Co-infections with human immunodeficiency virus type 1 (HIV-1) and human pegivirus (HPgV) are common in hepatitis C virus (HCV)-infected individuals. However, analysis on the evolutionary dynamics and transmission network profiles of these viruses among individuals with multiple infections remains limited. A total of 228 injecting drug users (IDUs), either HCV- and/or HIV-1-infected, were recruited in Kuala Lumpur, Malaysia. HCV, HIV-1 and HPgV genes were sequenced, with epidemic growth rates assessed by the Bayesian coalescent method. Based on the sequence data, mono-, dual- and triple-infection were detected in 38.8%, 40.6% and 20.6% of the subjects, respectively. Fifteen transmission networks involving HCV (subtype 1a, 1b, 3a and 3b), HIV-1 (CRF33_01B) and HPgV (genotype 2) were identified and characterized. Genealogical estimates indicated that the predominant HCV, HIV-1 and HPgV genotypes were introduced into the IDUs population through multiple sub-epidemics that emerged as early as 1950s (HCV), 1980s (HIV-1) and 1990s (HPgV). By determining the difference in divergence times between viral lineages (ΔtMRCA), we also showed that the frequency of viral co-transmission is low among these IDUs. Despite increased access to therapy and other harm reduction interventions, the continuous emergence and coexistence of new transmission networks suggest persistent multiple viral transmissions among IDUs.
The genus Flaviviridae comprises about 70 members, of which about 30 are found in southern, south-eastern and eastern Asia and Australasia. These include major pathogens such as Japanese encephalitis (JE), West Nile (WN), Murray Valley encephalitis (MVE), tick-borne encephalitis, Kyasanur Forest disease virus, and the dengue viruses. Other members are known to be associated with mild febrile disease in humans, or with no known disease. In addition, novel flaviviruses continue to be discovered, as demonstrated recently by New Mapoon virus in Australia, Sitiawan virus in Malaysia, and ThCAr virus in Thailand. About 19 of these viruses are mosquito-borne, six are tick-borne, and four have no known vector and represent isolates from rodents or bats. Evidence from phylogenetic studies suggest that JE, MVE and Alfuy viruses probably emerged in the Malaya-Indonesian region from an African progenitor virus, possibly a virus related to Usutu virus. WN virus, however, is believed to have emerged in Africa, and then dispersed through avian migration. Evidence suggests that there are at least seven genetic lineages of WN virus, of which lineage 1b spread to Australasia as Kunjin virus, lineages 1a and 5 spread to India, and lineage 6 spread to Malaysia. Indeed, flaviviruses have a propensity to spread and emerge in new geographic areas, and they represent a potential source for new disease emergence. Many of the factors associated with disease emergence are present in the region, such as changes in land use and deforestation, increasing population movement, urbanization, and increasing trade. Furthermore, because of their ecology and dependence on climate, there is a strong likelihood that global warming may significantly increase the potential for disease emergence and/or spread.
To reveal whether bats serve as an amplifying host for Yokose virus (YOKV), we conducted a serological survey and experimentally infected fruit bats with YOKV isolated from microbats in Japan. YOKV belongs to the Entebbe bat virus group of vector unknown group within the genus Flavivirus and family Flaviviridae. To detect antibodies against YOKV, we developed an enzyme-linked immunosorbent assay (ELISA) using biotinylated anti-bat IgG rabbit sera. Serological surveillance was conducted with samples collected in the Philippines and the sera supplied from Malaysia. One of the 36 samples from the Philippines (2.7%) and 5 of the 26 samples from Malaysia (19%) had detectable ELISA antibodies. In the experimental infections, no clinical signs of disease were observed. Moreover, no significant viral genome amplification was detected. These findings revealed that YOKV replicates poorly in the fruit bat, suggesting that fruit bats do not seem to serve as an amplifying host for YOKV.
