Complete genetic dissection and cell type-specific replication of old world alphaviruses, getah virus (GETV) and sagiyama virus (SAGV).

Getah virus (GETV), which was first isolated in Malaysia in 1955, and Sagiyama virus (SAGV), isolated in Japan in 1956, are members of the genus Alphavirus in the family Togaviridae. It is a consensus view that SAGV is a variant of GETV. In the present study, we determined the complete sequences of the prototype GETV MM2021 and SAGV M6-Mag132 genomic RNA extracted from plaque-purified viruses. The MM2021 genome was 11,692 nucleotides (nt) in length in the absence of 3' poly(A) tail, and the length of M6-Mag132 genome was 11,698 nt. Through sequence alignment of MM2021 and M6-Mag132, we located all the amino acid differences between these two strains, which were scattered in all the encoded proteins. Subsequently, we validated the close evolutionary relationship between GETV and SAGV by constructing phylogenetic trees based on either complete genomes or structural genomes. We eventually analyzed the growth kinetics of GETV and SAGV as well as other representative alphaviruses in various mammalian and insect cell lines. It was shown that human-oriented cell lines such as HEK-293T and Hela cells were relatively resistant to GETV and SAGV infection due to absence of proviral factors or species-specific barrier. On the other hand, both GETV and SAGV replicated efficiently in non-human cell lines. Our results provide essential genetic information for future epidemiological surveillance on Alphaviruses and lay the foundation for developing effective interventions against GETV and SAGV.

Genome Sequence Analysis of First Ross River Virus Isolate from Papua New Guinea Indicates Long-Term, Local Evolution.

Ross River virus (RRV) is the most medically significant mosquito-borne virus of Australia, in terms of human morbidity. RRV cases, characterised by febrile illness and potentially persistent arthralgia, have been reported from all Australian states and territories. RRV was the cause of a large-scale epidemic of multiple Pacific Island countries and territories (PICTs) from 1979 to 1980, involving at least 50,000 cases. Historical evidence of RRV seropositivity beyond Australia, in populations of Papua New Guinea (PNG), Indonesia and the Solomon Islands, has been documented. We describe the genomic characterisation and timescale analysis of the first isolate of RRV to be sampled from PNG to date. Our analysis indicates that RRV has evolved locally within PNG, independent of Australian lineages, over an approximate 40 year period. The mean time to most recent common ancestor (tMRCA) of the unique PNG clade coincides with the initiation of the PICTs epidemic in mid-1979. This may indicate that an ancestral variant of the PNG clade was seeded into the region during the epidemic, a period of high RRV transmission. Further epidemiological and molecular-based surveillance is required in PNG to better understand the molecular epidemiology of RRV in the general Australasian region.

Genome-Scale Phylogeny and Evolutionary Analysis of Ross River Virus Reveals Periodic Sweeps of Lineage Dominance in Western Australia, 1977-2014.

Ross River virus (RRV), an alphavirus of the Togaviridae family, is the most medically significant mosquito-borne virus of Australia. Past RRV phylogenetic and evolutionary analyses have been based on partial genome analyses only. Three geographically distinct RRV lineages, the Eastern, the Western, and the supposedly extinct North-Eastern lineage, were classified previously. We sought to expand on past phylogenies through robust genome-scale phylogeny to better understand RRV genetic diversity and evolutionary dynamics. We analyzed 106 RRV complete coding sequences, which included 13 genomes available on NCBI and 94 novel sequences derived for this study, sampled throughout Western Australia (1977-2014) and during the substantial Pacific Islands RRV epidemic (1979-1980). Our final data set comprised isolates sampled over 59 years (1959-2018) from a range of locations. Four distinct genotypes were defined, with the newly described genotype 4 (G4) found to be the contemporary lineage circulating in Western Australia. The prior geographical classification of RRV lineages was not supported by our findings, with evidence of geographical and temporal cocirculation of distinct genetic groups. Bayesian Markov chain Monte Carlo (MCMC) analysis revealed that RRV lineages diverged from a common ancestor approximately 94 years ago, with distinct lineages emerging roughly every 10 years over the past 50 years in periodic bursts of genetic diversity. Our study has enabled a more robust analysis of RRV evolutionary history and resolved greater genetic diversity that had been previously defined by partial E2 gene analysis.IMPORTANCE Ross River virus (RRV) causes the most common mosquito-borne infection in Australia and causes a significant burden of suffering to infected individuals as well as being a large burden to the Australian economy. The genetic diversity of RRV and its evolutionary history have so far only been studied using partial E2 gene analysis with a limited number of isolates. Robust whole-genome analysis has not yet been conducted. This study generated 94 novel near-whole-genome sequences to investigate the evolutionary history of RRV to better understand its genetic diversity through comprehensive whole-genome phylogeny. A better understanding of RRV genetic diversity will enable better diagnostics, surveillance, and potential future vaccine design.

