Development and validation of indirect ELISA for antibody detection against different protein antigens of porcine epidemic diarrhea virus in the colostrum and milk of sows.

The objectives of this study are to develop and optimize indirect ELISA based on three coating antigens of porcine epidemic diarrhea virus (PEDV), recombinant spike (S12), nucleocapsid (N), and whole viral (WV) proteins, for the detection of IgG and IgA antibodies in colostrum and milk and to evaluate the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay as a diagnostic method. Colostrum (n = 347) and milk (n = 272) samples from sows were employed in this assay. Indirect ELISA based on three coating antigens was assessed by receiver operating characteristic (ROC) curve analysis with a virus neutralization (VN) test as a reference method, and the cutoff value for calculating DSe and DSp was determined. S12-ELISA showed higher DSe and DSp of IgG and IgA detection compared to N- and WV-ELISA in both colostrum and milk samples. Moreover, S12-ELISA showed perfect agreement and a high correlation with the VN test, which was better than the N- and WV-ELISA for both IgG and IgA detection in colostrum and milk. In contrast, N-ELISA showed lower DSe and DSp compared to S12- and WV-ELISA, along with a correlation with VN and substantial agreement with the VN test. Nevertheless, our developed ELISAs have accuracy for repeatability in both inter- and intra-assay variation. Overall, this research demonstrates that S12-ELISA is more suitable than WV- and N-ELISA to detect IgG and IgA antibodies against PEDV from both colostrum and milk samples.

Evolutionary and epidemiological analyses based on spike genes of porcine epidemic diarrhea virus circulating in Thailand in 2008-2015.

Porcine epidemic diarrhea (PED) has been endemic causing sporadic outbreaks in Thailand since 2007. In 2014-2015, several herds had experienced severe PED outbreaks and the reason of the re-current outbreaks was unknown. Whether or not the introduction of exotic strains or continual evolution of existing PEDV, genetic analyses would provide a more understanding in its evolutionary pattern. In the study, 117 complete spike gene sequences of Thai PED virus (PEDV) collected from 2008 to 2015 were clustered along with 95 references of PEDV spike sequences, and analyzed with the US sequences dataset (n=99). The phylogenetic analysis demonstrated that Thai PEDV spike sequences were genetically diverse and had been influenced by multiple introduction of exotic strains. Although Thai PEDV have been evolved into 6 subgroups (TH1-6), Subgroup TH1 strains with the unique 9 nucleotides (CAA GGG AAT) insertion between 688th-689th position of spike (changing amino acid from N to TREY) insertion has become the dominant subgroup since 2014. Thai PEDV spike gene have higher evolutionary rate compare to that of the US sequences. One contributing factor would be the intra-recombination between subgroups. Thailand endemic strain should be assigned into new subclade of G2 (Thai pandemic variant).

The genetic diversity and complete genome analysis of two novel porcine deltacoronavirus isolates in Thailand in 2015.

Porcine deltacoronavirus (PDCoV) was identified in intestinal samples collected from piglets with diarrhea in Thailand in 2015. Two Thai PDCoV isolates, P23_15_TT_1115 and P24_15_NT1_1215, were isolated and identified. The full-length genome sequences of the P23_15_TT_1115 and P24_15_NT1_1215 isolates were 25,404 and 25,407 nucleotides in length, respectively, which were relatively shorter than that of US and China PDCoV. The phylogenetic analysis based on the full-length genome demonstrated that Thai PDCoV isolates form a new cluster separated from US and China PDCoV but relatively were more closely related to China PDCoV than US isolates. The genetic analyses demonstrated that Thai PDCoVs have 97.0-97.8 and 92.2-94.0% similarities with China PDCoV at nucleotide and amino acid levels, respectively, but share 97.1-97.3 and 92.5-93.0 similarity with US PDCoV at the nucleotide and amino acid levels, respectively. Thai PDCoV possesses two discontinuous deletions of five amino acids in ORF1a/b region. One additional deletion of one amino acid was identified in P23_15_TT_1115. The variation analyses demonstrated that six regions (nt 1317-1436, 2997-3096, 19,737-19,836, 20,277-20,376, 21,177-21,276, and 22,371-22,416) in ORF1a/b and spike genes exhibit high sequence variation between Thai and other PDCoV. The analyses of amino acid changes suggested that they could potentially be from different lineages.