Duck Tembusu virus (DTMUV), a newly emerging virus in ducks, was first reported in China in 2010. However, an unknown severe contagious disease associated with severe neurological signs and egg production losses in ducks, resembling to DTMUV infection, was observed in Thailand since 2007. To determine the presence of DTMUV in 2007, the clinical samples from affected ducks collected in 2007 were tested for DTMUV using pathological and virological analyses. Gross and histopathological lesions of affected ducks were mostly restricted to the ovary, brain and spinal cord, and correlated with the presence of flavivirus antigen in the brain and spinal cord samples. Subsequently, DTMUV was identified by RT-PCR and nucleotide sequencing of the polyprotein gene. Phylogenetic analysis of the polyprotein gene sequence revealed that the 2007 Thai DTMUV was a unique virus, belonged within DTMUV cluster 1, but distinctively separated from the Malaysian DTMUV, which was the most closely related DTMUV. It is interesting to note that the 2007 Thai DTMUV was genetically different from the currently circulating Thai and Chinese DTMUVs, which belonged to cluster 2. Our findings indicated that the 2007 Thai DTMUV emerged earlier from a common ancestor with the recently reported DTMUVs; however, it was genetically distinctive to any of the currently circulating DTMUVs. In conclusion, our data demonstrated the presence of DTMUV in the Thai ducks since 2007, prior to the first report of DTMUV in China in 2010. This study indicates that DTMUV may have circulated in the region long before 2010 and highlights high genetic diversity of DTMUVs in Asia.
A new virus named Sitiawan virus (SV) was isolated from sick broiler chicks in chicken embryos. The virus replicated well with cytopathogenic effect (CPE) in the chicken B-lymphocyte cell line LSCC-BK3. The virus was an enveloped RNA virus of approximately 41 nm in size with hemagglutinating activity (HA) to goose erythrocytes. It was cross-reactive with Japanese encephalitis virus (JEV), a member of flaviviruses by HA inhibition tests but not by cross-virus neutralization tests. The cDNA fragment of NS5 gene was amplified with primers corresponding to NS5 gene of flaviviruses. The nucleotide sequences were 92% homologous to Tembusu virus, a member of the mosquito-borne virus cluster of the genus Flavivirus. In cross-neutralization tests with Tembusu virus, antiserum to SV did not neutralize Tembusu virus, and antiserum to Tembusu virus neutralized more weakly to SV than against homologous virus. These results indicate that SV is a new virus which can be differentiated serologically from Tembusu virus but is otherwise similar with respect to nucleotide sequence. The virus causes encephalitis, growth retardation, and increased blood glucose levels in inoculated chicks.
A neurological disease of young Pekin ducks characterized by ataxia, lameness, and paralysis was observed at several duck farms in Malaysia in 2012. Gross pathological lesions were absent or inconsistent in most of the cases, but severe and consistent microscopic lesions were found in the brain and spinal cord, characterized by non-purulent panencephalomyelitis. Several virus isolates were obtained in embryonated duck eggs and in cell cultures (Vero and DF-1) inoculated with the brain homogenates of affected ducks. After exclusion of other viruses, the isolates were identified as a flavivirus by flavivirus-specific reverse transcription-polymerase chain reaction (RT-PCR) assays. Inoculation of 2-week-old Pekin ducks with a flavivirus isolate by the subcutaneous or intramuscular route resulted in typical clinical signs and histological lesions in the brain and spinal cord. The inoculated virus was detected by RT-PCR from organ samples of ducks with clinical signs and histological lesions. With a few days delay, the disease was also observed among co-mingled contact control birds. Phylogenetic analysis of NS5 and E gene sequences proved that the isolates were representatives of a novel phylogenetic group within clade XI (Ntaya virus group) of the Flavivirus genus. This Malaysian Duck Tembusu Virus (DTMUV), named Perak virus, has moderate genomic RNA sequence similarity to a related DTMUV identified in China. In our experiment the Malaysian strain of DTMUV could be transmitted in the absence of mosquito vectors. These findings may have implications for the control and prevention of this emerging group of flaviviruses.