Molecular epidemiology of porcine reproductive and respiratory syndrome viruses isolated from 1991 to 2013 in Taiwan.

Porcine reproductive and respiratory syndrome virus (PRRSV) was first identified in Taiwan in 1991, but the genetic diversity and evolution of PRRSV has not been thoroughly investigated over the past 20 years. The aim of this study was to bridge the gap in understanding of its molecular epidemiology. A total of 31 PRRSV strains were collected and sequenced. The sequences were aligned using the MUSCLE program, and phylogenetic analysis were performed by the maximum-likelihood method and the neighbor-joining method using MEGA 5.2 software. In the early 1990s, two prototype strains, WSV and MD001 of the North American genotype, were first identified. Over the years, both viruses evolved separately. The population dynamics of PRRSV revealed that the strains of the MD001 group were predominant in Taiwan. Evolution was manifested in changes in the nsp2 and ORF5 genes. In addition, a suspected newly invading exotic strain was recovered in 2013, suggesting that international spread is still taking place and that it is affecting the population dynamics. Overall, the results provide an important basis for vaccine development for the control and prevention of PRRS.

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.

Development of a reverse transcription multiplex real-time PCR for the detection and genotyping of classical swine fever virus.

A reverse transcription multiplex real-time PCR (RT-MRT-PCR) was developed for rapid detection and genotyping of classical swine fever virus (CSFV). The universal primers and specific TaqMan probes for each of the three genotypes, genotypes 1, 2, and 3, were designed within the 3'-UTR of the CSFV. Non-CSFV swine virus and clinical samples from specific pathogen-free (SPF) pigs were both demonstrated to be CSFV-negative by RT-MRT-PCR. The diagnostic sensitivity of RT-MRT-PCR was determined to be 1 viral copy/microl for each genotype of standard plasmid. For the analytical sensitivity experiment, 100 samples of 14 CSFV genotype 1 strains and 86 samples from CSFV outbreak farms were all detected as CSFV-positive by RT-MRT-PCR, and the genotype results were consistent with the results of sequencing from a previous study. The intra-assay and inter-assay variations of RT-MRT-PCR were below 3% in all experiments. The sensitivity of RT-MRT-PCR was the same as the reverse transcription nested PCR (RT-nPCR) and higher than reverse transcription PCR (RT-PCR) and viral isolation from clinical samples. This assay was used further to evaluate the duration of viremia of wild-type CSFV in vaccinated exposed pigs. The results indicated that pigs vaccinated with the E2 subunit vaccine had longer viremia than pigs given the C-strain vaccine, which is compatible with the findings of previous studies. Thus, the new RT-MRT-PCR is a rapid, reproducible, sensitive, and specific genotyping tool for CSFV detection.

Rapid detection and differentiation of wild-type and three attenuated lapinized vaccine strains of classical swine fever virus by reverse transcription polymerase chain reaction.

A simple one-step reverse transcription polymerase chain reaction (RT-PCR) method was developed based on T-rich insertions in the viral genome for simultaneous detection and differentiation of wild type and vaccine strains of Classical swine fever virus (CSFV). The CSFV-specific primers were designed to contain the sequences of the T-rich insertion sites that exist uniquely in the 3' nontranslated regions (3' NTR) of the genome of lapinized CSFV vaccine strains. By using a one-step RT-PCR or a nested PCR followed by an agarose gel electrophoresis or a multicapillary electrophoresis, the wild-type and lapinized vaccine strains of CSFV in clinical samples could be detected and accurately distinguished. These assays can be applied to at least 3 attenuated lapinized vaccine strains, lapinized Philippines Coronel (LPC), hog cholera lapinized virus (HCLV), and Chinese strain (C strain). The detection limit of the wild-type virus was 6.3 TCID(50) (50% tissue culture infective dose)/ml for RT-PCR and 0.63 TCID(50)/ml for nested PCR. In previous studies, notable T-rich insertions of 12-13 nucleotides (nt) were found in the 3' NTR of the genome of lapinized vaccine strains of CSFV. However, this study discovered that 2 T-rich insertions, 42 and 36 nt in length, are present in the viral genome of lapinized vaccine strains LPC/PRK (primary rabbit kidney) and LPC/TS (Tam-Sui), respectively. These T-rich insertions of 12, 36, and 42 nt length increases the size of PCR fragments, which are favorable genetic markers for rapid detection of and differentiation between wild-type and different lapinized vaccine strains of CSFV.

