avian hosts. However, reports of avipoxvirus-like viruses from reptiles such as the green sea turtle (Chelonia mydas) and crocodile tegu (Crocodilurus amazonicus) suggest that cross-species transmission, within avian species and beyond, may be possible. Here we report evidence for a possible host switching event with a fowlpox-like virus recovered from an endangered northern royal albatross (Diomodea sanfordi)-a species of Procellariiformes, unrelated to Galliformes, not previously known to have been infected with fowlpox-like viruses. Complete genome sequencing of this virus, tentatively designated albatrosspox virus 2 (ALPV2), contained many fowlpox virus-like genes, but also 63 unique genes that are not reported in any other poxvirus. The ALPV2 genome contained 296 predicted genes homologous to different avipoxviruses, 260 of which were homologous to an American strain of fowlpox virus (FWPV). Subsequent phylogenetic analyses indicate that ALPV2 likely originated from a fowlpox virus-like progenitor. These findings highlight the importance of host-switching events where viruses cross species barriers with the risk of disease in close and distantly related host populations.

Preclinical efficacy studies of influenza A haemagglutinin precursor cleavage loop peptides as a potential vaccine.

A universal influenza vaccine that does not require annual reformulation would have clear advantages over the currently approved seasonal vaccine. In this study, we combined the mucosal adjuvant alpha-galactosylceramide (αGalCer) and peptides designed across the highly conserved influenza precursor haemagglutinin (HA(0)) cleavage loop as a vaccine. Peptides designed across the HA(0) of influenza A/H3N2 viruses, delivered to mice via the intranasal route with αGalCer as an adjuvant, provided 100 % protection following H3N2 virus challenge. Similarly, intranasal inoculation of peptides across the HA(0) of influenza A/H5N1 with αGalCer completely protected mice against heterotypic challenge with H3N2 virus. Our data suggest that these peptide vaccines effectively inhibited subsequent influenza A/H3N2 virus replication. In contrast, only 20 % of mice vaccinated with αGalCer-adjuvanted peptides spanning the HA(0) of H5N1 survived homologous viral challenge, possibly because the HA(0) of this virus subtype is cleaved by intracellular furin-like enzymes. Results of these studies demonstrated that HA(0) peptides adjuvanted with αGalCer have the potential to form the basis of a synthetic, intranasal influenza vaccine.

Molecular analysis of H7 avian influenza viruses from Australia and New Zealand: genetic diversity and relationships from 1976 to 2007.

Full-genome sequencing of 11 Australian and 1 New Zealand avian influenza A virus isolate (all subtype H7) has enabled comparison of the sequences of each of the genome segments to those of other subtype H7 avian influenza A viruses. The inference of phylogenetic relationships for each segment has been used to develop a model of the natural history of these viruses in Australia. Phylogenetic analysis of the hemagglutinin segment indicates that the Australian H7 isolates form a monophyletic clade. This pattern is consistent with the long-term, independent evolution that is, in this instance, associated with geographic regions. On the basis of the analysis of the other H7 hemagglutinin sequences, three other geographic regions for which similar monophyletic clades have been observed were confirmed. These regions are Eurasia plus Africa, North America, and South America. Analysis of the neuraminidase sequences from the H7N1, H7N3, and H7N7 genomes revealed the same region-based relationships. This pattern of independent evolution of Australian isolates is supported by the results of analysis of each of the six remaining genomic segments. These results, in conjunction with the occurrence of five different combinations of neuraminidase subtypes (H7N2, H7N3, H7N4, H7N6, H7N7) among the 11 Australian isolates, suggest that the maintenance host(s) is nearly exclusively associated with Australia. The single lineage of Australian H7 hemagglutinin sequences, despite the occurrence of multiple neuraminidase types, suggests the existence of a genetic pool from which a variety of reassortants arise rather than the presence of a small number of stable viral clones. This pattern of evolution is likely to occur in each of the regions mentioned above.

Genomic characterisation of a novel avipoxvirus, magpiepox virus 2, from an Australian magpie (Gymnorhina tibicen terraereginae).