Epidemiologic, Entomologic, and Virologic Factors of the 2014-15 Ross River Virus Outbreak, Queensland, Australia.

Australia experienced its largest recorded outbreak of Ross River virus (RRV) during the 2014-15 reporting year, comprising >10,000 reported cases. We investigated epidemiologic, entomologic, and virologic factors that potentially contributed to the scale of the outbreak in Queensland, the state with the highest number of notifications (6,371). Spatial analysis of human cases showed that notifications were geographically widespread. In Brisbane, human case notifications and virus detections in mosquitoes occurred across inland and coastal locations. Viral sequence data demonstrated 2 RRV lineages (northeastern genotypes I and II) were circulating, and a new strain containing 3 unique amino acid changes in the envelope 2 protein was identified. Longitudinal mosquito collections demonstrated unusually high relative abundance of Culex annulirostris and Aedes procax mosquitoes, attributable to extensive freshwater larval habitats caused by early and persistent rainfall during the reporting year. Increased prevalence of these mosquitoes probably contributed to the scale of this outbreak.

The non-human reservoirs of Ross River virus: a systematic review of the evidence.

Understanding the non-human reservoirs of zoonotic pathogens is critical for effective disease control, but identifying the relative contributions of the various reservoirs of multi-host pathogens is challenging. For Ross River virus (RRV), knowledge of the transmission dynamics, in particular the role of non-human species, is important. In Australia, RRV accounts for the highest number of human mosquito-borne virus infections. The long held dogma that marsupials are better reservoirs than placental mammals, which are better reservoirs than birds, deserves critical review. We present a review of 50 years of evidence on non-human reservoirs of RRV, which includes experimental infection studies, virus isolation studies and serosurveys. We find that whilst marsupials are competent reservoirs of RRV, there is potential for placental mammals and birds to contribute to transmission dynamics. However, the role of these animals as reservoirs of RRV remains unclear due to fragmented evidence and sampling bias. Future investigations of RRV reservoirs should focus on quantifying complex transmission dynamics across environments.

La enfermedad producida por el virus chikungunya. ¿Qué esperar luego del estadio agudo? / Chikungunya virus (CHIKV): what can be expected after the acute phase?

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Ross River virus infection surveillance in the Greater Perth Metropolitan area--has there been an increase in cases in the winter months?

An increase in off-season (June to September) Ross River virus (RRV) notifications from the greater Perth metropolitan area was observed from 2006 to 2009. We investigated the increase to determine whether it is likely to have reflected a true increase in off-season cases. A single positive RRV IgM test result is sufficient for RRV notification but where follow-up testing was performed, the positive predictive value of an IgM test where IgG was negative was very low in the off-season and also in the season when using the only commercially available test kit. The increase in off-season notifications was not associated with an increase in off-season testing. Some Perth laboratories use more stringent notification criteria than the nationally agreed RRV case definition, and the geographical distribution of samples tested varies between laboratories. Our findings make a strong case to change the nationally agreed case definition for RRV to not accept a single IgM positive test result as laboratory definitive evidence where the IgG is negative. Our study also identified a range of challenges in interpreting changes in seasonal patterns and geographical distribution of RRV. Any such observed changes should be investigated through further data analysis and/or mosquito trapping and testing in order to assess validity.

Persistence of multiple genetic lineages within intrahost populations of Ross River virus.

We examined the structure and extent of genetic diversity in intrahost populations of Ross River virus (RRV) in samples from six human patients, focusing on the nonstructural (nsP3) and structural (E2) protein genes. Strikingly, although the samples were collected from contrasting ecological settings 3,000 kilometers apart in Australia, we observed multiple viral lineages in four of the six individuals, which is indicative of widespread mixed infections. In addition, a comparison with previously published RRV sequences revealed that these distinct lineages have been in circulation for at least 5 years, and we were able to document their long-term persistence over extensive geographical distances.