Genetic variation in open reading frame 5 gene of porcine reproductive and respiratory syndrome virus in Taiwan.

In an effort to understand the genetic variation in porcine reproductive and respiratory syndrome virus (PRRSV) isolates in Taiwan, 40 isolates obtained between 2004 and 2006 were analyzed for their sequences of open reading frame 5. After reverse transcription polymerase chain reaction, the amplified open reading frame 5 fragments were analyzed by restriction fragment length polymorphism and sequence comparison. The results showed that all the Taiwanese isolates belonged to the North American genotype. Multiple patterns obtained from restriction fragment length polymorphism, 83-99% nucleotide similarity and 84-99% deduced amino acid similarity suggested a high level of genetic variation and PRRSV was not a single invasion to Taiwan. Moreover, vaccine-like isolates were isolated from the field, implying that some field isolates might originate from vaccine virus.

Study on the porcinophilic foot-and-mouth disease virus I. production and characterization of monoclonal antibodies against VP1.

Monoclonal antibodies (MAbs) reported here were produced against the porcinophilic foot-and-mouth disease virus (FMDV) that caused the devastating swine disease on 1997 in Taiwan. A panel (25) of MAbs were found to react with VP1 of O/Taiwan/97 (O/97) by ELISA with various potencies. The biological identities of these VP1 reacting MAbs, such as neutralization activity, isotype and capability to distinguish between two serotype O FMDVs, O/97 and O/Taiwan/KM1/99 (O/99), were further analyzed. Eleven out of the total eighteen O/97 neutralizing MAbs were able to neutralize heterologous O/99. Eight O/97 neutralizing and five non-neutralizing MAbs could differentiate two serotype O FMDVs by immunofluorescence assay (IFA) implied that these thirteen MAbs recognized O/97 specific epitope(s). Furthermore, reactivities of the VP1 reacting MAbs with a 29 amino acids synthetic peptide (P29) representing the betaG-betaH loop of VP1 were analyzed by ELISA and fourteen were found positive. MAb clone Q10E-3 reacting strongest with VP1 and P29, neutralizing both but not differentiating two serotype O viruses suggested that the antibody binding site might involve the RGD motif and its C terminal conserved region on betaG-betaH loop. MAbs with diverse characters presented in this study were the first raised against porcinophilic FMDV. The complete set of MAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.

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.

Characterization of porcine circovirus type 2 in Taiwan.

In an effort to understand the genetic diversity of porcine circovirus type 2 (PCV2) and the prevalence of PCV2 infection in Taiwanese herds, we have sequenced the complete genomes from PCV2-infected specimens and individually measured the antibody titer against PCV2 from pigs reared in Taiwan between the years 2000 and 2002. A total of 623 specimens originating from pigs displaying varied clinical signs were screened with the polymerase chain reaction (PCR). Results showed that 309 pigs (49.6%) tested positive for PCV2. Eight of the positive specimens were used for the amplification of the complete viral genome. Sequence comparison of the complete genomes indicated that the 8 Taiwanese PCV2 isolates shared 95-99% similarity. Phylogenetic analysis of all 40 PCV2 isolates from North America, Europe, Asia and Taiwan revealed that those isolates were grouped together in one large group containing two minor subgroups. The Taiwanese PCV2 isolates were classified into the two minor subgroups. The prevalence of serum antibodies to PCV2 in pigs was investigated, and results showed that approximately 83.5% of the pigs in Taiwan were seropositive. Finishing pigs possess the highest titers of antibodies, while 9-week-old pigs contained the lowest titers for specific antibodies. Our results suggest that PCV2 infections have become common in Taiwanese pig farms.

Natural infections of pigs with akabane virus.

Akabane (AKA) virus is considered a pathogen of herbivores in nature. However, we found that pig populations in fields were infected in Taiwan. An isolate (NT-14) of AKA virus was obtained from pigs. The NT-14 virus was able to infect pigs by the oronasal route. Subsequently, low levels of infectious virus particles were excreted into the oronasal discharge during the stage of viremia but they were not sufficient to infect new porcine hosts via contact transmission. The prevalence of serum neutralizing antibodies to AKA virus in pig populations was investigated, indicating that approximately 75% of pigs in Taiwan were seropositive. Sows and newborn piglets have the highest titers of neutralizing antibodies. Contrarily, fattening pigs aged at approximately 20 weeks old contained the lowest titers of specific antibodies. Our results suggest that pigs in natural situations are part of the AKA virus transmission cycle.