Avipoxviruses are large, double-stranded DNA viruses and are considered significant pathogens that may impact on the conservation of numerous bird species. The vast majority of avipoxviruses in wild birds remain uncharacterised and their genetic variability is unclear. Here, we fully sequenced a novel avipoxvirus, magpiepox virus 2 (MPPV2), which was isolated 62 years ago (in 1956) from an Australian black-backed magpie. The MPPV2 genome was 298,392 bp in length and contained 419 predicted open-reading frames (ORFs). While 43 ORFs were novel, a further 24 ORFs were absent compared with another magpiepox virus (MPPV) characterised in 2018. The MPPV2 genome contained an additional ten genes that were homologs to shearwaterpox virus 2 (SWPV2). Subsequent phylogenetic analyses showed that the novel MPPV2 was most closely related to other avipoxviruses isolated from passerine and shearwater bird species, and demonstrated a high degree of sequence similarity (95.0%) with MPPV.

Genomic Characterisation of a Novel Avipoxvirus Isolated from an Endangered Yellow-Eyed Penguin (Megadyptes antipodes).

Emerging viral diseases have become a significant concern due to their potential consequences for animal and environmental health. Over the past few decades, it has become clear that viruses emerging in wildlife may pose a major threat to vulnerable or endangered species. Diphtheritic stomatitis, likely to be caused by an avipoxvirus, has been recognised as a significant cause of mortality for the endangered yellow-eyed penguin (Megadyptes antipodes) in New Zealand. However, the avipoxvirus that infects yellow-eyed penguins has remained uncharacterised. Here, we report the complete genome of a novel avipoxvirus, penguinpox virus 2 (PEPV2), which was derived from a virus isolate obtained from a skin lesion of a yellow-eyed penguin. The PEPV2 genome is 349.8 kbp in length and contains 327 predicted genes; five of these genes were found to be unique, while a further two genes were absent compared to shearwaterpox virus 2 (SWPV2). In comparison with penguinpox virus (PEPV) isolated from an African penguin, there was a lack of conservation within the central region of the genome. Subsequent phylogenetic analyses of the PEPV2 genome positioned it within a distinct subclade comprising the recently isolated avipoxvirus genome sequences from shearwater, canary, and magpie bird species, and demonstrated a high degree of sequence similarity with SWPV2 (96.27%). This is the first reported genome sequence of PEPV2 from a yellow-eyed penguin and will help to track the evolution of avipoxvirus infections in this rare and endangered species.

Genomic Characterisation of a Novel Avipoxvirus Isolated from an Endangered Northern Royal Albatross (Diomedea sanfordi).

Marine bird populations have been declining globally with the factors driving this decline not fully understood. Viral diseases, including those caused by poxviruses, are a concern for endangered seabird species. In this study we have characterised a novel avipoxvirus, tentatively designated albatrosspox virus (ALPV), isolated from a skin lesion of an endangered New Zealand northern royal albatross (Diomedea sanfordi). The ALPV genome was 351.9 kbp in length and contained 336 predicted genes, seven of which were determined to be unique. The highest number of genes (313) in the ALPV genome were homologs of those in shearwaterpox virus 2 (SWPV2), while a further 10 were homologs to canarypox virus (CNPV) and an additional six to shearwaterpox virus 1 (SWPV1). Phylogenetic analyses positioned the ALPV genome within a distinct subclade comprising recently isolated avipoxvirus genome sequences from shearwater, penguin and passerine bird species. This is the first reported genome sequence of ALPV from a northern royal albatross and will help to track the evolution of avipoxvirus infections in this endangered species.

Extended sequencing of vaccine and wild-type capripoxvirus isolates provides insights into genes modulating virulence and host range.