Lineage replacement accompanying duplication and rapid fixation of an RNA element in the nsP3 gene in a species of alphavirus.

A sequence of thirty-six nucleotides in the nsP3 gene of Ross River virus (RRV), coding for the amino acid sequence HADTVSLDSTVS, was duplicated some time between 1969 and 1979 coinciding with the appearance of a new lineage of this virus and with a major outbreak of Epidemic Polyarthritis among residents of the Pacific Islands. This lineage of RRV continues to circulate throughout Australia and both earlier lineages, which lacked the duplicated element, now are extinct. Multiple copies of several other elements also were observed in this region of the nsP3 gene in all lineages of RRV. Multiple copies of one of these, coding for the amino acid sequence P*P*PR, were detected in the C-terminal region of the nsP3 protein of all alphaviruses except those of African origin. The fixation of duplications and insertions in 3' region of nsP3 genes from all lineages of alphaviruses, suggests they provide some fitness advantage.

Molecular evolutionary dynamics of Ross River virus and implications for vaccine efficacy.

Ross River virus (RRV) is a mosquito-borne member of the genus Alphavirus that causes epidemic polyarthritis in humans, costing the Australian health system at least US$10 million annually. Recent progress in RRV vaccine development requires accurate assessment of RRV genetic diversity and evolution, particularly as they may affect the utility of future vaccination. In this study, we provide novel RRV genome sequences and investigate the evolutionary dynamics of RRV from time-structured E2 gene datasets. Our analysis indicates that, although RRV evolves at a similar rate to other alphaviruses (mean evolutionary rate of approx. 8x10(-4) nucleotide substitutions per site year(-1)), the relative genetic diversity of RRV has been continuously low through time, possibly as a result of purifying selection imposed by replication in a wide range of natural host and vector species. Together, these findings suggest that vaccination against RRV is unlikely to result in the rapid antigenic evolution that could compromise the future efficacy of current RRV vaccines.

Vector competence of three Australian mosquitoes, Verrallina carmenti, Verraullina lineata, and Mansonia septempunctata (Diptera: Culicidae), for Ross River virus.

The vector competence of Verrallina carmenti (Edwards), Verrallina lineata (Taylor), and Mansonia septempunctata (Theobald) (Diptera: Culicidae) from north Queensland, Australia, was tested using two isolates of Ross River virus (family Togaviridae, genus Alphavirus, RRV). All three species were tested using RRV isolate RR102MP (ex-Cairns, Queensland, Australia), whereas Ve. carmenti and Ve. lineata also were tested using RR2186 (ex-Bourke, New South Wales, Australia). Transmission was tested using the capillary tube method, with infection of mosquito bodies and saliva determined using cell culture. Infection with RR102MP resulted in 27.5% of Ve. carmenti, 19.2% of Ve. lineata and 13.3% of Ma. septempunctata transmitting virus. When Ve. carmenti and Ve. lineata were infected with RR2186, transmission rates for both species were generally < 10%, although a transmission rate of 25% was recorded for Ve. lineata exposed to high titer virus. These results indicated that the three mosquito species have the potential to contribute to local transmission cycles.

Isolation and characterization of a Sagiyama virus from domestic pigs.

In 2002, a strain of Sagiyama virus (SAGV) designated ML/Taiwan/02 was isolated from farmed pigs in Taiwan. The nsP1 and E1 gene sequences of the ML/Taiwan/02 strain shared 98.6 and 96.7% homology, respectively, with corresponding genes of a Japanese strain of SAGV. Nucleotide and amino acid sequence comparison revealed this strain of SAGV to be most closely related to Getah virus, as opposed to its current classification as a subtype of Ross River virus. To investigate the seroprevalence of SAGV infection in Taiwan, a total of 586 pig sera collected from 11 of 17 Taiwanese districts were tested for serum neutralizing antibodies (SNA) against SAGV. Results indicated that 51% of the samples had SNA titer > or = 4, and 40% had SNA titer > or = 48, indicative of repeated exposure to SAGV in the field. To study the pathogenicity of the ML/Taiwan/02 strain, this strain was experimentally inoculated into 4-week-old specific-pathogen-free pigs that were seronegative for SAGV. Viremia was detected during postinoculation days (PID) 2-4, when the SNA titer was < or = 16. By PID 7, viremia was no longer detectable, coinciding with the increase of SNA titer to > or = 48. Clinical illnesses or remarkable lesions were not observed. To the authors' knowledge, this is the first reported isolation of a strain of SAGV from pigs in the field. The virus is experimentally nonpathogenic to pigs but is moderately widespread, most likely via repeated exposure to virus-carrying mosquitoes.

