Fan Lee

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.

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.

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.

Subclinical bluetongue virus infection in domestic ruminants in Taiwan

Bluetongue is an arthropod-borne viral disease affecting domestic and wild ruminants. Taiwan, with the Tropic of Cancer crossing through it, was considered free of bluetongue virus (BTV) before 2001. The goals of this study are to identify the serotype and phylogeny of Taiwan BTV isolates and to understand the serological status and chronology of BTV infection. Analysis of the S10 gene segment revealed that Taiwan BTV isolates are closely related to Chinese strains. Seropositive results were found in 32.7% of the cattle and 8.2% of the goats by head, and 90.7% of the cattle herds and 28.9% of the goat flocks. Anti-BTV antibodies have existed in goat sera since 1989 and in bovine sera since 1993, and over the years, the seropositive rates in rapidly urbanized districts have decreased, most likely due to the loss of vector habitats. Seropositive rates for sheep were variable, due to a small sample size and a small sheep population. Thus far, all natural BTV infections have been subclinical, consistent with experimental sheep inoculation, revealing that the Taiwan isolate is of low virulence.

Development of a Luminex assay for the detection of swine antibodies to non-structural proteins of foot-and-mouth disease virus

Abstract Foot-and mouth disease (FMD), swine vesicular disease (SVD), and vesicular stomatitis (VS) are highly contagious vesicular diseases of swine but are not easy to differentiate clinically. For the purpose of instant detecting of FMD and differentiating it from the other vesicular diseases, a Luminex assay was developed. Sera from 64 infected, 307 vaccinated, and 280 naïve pigs were tested by the Luminex assay. Diagnostic sensitivity of the assay was 100%. Diagnostic specificity of the assay was 98.7% in vaccinated pigs and 97.5% to 100% in naïve pigs. Agreement between the results from the Luminex assay and those from a 3ABC polypeptide blocking ELISA was 96.3% with kappa statistics of 0.92. The Luminex assay can detect the immune response to NSP-3ABC in swine as early as eight days post-infection. Moreover, all of the 15 vaccinated but unprotected pigs were all detected by the Luminex assay. The results indicated that the Luminex assay has potential with specificity in detecting antibodies to FMDV 3ABC NSP and in distinguishing FMDV-infected pigs from with either SVDV or VSV.

Analysis of Bluetongue Virus mRNA in Baby Hamster Kidney Cells and Sheep Tissues by Real-time Reverse Transcription Polymerase Chain Reaction

Bluetongue virus (BTV), a member of the genus Orbivirus of the family Reoviridae, is the causative agent of bluetongue. The present study further characterizes the replication of Taiwan prototype BTV2/KM/2003 in baby hamster kidney cell line BHK-21 and in formalin-fixed paraffin-embedded tissues of an infected sheep. Messenger RNA transcribed from the BTV genes was measured by a real-time reverse transcription polymerase chain reaction. In the infected BHK-21 cells, the three most transcribed genes were NS2, VP4, and VP7 genes. The abundance of BTV mRNA species in the tissues of infected sheep demonstrated a different pattern, wherein NS1-3 genes were most frequently transcribed. Among all tissues examined, NS1-3 genes were detected at relatively higher levels, indicated by the lower Ct values, in the tongue, haired lip, ear pinna and cerebrum, consistent with the low virulence of the Taiwan BTV2/KM/2003 to Corriedal sheep as previously characterized. The tongue, haired lip, ear, and cerebrum are located primarily in the craniofacial regions and are closer to the inoculation site of contralateral ear pinna. The inoculated BTV should have low or moderate penetration capability, so that the viral gene expressions in the more distant tissues were relatively low, despite the establishment of viremia and the permissiveness of tissues to the virus. Alternatively BTV may have higher tropism for these craniofacial tissues.

Reassortant Clade 2.3.4.4 of Highly Pathogenic Avian Influenza A(H5N6) Virus, Taiwan, 2017

A highly pathogenic avian influenza A(H5N6) virus of clade 2.3.4.4 was detected in a domestic duck found dead in Taiwan during February 2017. The endemic situation and continued evolution of various reassortant highly pathogenic avian influenza viruses in Taiwan warrant concern about further reassortment and a fifth wave of intercontinental spread.

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.

Development of a multiplex Luminex assay for detecting swine antibodies to structural and nonstructural proteins of foot-and-mouth disease virus in Taiwan

BACKGROUND/PURPOSE(S) Foot-and-mouth disease (FMD) and swine vesicular disease (SVD) are serious vesicular diseases that have devastated swine populations throughout the world. The aim of this study was to develop a multianalyte profiling (xMAP) Luminex assay for the differential detection of antibodies to the FMD virus of structural proteins (SP) and nonstructural proteins (NSP). METHODS After the xMAP was optimized, it detected antibodies to SP-VP1 and NSP-3ABC of the FMD virus in a single serum sample. These tests were also compared with 3ABC polypeptide blocking enzyme-linked immunosorbent assay (ELISA) and virus neutralization test (VNT) methods for the differential diagnosis and assessment of immune status, respectively. RESULTS To detect SP antibodies in 661 sera from infected naïve pigs and vaccinated pigs, the diagnostic sensitivity (DSn) and diagnostic specificity (DSp) of the xMAP were 90.0-98.7% and 93.0-96.5%, respectively. To detect NSP antibodies, the DSn was 90% and the DSp ranged from 93.3% to 99.1%. The xMAP can detect the immune response to SP and NSP as early as 4 days postinfection and 8 days postinfection, respectively. Furthermore, the SP and NSP antibodies in all 15 vaccinated but unprotected pigs were detected by xMAP. A comparison of SP and NSP antibodies detected in the sera of the infected samples indicated that the results from the xMAP had a high positive correlation with results from the VNT and a 3ABC polypeptide blocking ELISA assay. However, simultaneous quantitation detected that xMAP had no relationship with the VNT. Furthermore, the specificity was 93.3-94.9% with 3ABC polypeptide blocking ELISA for the FMDV-NSP antibody. CONCLUSION The results indicated that xMAP has the potential to detect antibodies to FMDV-SP-VP1 and NSP-3ABC and to distinguish FMDV-infected pigs from pigs infected with the swine vesicular disease virus.

First Finding of Southeast Asia Topotype of Foot-and-Mouth Disease Virus in Kinmen, Taiwan, in the 2012 Outbreak

ABSTRACT Foot-and-mouth disease virus, a member of genus Aphthovirus within the family Picornaviridae, affects cloven-hoofed animals, causing foot-and-mouth disease characterized by vesicle development. The Southeast Asia topotype, one of the topotypes within serotype O of the virus, is prevalent in some Asian countries, but had not previously been found in Taiwan. The topotype was first found in pigs in Kinmen Island, Taiwan, in 2012 and identified by nucleotide sequence comparison and phylogenetic analysis. Outbreaks were reported at 4 farms, resulting in the culling of 628 pigs and 1 cattle. Pigs were the only species infected during the outbreak. The incursion of Southeast Asia topotype into Taiwan implies the expansion of the topotype in East Asia.