Humoral immune responses and viral shedding following vaccination with modified live porcine reproductive and respiratory syndrome virus vaccines.

The antibody response and pattern of shedding of vaccine virus following vaccination with modified live genotype I or II porcine reproductive and respiratory syndrome virus (PRRSV) vaccines (MLVs) were investigated. Ninety PRRSV-free pigs were divided randomly seven, groups including the NEG, EU1, EU2, US1, US2, US3 and US4 groups. The NEG group was unvaccinated. The EU1, EU2, US1, US2, US3 and US4 groups were vaccinated with the following MLVs: AMERVAC® PRRS, Porcillis® PRRS, Fostera™ PRRS, Ingelvac® PRRS MLV, Ingelvac® PRRS ATP, and PrimePac™ PRRS+ , respectively. Sera were quantitatively assayed for viral RNA using qPCR. Antibody responses were measured using Idexx ELISA and serum neutralization (SN). Shedding of vaccine virus was investigated using sentinel pigs and by detection of viral RNA in tonsil scrapings. Antibody responses were detected by ELISA at 7-14 days post-vaccination (DPV) and persisted at high titers until 84 DPV in all MLV groups. The SN titers were delayed and isolate-specific. SN titers were higher for the homologous virus than for heterologous viruses. Age-matched sentinel pigs introduced into the EU2, US2 and US3 groups at 60 DPV seroconverted. In contrast, sentinel pigs introduced at 84 DPV remained negative in all of the MLV groups. Vaccine viral RNA was detected in tonsil scrapings from the EU2, US2 and US3 groups at 84-90 DPV. No viral RNA was detected beyond 70 DPV in the EU1, US1 and US4 groups. In conclusion, all MLV genotypes induced rapid antibody responses, which were measured using ELISA. The development of SN antibodies was delayed and isolate-specific. However, the shedding pattern was variable and depended on the by virus isolate used to manufacture the vaccine.

The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR.

Porcine deltacoronavirus (PDCoV) has been reported in many countries, including Hong Kong, the United States, South Korea, China and Thailand. In January 2016, clinical diarrhea similar to that of porcine epidemic diarrhea virus (PEDV) with a lower mortality rate was reported on a swine farm in Lao PDR. Intestine samples were collected from 3-day-old pigs with clinical diarrhea and assayed for the presence of swine enteric coronaviruses. The PCR results were positive for PDCoV but negative for PEDV and TGEV. A phylogenetic tree demonstrated that PDCoV from Lao PDR was grouped separately from PDCoV isolates from China and the USA, but was more closely related to the Chinese isolates than to the US isolates. The full-length genome sequence of the novel PDCoV isolate P1_16_BTL_0116 was determined.

Full-length genome analysis of two genetically distinct variants of porcine epidemic diarrhea virus in Thailand.

