Evolutionary history of H5 highly pathogenic avian influenza viruses (clade 2.3.4.4c) circulating in Taiwan during 2015-2018.

The highly pathogenic avian influenza (HPAI) virus A/goose/Guangdong/1/96 H5N1 (Gs/GD) lineage has been transmitted globally and has caused deaths in wild birds, poultry, and humans. Clade 2.3.4.4c, one of the subclades of the Gs/GD lineage, spread through Taiwan in late 2014 and become an endemic virus. We analyzed 239 newly sequenced HPAI clade H5Nx isolates to explore the phylogenetic relationships, divergence times, and evolutionary history of Taiwan HPAI H5Nx viruses from 2015 to 2018. Overall, 15 reassortant genotypes were identified among H5N2, H5N3, and H5N8 viruses. Maximum likelihood and Bayesian phylogenies based on homologous hemagglutinin (HA) and matrix protein (MP) genes suggest that Taiwan HPAI H5Nx viruses share a most recent common ancestor that has diversified since October 2014 and is closely related to two HPAI H5N8 viruses identified from wild birds in Japan. Two waves of HPAI caused by multiple reassortants were identified, the first occurring in late 2014 and the second beginning in late 2016. The first wave consisted of seven H5Nx reassortants that spread through Taiwan. In the second wave, eight novel reassortants were detected which had newly introduced internal genes, mostly derived from the avian influenza virus gene pool maintained in wild birds in Asia. Phylodynamic reconstruction using the Bayesian Skygrid model revealed varied fluctuating patterns of relative genetic diversity among reassortants. The mean evolutionary rate also varied among reassortants and subtypes. The neuraminidase (NA) gene evolved faster than the HA gene in H5N2 viruses, while HA evolved faster than NA in H5N8 viruses. The HA mean evolutionary rate ranged from 6.10 × 10-3 to 7.73 × 10-3 and from 5.81 × 10-3 to 9.45 × 10-3 substitutions/site/year for H5N2 and H5N8 viruses, respectively. The continuous circulation of HPAI H5Nx variants and the emergence of novel reassortants in Taiwan highlight that the surveillance, biosecurity, and management systems of poultry farms need to be improved and carefully executed.

A case report of reptile-associated nidovirus (serpentovirus) in a ball python (Python regius) in Taiwan.

Reptile-associated nidoviruses (serpentoviruses) have been reported to cause proliferative interstitial pneumonia in pythons and other reptile species. A captive, younger than 2 years old, intact female ball python (Python regius) showed increased oral mucus, wheezing, and audible breathing with weight loss. Gross and microscopic examination revealed large amounts of mucus in the esophagus and proliferative interstitial pneumonia. Serpentovirus genes were detected from the lung tissues by polymerase chain reaction. The current serpentoviruses was phylogenetically grouped with the serpentovirus previously identified in the US. No case of serpentovirus infection has been reported in Asia. The present report provides information of complete genome sequence and global distribution of serpentovirus.

Fatal canine parvovirus-2 (CPV-2) infection in a rescued free-ranging Taiwanese pangolin (Manis pentadactyla pentadactyla).

Carnivore protoparvovirus 1 includes feline parvovirus (FPV), variants of canine parvovirus-2 (CPV-2), mink enteritis virus, and raccoon parvovirus, important pathogens affecting both wild and domestic carnivores. In this report, we described a fatal CPV-2 infection in a rescued Taiwanese pangolin, which provides the first evidence of CPV-2 infection in a non-carnivore. Post-rescue, the Taiwanese pangolin died from complications resulting from a severe panleucocytopenia and bloody diarrhoea. A full autopsy was performed and microscopic examination of the tissues revealed ulcerative, necrotizing, and haemorrhagic glossitis, esophagitis and enteritis. The results of transmission electronic microscopy, polymerase chain reaction and in situ hybridization provided confirmatory evidence that the lesions in the tongue, oesophagus and intestine were associated with a protoparvovirus. Phylogenetic comparison of the whole VP2 gene from the current pangolin protoparvovirus strain showed close clustering with the CPV-2c strains from domestic dogs in Taiwan, China and Singapore. The amino acid sequence of the pangolin protoparvovirus showed 100% identity to the CPV-2c strains from domestic dogs in China, Italy, and Singapore. The current findings highlight that pangolins are susceptible to protoparvoviruses. The potential of cross-species transmission of protoparvoviruses between Carnivora and Pholidota should be considered when housing pangolins in close proximity to carnivores and adopting strict biosecurity measures to avoid cross-species transmission in rescue facilities and zoos.

Detection of reassortant H5N6 clade 2.3.4.4 highly pathogenic avian influenza virus in a black-faced spoonbill (Platalea minor) found dead, Taiwan, 2017.

A H5N6 highly pathogenic avian influenza virus (HPAIV) was detected in a black-faced spoonbill (Platalea minor) found dead in Taiwan during December 2017. Genome sequencing and phylogenetic analyses suggest the hemagglutinin gene belongs to H5 clade 2.3.4.4 Group B. All genes except neuraminidase gene shared high levels of nucleotide identity with H5N8 HPAIV identified from Europe during 2016-2017. Genetically similar H5N6 HPAIV was also identified from Japan during November 2017. Enhanced surveillance is required in this region.

Lyssavirus in Japanese Pipistrelle, Taiwan.

A putative new lyssavirus was found in 2 Japanese pipistrelles (Pipistrellus abramus) in Taiwan in 2016 and 2017. The concatenated coding regions of the virus showed 62.9%-75.1% nucleotide identities to the other 16 species of lyssavirus, suggesting that it may be representative of a new species of this virus.