Isolation of arboviruses from mosquitoes (Diptera: Culicidae) collected from the Gulf Plains region of northwest Queensland, Australia.

As part of investigations into Japanese encephalitis (JE) virus and related flaviviruses in northern Australia, 153,529 mosquitoes were collected and processed for virus isolation from the Gulf Plains region of northwest Queensland. Collections from within 30 km of each of the townships of Croydon, Normanton and Karumba yielded 3,087 (2.0%), 66,009 (43.0%), and 84,433 (55.0%) mosquitoes, respectively, from which 16 viruses were isolated. Four isolates of Murray Valley encephalitis (MVE), two of Kunjin (KUN), three of Ross River (RR), and one of Sindbis (SIN) viruses were obtained from Culex sitiens subgroup mosquitoes. Molecular identification of the mosquito species composition of these virus positive pools revealed that most isolates were from pools containing mainly Culex annulirostris Skuse and low numbers of Culex palpalis (Taylor). Only three pools, one each of MVE, KUN, and RR, were from mosquitoes identified exclusively as Cx. annulirostris. Other viruses isolated include one Edge Hill virus from Ochlerotatus normanensis (Taylor), an isolate of SIN from Anopheles meraukensis Venhuis, two isolates of RR from Anopheles amictus Edwards, and single isolates of RR from Anopheles bancroftii Giles andAedes lineatopennis (Ludlow). The isolate of RR from Ae. lineatopennis was the first reported from this species. The public health implications of these isolations in the Gulf Plains region are discussed briefly.

Definition of Ross River virus vectors at Maroochy Shire, Australia.

Evidence of Ross River (RR) virus infection in field-collected mosquitoes and data from laboratory vector competence experiments incriminated a range of mosquito species as important vectors of RR virus in Maroochy Shire, Queensland, Australia. Nine RR and 2 Barmah Forest virus isolates were recovered from 27,529 mosquitoes collected in light traps from Maroochy Shire during 1996. Nine of the 10 most abundant mosquito species collected in light traps were fed on blood containing the B94/20 strain RR isolated from Queensland in 1994. All species except for Culex sitiens Wiedemann were susceptible to experimental infection. Evidence of RR virus transmission to mice was found with Aedes vigilax (Skuse), Aedes funereus (Theobald), Aedes procax (Skuse), Culex annulirostris Skuse, Mansonia uniformis (Theobald) and Culex australicus Dobrotworsky & Drummond. Aedes notoscriptus (Skuse) and Aedes multiplex (Theobald) were susceptible to RR virus infection, although there was no evidence of virus transmission. Based on adult abundance and vector competence results, freshwater species such as Cx. annulirostris, Ae. procax, and Ae. funereus, and saltmarsh Ae. vigilax, appear to be important vectors of RR virus in Maroochy Shire and control programs should be revised to include these species.

Genetic heterogeneity among isolates of Ross River virus from different geographical regions.

The RNase T1 maps of 80 isolates of Ross River virus from different regions of mainland Australia and the Pacific Islands were compared. Four different clusters of isolates with greater than an estimated 5 to 6% diversity at the nucleotide level were found. There was a pattern of differences between eastern and western Australian strains; however, the pattern was disturbed by overlaps and incursants. Pacific Islands isolates belonged to the eastern Australian topotype. Our findings suggest that certain genetic types of Ross River virus predominate in different geographical regions. In contrast, populations of other important Australian arboviruses (Murray Valley encephalitis, Kunjin, and Sindbis viruses) are distributed across the Australian continent as minor variants of one strain. Our data also show that in one region, strains of Ross River virus with identical RNase T1 maps circulate during both years when epidemics occur and years when they do not. This finding suggests that Ross River virus epidemics are not dependent on the introduction or evolution of new strains of the virus. Two strains, belonging to the eastern Australian topotype, were isolated in Western Australia. It is likely that viremic humans or possibly domestic livestock travelling by aircraft were responsible for this movement.