Porcine epidemic diarrhea virus (PEDV) has continued to cause sporadic outbreaks in Thailand since 2007 and a pandemic variant containing an insertion and deletion in the spike gene was responsible for outbreaks. In 2014, there were further outbreaks of the disease occurring within four months of each other. In this study, the full-length genome sequences of two genetically distinct PEDV isolates from the outbreaks were characterized. The two PEDV isolates, CBR1/2014 and EAS1/2014, were 28,039 and 28,033 nucleotides in length and showed 96.2% and 93.6% similarities at nucleotide and amino acid levels respectively. In total, we have observed 1048 nucleotide substitutions throughout the genome. Compared to EAS1/2014, CBR1/2014 has 2 insertions of 4 ((56)GENQ(59)) and 1 ((140)N) amino acid positions 56-59 and 140, and 2 deletions of 2 ((160)DG(161)) and 1 ((1199)Y) amino acid positions 160-161 and 1199. The phylogenetic analysis based on full-length genome of CBR1/2014 isolate has grouped the virus with the pandemic variants. In contrast, EAS1/2014 isolate was grouped with CV777, LZC and SM98, a classical variant. Our findings demonstrated the emergence of EAS1/2014, a classical variant which is novel to Thailand and genetically distinct from the currently circulating endemic variants. This study warrants further investigations into molecular epidemiology and genetic evolution of the PEDV in Thailand.

Complete Genome Sequence of Porcine Deltacoronavirus Isolated in Thailand in 2015.

In Thailand, porcine deltacoronavirus (PDCoV) was first identified in November 2015. The virus was isolated from piglets experiencing diarrhea outbreak. Herein, the full-length genome sequence of the Thai PDCoV isolate P23_15_TT_1115 is reported. The results provide a clearer understanding of the molecular characteristics of PDCoV in Thailand.

Complete Genome Sequences of Two Genetically Distinct Variants of Porcine Epidemic Diarrhea Virus in the Eastern Region of Thailand.

Porcine epidemic diarrhea virus (PEDV) has continued to cause sporadic outbreaks in Thailand since 2007. Previously, PEDV in Thailand was a new variant containing an insertion and deletion in the spike gene. Herein, full-length genome sequences are reported for two variants of PEDV isolates from pigs displaying diarrhea in Thailand.

Complete genome characterization of porcine epidemic diarrhea virus in Vietnam.

Porcine epidemic diarrhea virus (PEDV) first emerged in Vietnam in 2009. In this study, the complete genomes of three Vietnamese PEDV isolates were characterized. These three isolates were isolated from 3-day-old pigs experiencing diarrhea. Two isolates were from swine farms in the south, and the other was from northern Vietnam. The whole genome sequences of these isolates are 28,035 nucleotides in length and have characteristics similar to those of other PEDV isolates. All three Vietnamese PEDV isolates share 99.8 % and 99.6 % sequence identity at the nucleotide and amino acid level, respectively, and have insertions of four amino acids (GENQ) and one amino acid (N) at positions 56-59 and 140, respectively, and one deletion of two amino acids (DG) at positions 160-161. Phylogenetic analysis based on the whole genome revealed that the three Vietnamese PEDV isolates are grouped together with new variants from China from 2011 to 2012 and are genetically distinct from US isolates and the classical PEDV variant. The results suggest that Vietnamese PEDV isolates are new variants, as evidenced by their genetic composition of insertions and a deletion in the spike gene, and they might have originated from the same ancestor as the Chinese PEDV strain. This study provides a better understanding of the molecular characteristics of PEDV in Vietnam.

Dynamics and evolution of highly pathogenic porcine reproductive and respiratory syndrome virus following its introduction into a herd concurrently infected with both types 1 and 2.

Since its first emergence in Thailand in late 2010, highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) has caused sporadic outbreaks on Thai swine farms. The objective of this study was to investigate the dynamics and evolution of PRRSV in a herd experiencing an HP-PRRSV outbreak. Following its introduction, HP-PRRSV caused severe outbreaks and subsequently established persistent infection in the herd, resulting in the emergence of a novel cluster of type 2 (North American, NA) isolates. HP-PRRSV co-existed with type 1 (European, EU) isolates without influencing their development. In contrast, HP-PRRSV influenced the evolution of the type 2 (NA) isolates by increasing diversity through the addition of a novel cluster and influencing the evolution of other viral clusters previously existing in the herd. Recombination between the endemic and emerging isolates was observed. The recombinants, however, disappeared and were not able to survive in the herd. The results of this study suggest that the introduction of HP-PRRSV to a herd results in an increased diversity of genetically related isolates and persistent HP-PRRSV infection.