The genus Capripoxvirus in the subfamily Chordopoxvirinae, family Poxviridae, comprises sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV), which cause the eponymous diseases across parts of Africa, the Middle East and Asia. These diseases cause significant economic losses and can have a devastating impact on the livelihoods and food security of small farm holders. So far, only live classically attenuated SPPV, GTPV and LSDV vaccines are commercially available and the history, safety and efficacy of many have not been well established. Here, we report 13 new capripoxvirus genome sequences, including the hairpin telomeres, from both pathogenic field isolates and vaccine strains. We have also updated the genome annotations to incorporate recent advances in our understanding of poxvirus biology. These new genomes and genes grouped phenetically with other previously sequenced capripoxvirus strains, and these new alignments collectively identified several recurring alterations in genes thought to modulate virulence and host range. In particular, some of the many large capripoxvirus ankyrin and kelch-like proteins are commonly mutated in vaccine strains, while the variola virus B22R-like gene homolog has also been disrupted in many vaccine isolates. Among these vaccine isolates, frameshift mutations are especially common and clearly present a risk of reversion to wild type in vaccines bearing these mutations. A consistent pattern of gene inactivation from LSDV to GTPV and then SPPV is also observed, much like the pattern of gene loss in orthopoxviruses, but, rather surprisingly, the overall genome size of ~150 kbp remains relatively constant. These data provide new insights into the evolution of capripoxviruses and the determinants of pathogenicity and host range. They will find application in the development of new vaccines with better safety, efficacy and trade profiles.

Microarray-based detection of viruses causing vesicular or vesicular-like lesions in livestock animals.

Definitive diagnosis of vesicular or vesicular-like lesions in livestock animals presents challenges both for veterinary clinicians and diagnostic laboratories. It is often impossible to diagnose the causative disease agent on a clinical basis alone and difficult to collect ample vesicular epithelium samples. Due to restrictions of time and sample size, once laboratory tests have ruled out foot-and-mouth disease, vesicular stomatitis and swine vesicular disease a definitive diagnosis may remain elusive. With the ability to test a small quantity of sample for a large number of pathogens simultaneously, DNA microarrays represent a potential solution to this problem. This study describes the application of a long oligonucleotide microarray assay to the identification of viruses known to cause vesicular or vesicular-like lesions in livestock animals. Eighteen virus isolates from cell culture were successfully identified to genus level, including representatives of each foot-and-mouth disease virus serotype, two species of vesicular stomatitis virus (VSV), swine vesicular disease virus, vesicular exanthema of swine virus (VESV), bovine herpesvirus 1, orf virus, pseudocowpox virus, bluetongue virus serotype 1 and bovine viral diarrhoea virus 1. VSV and VESV were also identified in vesicular epithelium samples, with varying levels of sensitivity. The results indicate that with further development this microarray assay could be a valuable tool for the diagnosis of vesicular and vesicular-like diseases.

Evaluation of an ovine testis cell line (OA3.Ts) for propagation of capripoxvirus isolates and development of an immunostaining technique for viral plaque visualization.

An ovine testis cell line (OA3.Ts) was evaluated and compared with primary lamb kidney (LK) cells for its utility in capripoxvirus propagation and titration. A comparison of OA3.Ts cell growth kinetics and morphology at low (<33) and high (34-36) passage levels indicated a difference in both characteristics. However, viral titers determined in low and high passage OA3.Ts cells were comparable with those obtained using LK cells. Capripoxvirus infection of OA3.Ts and LK cells resulted in a similar cytopathic effect, which allowed for the detection of discrete viral plaques following immunostaining with capripoxvirus-specific antiserum.

Detection and quantitative pathogenesis study of classical swine fever virus using a real time RT-PCR assay.

A real time reverse transcription (RT) TaqMan PCR assay for the detection of classical swine fever virus (CSFV) previously described for use on a SmartCycler was validated on the Applied Biosystems AB 7700 Sequence Detection System using the Roche MagNA pure instrument for nucleic acid extraction and reaction set up. The primers and probe were specific for the CSFV strains (NSW, Baker and Weybridge) and did not react with other pestiviruses (BDV Tobias, BDV #327, BVDV non-CPE and BVDV C24V). Analysis of blood samples collected from pigs 1-6 and 8 days post-oronasal infection showed that over >10(6) range there was a linear relationship between log10TCID50ml-1 blood and the log10 normalised genetic load measured by quantitative TaqMan assay. The assay was used to assess CSFV shedding from infected pigs by quantitative TaqMan assay of virus genetic loads in tonsil, nasal and rectal swabs. Infection of tonsils was detected as early as 1 day post-inoculation. Shedding of virus detected by nasal and rectal swabs commenced on the third day post-inoculation. Quantitative TaqMan was used to analyse virus genetic load in tissues collected from pigs killed on days 1-3, 5 and 8 post-infection. Virus infection appeared first in tonsil (day 1), then submandibular lymph node, spleen, ileum and mesenteric lymph node (by day 3). Thereafter, virus spread to the visceral organs and finally to the pancreas and brain. Tonsil, nasal and rectal swabs as well as whole blood were found to be suitable samples for the rapid detection of CSFV using the TaqMan assay and automated nucleic acid extraction and reaction set up.

