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.

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.

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.

First finding of Southeast Asia topotype of foot-and-mouth disease virus in Kinmen, Taiwan, in the 2012 outbreak.

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.

Comparison of primer sets and one-step reverse transcription polymerase chain reaction kits for the detection of bluetongue viral RNA.

Bluetongue virus is the etiological agent of bluetongue, one of the most important insect-transmitted animal diseases in the world. To establish a feasible diagnostic procedure for detecting the viral RNA, seven commercially available one-step RT-PCR kits in combination with three primer sets were evaluated. Results of this study showed remarkable differences in analytical sensitivity between the examined RT-PCR kits. In addition, it was found that a World Organization for Animal Health-recommended primer set may not be effective in detecting most BTV RNA.

Development and evaluation of a loop-mediated isothermal amplification method for rapid detection and differentiation of two genotypes of porcine circovirus type 2.

BACKGROUND: Porcine circovirus type 2 (PCV2) is one of the major swine viral diseases and has caused significant economic loss to pig producers. PCV2 has been divided into two major genotypes: PCV2a, PCV2b. A loop-mediated isothermal amplification (LAMP) method was developed for the detection and differentiation of PCV2a and PCV2b in clinical samples. METHODS: LAMP-specific primer sets were designed based on six PCV2a and six PCV2b reference isolates. To determine the analytical specificity of LAMP, DNA samples extracted from 36 porcine virus isolates were tested by LAMP, including eight PCV2a, 11 PCV2b, four PCV type 1, two porcine parvovirus, three pseudorabies virus, and eight porcine reproductive and respiratory virus. To evaluate the analytical sensitivity of the assay, 10-fold serial dilutions of PCV2a and PCV2b recombinant plasmids were performed to prepare the dilutions at concentration from 10(6) to 1 copy(ies)/µL, and each dilution was tested by both LAMP and nested polymerase chain reaction (nested PCR). A total of 168 clinical samples were analyzed by both LAMP and nested PCR, and the relative sensitivity and specificity of LAMP compared to nested PCR were calculated. RESULTS: Using different primer sets of LAMP, LAMP could be completed within 50 minutes. This method was found to be highly analytically specific for PCV2a and PCV2b; only the target gene was detected without cross-reaction. The analytical sensitivity of LAMP for PCV2a and PCV2b were 10 copies/µL, demonstrating analytical sensitivity comparable to that obtained using nested PCR. In addition, the sensitivity and specificity of LAMP relative to those of nested PCR were 97.7% and 100.0%, respectively. The percentage of observed agreement was 98.2%, and the κ statistic was 0.949. CONCLUSION: LAMP is a rapid, specific, and sensitive diagnostic method for the detection and differentiation of PCV2a and PCV2b in clinical samples.

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

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.

Prevalence and genetic variation of porcine circovirus type 2 in Taiwan from 2001 to 2011.

Porcine circovirus type 2 (PCV2) is the major causative agent of postweaning multisystemic wasting syndrome (PMWS) in Taiwanese pig farms. We analyzed the complete genomes of 571 Taiwanese PCV2 isolates in Taiwan from 2001 to 2011 and divided the isolates into 2 distinct genotypes (PCV2a and PCV2b) with 6 clusters (1A, 1B, 1C, 2B, 2D, and 2E). Of the 571 Taiwanese PCV2 isolates, 22.9% (131/571) belonged to PCV2a and 77.1% (440/571) to PCV2b. In this study, PCV2a isolates were the most common in 2001, and then PCV2b isolates became predominate thereafter and widely distributed in pig farms since 2003. Sequence comparisons among the 571 isolates indicated that 89.6-100% had nucleotide identity for complete genome and 87.3-100% for open reading frames 2 (ORF2). The results suggest that a higher genetic variation and shift occurred among PCV2 isolates collected from 2001 to 2011 in Taiwan.

Identification of conformational epitopes and antigen-specific residues at the D/A domains and the extramembrane C-terminal region of E2 glycoprotein of classical swine fever virus.

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. Previous studies revealed that both conformation-dependent and linear epitopes are most present within domains B/C/D/A in the N-terminal half of E2. However, studies of antigenicity beyond the B/C domains remain limited. This study revealed that conformational epitopes were present on the D/A domains as well as the proximal C-terminal of E2, since the mutation of cysteine abrogated their bindings to monoclonal antibodies (mAbs). The residue R845 at domain A and E902 at the C-terminal region were critical for specific binding to mAbs, further supporting the presence of antigenic determinants on these regions. Substitutions of cysteines in domains D/A not only abrogated the binding to mAbs directed to D/A, but also affected the binding of the downstream C-terminal region to its specific mAbs, suggesting a close interaction between the two conformational epitopes. Mutations on the five proximal cysteines at positions 869, 877, 893, 896 and 930 in the C-terminal region only affected the binding to its specific mAbs binding sites. In addition, mutation on the three distal C-terminal cysteines at positions 945, 966, and 983 resulted in loss of E2 homodimerization. This study demonstrates new antigenic epitopes on D/A domains and C-terminal of E2 that have not been reported before, and that the nine cysteines in the C-terminal function differently in either maintaining the antigenic structure or in intermolecular dimerization of E2.