Genetic diversity of ORF3 and spike genes of porcine epidemic diarrhea virus in Thailand.

Porcine epidemic diarrhea virus (PEDV) has become endemic in the Thai swine industry, causing economic losses and repeated outbreaks since its first emergence in 2007. In the present study, 69 Thai PEDV isolates were obtained from 50 swine herds across Thailand during the period 2008-2012. Both partial and complete nucleotide sequences of the spike (S) glycoprotein and the nucleotide sequences of ORF3 genes were determined to investigate the genetic diversity and molecular epidemiology of Thai PEDV. Based on the analysis of the partial S glycoprotein genes, the Thai PEDV isolates were clustered into 2 groups related to Korean and Chinese field isolates. The results for the complete spike genes, however, demonstrated that both groups were grouped in the same cluster. Interestingly, both groups of Thai PEDV isolates had a 4-aa (GENQ) insertion between positions 55 and 56, a 1-aa insertion between positions 135 and 136, and a 2-aa deletion between positions 155 and 156, making them identical to the Korean KNU series and isolates responsible for outbreaks in China in recent years. In addition to the complete S sequences, the ORF3 gene analyses suggested that the isolates responsible for outbreaks in Thailand are not vaccine related. The results of this study suggest that the PEDV isolates responsible for outbreaks in Thailand since its emergence represent a variant of PEDV that was previously reported in China and Korea.

Dynamics and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 following modified live PRRSV vaccination in a PRRSV-infected herd.

The objective of this study was to investigate the dynamics and evolution of porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 following the use of a modified live PRRSV (MLV) vaccine. A PRRSV-positive farm with coexistence of types 1 and 2 and no history of MLV vaccination was investigated. Vaccination with a type 2 MLV (Ingelvac PRRS MLV, Boehringer Ingelheim, USA) was implemented. All sows were vaccinated at monthly intervals for two consecutive months and then every third month. Piglets were vaccinated once at 7-10 days of age and weaned to nursery facilities at 21-23 days of age. Serum samples were collected monthly before and after vaccination from four population groups, including replacement gilts and suckling, nursery and finishing pigs, and assayed by PCR. After a year of blood collection, amplified products were sequenced, resulting in 277 complete ORF5 gene sequences from 145 type 1 and 132 type 2 isolates. Prior to and following vaccination, both type 1 and type 2 PRRSV were isolated and found to coexist in an individual pig. Each genotype evolved separately without influencing the strain development of the other. Although the substitution rates of both genotypes were relatively similar, MLV vaccination appears to increase the heterogenicity of type 2 PRRSV, resulting in the emergence of three novel type 2 PRRSV clusters in the herd, including an MLV-like cluster, which disappeared within the month following whole-herd vaccination. Two additional clusters included one related to the MLV vaccine and one related to the endemic cluster of the herd.

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus (PRRSV) genotypes I and II in Thailand.

To investigate the genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) in Thailand, 279 ORF5 sequences of PRRSV collected during 2010-2011 from 102 swine herds in five swine-producing areas were analyzed. The co-existence of European (EU) and North American (NA) genotypes was observed in 98 % of herds investigated and was evident at the pig level. Both genotypes have evolved separately with a temporal influence on strain development. Novel introductions influence the genetic diversity of the NA genotype. Although Thai EU and NA isolates develop their own clusters that are separate from those of other countries, there was no geographic influence on strain development within Thailand.

Porcine reproductive and respiratory syndrome virus, Thailand, 2010-2011.

Characterization of porcine reproductive and respiratory syndrome virus (PRRSV) isolates from pigs in Thailand showed 30-aa discontinuous deletions in nonstructural protein 2, identical to sequences for highly pathogenic PRRSV. The novel virus is genetically related to PRRSV from China and may have spread to Thailand through illegal transport of infectious animals from bordering countries.