Highly pathogenic avian influenza viruses H5N2, H5N3, and H5N8 in Taiwan in 2015.

A severe epidemic, affecting mainly goose populations, broke out in early January 2015. The causative agents were identified as novel H5 avian influenza viruses carrying N2, N3, and N8 subtypes of the neuraminidase gene. From January 8 to February 11, 766 waterfowl and poultry farms were invaded by the H5 viruses, and more than 2.2 million geese died or were culled. Phylogenetic analysis suggested that these avian influenza viruses derived from the H5 viruses of clade 2.3.4.4 which were emerging in 2014 in East Asia, West Europe, and North America.

Invasion of exotic bovine ephemeral fever virus into Taiwan in 2013-2014.

Bovine ephemeral fever virus is a member of the family Rhabdoviridae and bovine ephemeral fever has frequently affected cattle population in Taiwan since 1967. During the outbreaks in 2013 and 2014, exotic bovine ephemeral fever viruses were detected by reverse transcription polymerase chain reaction and nucleotide sequencing. Sequence comparison showed that the exotic viruses shared 99.0-99.4% nucleotide identities (99.4-100.0% amino acid identities) with Chinese viruses and, on the contrary, 96.2-97.2% nucleotide identities (97.8-98.6% amino acid identities) with indigenous Taiwanese viruses. Additionally, our phylogenetic analysis also supported that the newly invaded bovine ephemeral fever viruses were closely related to the Chinese strains. These exotic 2013-2014 viruses have become prevalent and displaced indigenous virus strains since their appearance.

Relationships of bovine ephemeral fever epizootics to population immunity and virus variation.

Bovine ephemeral fever is an arthropod-borne bovine viral disease caused by infection with bovine ephemeral fever virus which belongs to genus Ephemerovirus within the family Rhabdoviridae. In this study, serological data and virological information about the disease and the virus, spanning from 2001 to 2013, were employed to analyze the relationships of bovine ephemeral fever epizootics to population immunity and virus variation. National and regional surveillance data indicated that 2 of the 3 major epizootics and 87% regional outbreaks were associated with lower neutralizing antibody titers and immunity coverage, reflecting the importance of population immunity for the control of bovine ephemeral fever. Phylogenetic analysis and sequence comparison demonstrated that Taiwanese bovine ephemeral fever viruses were >96.0% and >97.6% similar to the East Asian isolates in nucleotide and amino acid sequences, respectively. These analyses supported that the Taiwanese viruses shared the same gene pool with the strains of the other East Asian countries, mainly Japan.

Genetic analysis of two Taiwanese bluetongue viruses.

BTV2/KM/2003 and BTV12/PT/2003 are the first identified bluetongue viruses in Taiwan. The prototype virus BTV2/KM/2003 was previously characterized in various respects as low virulent. In the present study, nucleotide sequences of the ten genome segments and their coding regions of the Taiwan strains were determined and analyzed. The two strains had >96.8% nucleotide and >97.9% deduced amino acid identities to each other, except for the VP2 genes. Their genome sequences, except for NS1 and VP2 genes, clustered overall in the Asian lineage, and were closely related to strains from China, India, Indonesia, and Japan. The phylogenetic trees and nucleotide identities of six BTV genes were suggestive of the geographical origin of the bluetongue virus strains analyzed, with a few exceptions. To examine which genes better distinguished strains from different origins (topography), the distribution of and the levels of differences in nucleotide identities were analyzed, revealing that VP3, NS2, and NS3 genes were more suitable for topotyping of BTVs. Analysis of ratios of non-synonymous/synonymous substitutions (dN/dS values) between putative ancestry and their descendant strains suggested that most BTV genes evolved under a negative selection, whereas the VP7 gene evolved under positive selection, and its non-synonymous substitutions accumulated more rapidly in strains from the Mediterranean region.

Isolation and characterization of potentially pathogenic H5N2 influenza virus from a chicken in Taiwan in 2008.

During the surveillance of avian influenza, an H5N2 influenza A virus was isolated from a cloacal swab sample of an apparently healthy chicken in Taiwan in October 2008. It was found that the HA of the virus had a pair of dibasic amino acid residues at the cleavage site, which might be a marker of highly pathogenic avian influenza virus. However, the intravenous pathogenicity index of the isolate was 0.89, indicating that the virus was approaching high pathogenicity in chickens. Virus isolation was negative in 2916 birds from 146 farms in a 3-km radius around the farm where the virus was isolated. Genetic analysis of the eight segments of the isolate indicated that the isolated virus was a reassortant whose HA and NA gene segments belonged to the American lineage and internal genes to the Eurasian lineage.

The identification of frequent variations in the fusion protein of canine distemper virus.

Canine distemper (CD) is a highly contagious disease with a worldwide distribution. Genetic diversity in genes encoding the haemagglutinin (H) and fusion (F) virus envelope proteins have been implicated in the increasing incidence of CD. Unlike the H gene, little is known about the genetic variability of the F gene in this virus. In the present study sequence analysis of the complete coding region of the F protein from CD virus isolates from Taiwan were carried out. Phylogenetic analysis demonstrated that the majority of isolates were similar to those found in neighbouring China and Japan, but were genetically distinct from vaccine strains. Remarkable variations were found scattered throughout the pre-peptide region (residues 1-135). The sequence identity of this region between locally sourced strains and between these strains and vaccine strains was 89% and 64 to 67%, respectively. Analysis suggested a novel strain of distant genetic lineage was present in dogs in the geographically isolated city of Hualien.