Antigenic mimicking with cysteine-based cyclized peptides reveals a previously unknown antigenic determinant on E2 glycoprotein of classical swine fever virus.

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. The conformational epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779 (Chang et al., 2010a). To mimic the conformational epitopes, a set of synthetic cyclized peptides spanning the B/C domains of E2 were used to react with monoclonal antibodies (mAbs) against E2 and with swine anti-CSFV polyclonal sera. All antibodies recognized a highest common element, (753)RYLASLHKKALPTSV(767), on the double-looped peptides. This epitope region has not been revealed previously in the literature. Both substitution-scanning of residues (753)RYLASLHKKALPTSV(767) on a double-looped peptide and site-directed mutagenesis of expressed E2 demonstrated that residues (761)K, (763)L and (764)P were critical for the reactivity with mAbs. In addition, the up- and downstream residues (753)R, (754)Y, (755)L and (765)T were also crucial. Alignment showed that this stretch of amino acids was relatively conserved among various CSFVs. Thus, we identified a motif (753)RYLASLHKKALPT(765), which may be part of group-specific antigen and important for the structural integrity of conformational epitope recognition.

Detection of porcine circovirus type 1 in commercial porcine vaccines by loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1. No cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. The analytical sensitivity of the LAMP for PCV1 DNA was 10 copies/µl in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction (nested PCR). Furthermore, 25 commercial swine vaccines were tested by both the LAMP and the nested PCR, and three of them were tested positive for PCV1 DNA. These results indicate that PCV1 DNA can be real-time detected by the LAMP; the method was highly specific, sensitive, and rapid for the detection of PCV1 DNA, particularly in commercial swine vaccines.

Differentiation of foot-and-mouth disease-infected pigs from vaccinated pigs using antibody-detecting sandwich ELISA.

The presence of serum antibodies for nonstructural proteins of the foot-and-mouth disease virus (FMDV) can differentiate FMDV-infected animals from vaccinated animals. In this study, a sandwich ELISA was developed for rapid detection of the foot-and-mouth disease (FMD) antibodies; it was based on an Escherichia coli-expressed, highly conserved region of the 3ABC nonstructural protein of the FMDV O/TW/99 strain and a monoclonal antibody derived from the expressed protein. The diagnostic sensitivity of the assay was 98.4%, and the diagnostic specificity was 100% for naïve and vaccinated pigs; the detection ability of the assay was comparable those of the PrioCHECK and UBI kits. There was 97.5, 93.4 and 66.6% agreement between the results obtained from our ELISA and those obtained from the PrioCHECK, UBI and CHEKIT kits, respectively. The kappa statistics were 0.95, 0.87 and 0.37, respectively. Moreover, antibodies for nonstructural proteins of the serotypes A, C, Asia 1, SAT 1, SAT 2 and SAT 3 were also detected in bovine sera. Furthermore, the absence of cross-reactions generated by different antibody titers against the swine vesicular disease virus and vesicular stomatitis virus (VSV) was also highlighted in this assay's specificity.

Genetic and antigenic characterization of foot-and-mouth disease viruses isolated in Taiwan between 1998 and 2009.

A devastating outbreak of foot-and-mouth disease (FMD), caused by a porcinophilic serotype O virus, occurred in Taiwan in March 1997. This outbreak was brought under control by means of a stamping-out policy and vaccination. Although mandatory vaccination was conducted in Taiwan between 1997 and 2007, sporadic outbreaks of FMD occurred between 1998 and 2009; however, the viruses that caused these outbreaks remain uncharacterized. This article reports the genetic and antigenic characterization of FMD viruses isolated in Taiwan during this period. Sequence analysis of the VP1 coding region showed that the viruses isolated in Taiwan between 1998 and 2009 were most similar to viruses isolated in Taiwan in 1997 and to viruses isolated from Hong Kong and Vietnam in 1991-1996. The results of phylogenetic analysis suggested that the viruses isolated in Taiwan in 1998-2009 were derived from the viruses isolated in Taiwan in 1997. However, substantial mutations were found in the viruses isolated in 2009, and some of these changes may have resulted from vaccine pressure in the field. Serum neutralization tests confirmed that viruses isolated in 2009 showed a significant change in antigenicity. This is the first report of changes in the VP1 sequence and antigenicity of porcinophilic FMD viruses isolated from an area in which long-term mandatory vaccination against FMD was practiced.

Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes.

BACKGROUND: Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV), that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. METHODS: We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s) of the capsid protein VP1 of foot-and-mouth disease virus (FMDV). The recombinant BaMV plasmid (pBVP1) was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164) of FMDV VP1. RESULTS: The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-gamma. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. CONCLUSION: Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

Comparison of ELISA for the detection of porcine serum antibodies to non-structural proteins of foot-and-mouth disease virus.

Three foot-and-mouth disease virus non-structural protein antibody detection kits, CHEKIT FMD-3ABC, UBI FMD NS EIA and DVIVR NSP ELISA, were compared in the study. The results showed that the specificity of the kits ranged from 96.7 to 100% in nai;ve pigs and from 93.6 to 98.1% in vaccinated pigs, and that the DVIVR kit had the highest analytical sensitivity. The kappa statistics for the detection of 612 sera were 0.582, 0.447 and 0.658 for CHEKIT/UBI, CHEKIT/DVIVR and UBI/DVIVR, respectively. This study also revealed that measurable non-structural protein specific antibodies in some of infected pigs were sustained either for shorter periods or in intermittent patterns, thus aggravating the difficulties associated with the removal of pre-exposed pigs in the field.

Induction of immunity in swine by purified recombinant VP1 of foot-and-mouth disease virus.

VP1, a capsid protein of foot-and-mouth disease virus (FMDV), contains neutralizing epitopes of the virus. Due to its poor water solubility, recombinant Escherichia coli derived VP1 (rVP1) has previously been used mainly in a denatured form and is not well characterized. Here, using SDS to assist protein refolding and then removing SDS with a detergent removing column, we have successfully purified rVP1 in two aqueous-soluble forms, i.e. monomer and dimer. Studies showed that dimerization occurs by an inter-molecular disulfide bond between two cysteine residues at position 187 of each monomer. Heat treatment revealed that rVP1 dimer exhibited a more thermal-stable conformation than the monomeric form. Both monomeric and dimeric rVP1 reacted with anti-FMDV antibodies. Immunization studies demonstrated that vaccination of swine with either forms of rVP1 was effective in generating immune responses and protecting them from viral challenge.

Effective synthetic peptide vaccine for foot-and-mouth disease in swine.

We have designed a peptide-based vaccine for foot-and-mouth disease (FMD) effective in swine. The peptide immunogen has a G-H loop domain from the VP1 capsid protein of foot-and-mouth disease virus (FMDV) and a novel promiscuous T helper (Th) site for broad immunogenicity in multiple species. The G-H loop VP1 site was optimised for cross-reactivity to FMDV by the inclusion into the peptide of cyclic constraint and adjoining sequences. The incorporation of consensus residues into the hypervariable positions of the VP1 site provided for broad immunogenicity. The vaccine protected 20 out of 21 immunised pigs from infectious challenge by FMDV O1 Taiwan using peptide doses as low as 12.5 microg, and a mild adjuvant that caused no lesions. A safe chemically-defined product would have considerable advantages for vaccination against FMD.

Anti-3AB antibodies in the Chinese yellow cattle infected by the O/Taiwan/99 foot-and-mouth disease virus.

The O/Taiwan/99 foot-and-mouth disease virus (FMDV), a South Asian topotype of serotype O, was introduced into Taiwan in 1999. The Chinese yellow cattle infected by the virus did not develop clinical lesions under experimental and field conditions. A blocking enzyme-linked immunosorbent assay (ELISA) kit with the 3AB antigen, a polypeptide of FMDV non-structural (NS) proteins, was used to evaluate the development and duration of anti-3AB antibodies, proving active viral replication, in the Chinese yellow cattle. The specificity of the assay was 99%, as was established with negative sera from regularly vaccinated and from naïve cattle. The sensitivity tested with sera from naturally infected animals was approximately 64% and it was lower than that obtained by serum neutralization (SN) test. Under experimental infection, the Chinese yellow cattle developed lower anti-3AB antibodies than that developed in other species. Duration of anti-3AB antibodies was traced in two herds of naturally infected animals, indicating that anti-3AB antibodies persisted for approximately 6 months after outbreaks. On the basis of this study, we propose that the Chinese yellow cattle may have natural resistance, which limits viral replication and reduces the development of anti-3AB antibodies.