Prime-boost strategies in DNA vaccines.

Induction of HIV-specific T-cell responses by vaccines may facilitate efficient control of HIV replication. Plasmid DNA vaccines and recombinant fowlpox virus (rFPV) vaccines are promising HIV-1 vaccine candidates, although delivering either vaccine alone may be insufficient to induce sufficient T-cell responses. A consecutive immunization strategy, known as "prime-boost," involving priming with DNA and boosting with rFPV vaccines encoding multiple common HIV antigens, is used to induce broad and high-level T-cell immunity and ameliorate AIDS in macaques. This vaccine strategy is proceeding to clinical trials. This chapter describes the use of prime-boost vaccines to induce T-cell responses against HIV-1 and protective immunity against AIDS in macaques. Methods for the construction of the vaccines, the use of animal models, and the detection of immune responses are described.

Genomic characterisation of three Mapputta group viruses, a serogroup of Australian and Papua New Guinean bunyaviruses associated with human disease.

The Mapputta serogroup tentatively contains the mosquito-associated viruses Mapputta, Maprik, Trubanaman and Gan Gan. Interestingly, this serogroup has previously been associated with an acute epidemic polyarthritis-like illness in humans; however, there has been no ensuing genetic characterisation. Here we report the complete genome sequences of Mapputta and Maprik viruses, and a new Mapputta group candidate, Buffalo Creek virus, previously isolated from mosquitoes and detected by serology in a hospitalised patient. Phylogenetic analyses indicate that the group is one of the earliest diverged groups within the genus Orthobunyavirus of the family Bunyaviridae. Analyses show that these three viruses are related to the recently sequenced Australian bunyaviruses from mosquitoes, Salt Ash and Murrumbidgee. A notable feature of the Mapputta group viruses is the absence of the NSs (non-structural) ORF commonly found on the S segment of other orthobunyaviruses. Viruses of the Mapputta group have been isolated from geographically diverse regions ranging from tropical Papua New Guinea to the semi-arid climate of south-eastern Australia. The relevance of this group to human health in the region merits further investigation.

Construction of recombinant fowlpox viruses carrying multiple vaccine antigens and immunomodulatory molecules.

Here we describe plasmid vectors and selection protocols developed to allow the construction of recombinant fowlpox viruses (rFPVs) with up to three insertions of foreign DNA in the viral genome. Transient dominant selection allows the construction of recombinant viruses that do not retain the selection markers and can therefore be used for the insertion of additional genes at other sites in the viral genome. A SYBR Green real-time PCR sequence detection assay was applied to the identification of recombinant viruses from individual plaques, eliminating the need for amplification and hybridization from the transient dominant protocol and resulting in significant savings in time at each round of plaque purification. Dominant selection techniques allow more rapid recombinant virus construction; however, as the markers are retained along with the gene of interest, they can only be used to generate the final recombinant. rFPV vaccines constructed using these techniques have reached preclinical nonhuman primate and phase I human clinical trials in prime/boost vaccination studies as human immunodeficiency virus (HIV) therapeutic andprophylactic vaccines.

Phylogenetic analysis of the E2 gene of classical swine fever viruses from Lao PDR.

The E2 genes of 21 classical swine fever viruses (CSFV) were genetically characterized and compared with reference CSF viruses. The viruses originated from CSF outbreaks that occurred in the Lao People's Democratic Republic (Lao PDR) during 1997 though to 1999. All viruses characterized belonged to genogroup 2 and were members of subgroups 2.1 and 2.2. Results demonstrated a geographic delineation between subgroups 2.1 that was only found in the North-Central region, and subgroup 2.2 that was mostly found in the South-Central regions of Lao PDR. Although it was not possible to determine the origin of these viruses, it is probable that they may have been introduced to Lao PDR following cross-border trade. Alternatively, they have evolved independently of other viruses in the region.