Angkana Tantituvanont

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.

Pharmacokinetics of ceftiofur hydrochloride in pigs infected with porcine reproductive and respiratory syndrome virus

OBJECTIVES To compare the pharmacokinetic profile of ceftiofur hydrochloride (ceftiofur) administered intramuscularly at 3 mg/kg body weight (BW) in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV) versus clinically healthy pigs. METHODS Sixteen 3- to 4-week-old PRRSV-negative pigs were randomly assigned to two groups (A and B), with eight pigs per group. Pigs in Group A were uninfected controls and pigs in Group B were intranasally challenged with a PRRSV isolate of Thai origin. Pigs in both groups were intramuscularly administered ceftiofur at 3 mg/kg BW at 7 days post-infection. Blood samples were serially collected up to 72 h post-injection. Plasma was analysed for ceftiofur and its related metabolites using HPLC. Pharmacokinetic parameters of ceftiofur were calculated based on non-compartmental analysis. RESULTS Pharmacokinetic parameters of ceftiofur revealed statistically significant differences (P < 0.01) in maximum concentration (C(max)), AUC, volume of distribution at the terminal phase over bioavailability (V(z)/F), clearance over bioavailability (CL/F) and the terminal half-life (t(1/2z)) between Groups A and B. PRRSV-infected pigs had a V(z)/F and CL/F of ceftiofur significantly higher than in the non-infected pigs (116% increase in V(z)/F, 234% increase in CL/F). The C(max) and AUC of the infected pigs decreased by 54% and 70%, respectively, compared with the non-infected pigs. The t(1/2z) of the infected pigs and the non-infected pigs was 13.1 and 21.0 h, respectively. CONCLUSIONS The pharmacokinetic profile of ceftiofur is altered in PRRSV-infected pigs due to the decreased plasma ceftiofur concentration compared with clinically healthy pigs.

Different Lineage of Porcine Deltacoronavirus in Thailand, Vietnam and Lao PDR in 2015.

&NA; Porcine deltacoronavirus (PDCoV) was detected by RT‐PCR in 12 of 97 (12.4%) intestinal samples collected during 2015 from piglets with diarrhoea in Thailand, Vietnam and Lao PDR. Spike, membrane and nucleocapsid genes were characterized, and phylogenetic analyses demonstrated that PDCoV isolates from Thai and Lao PDR form a novel cluster, separated from US and China isolates, but relatively were more closely related to China PDCoV than US isolates. Vietnam PDCoVs, however, were grouped together with US PDCoV. The analyses of amino acid changes suggested that they were from different lineage.

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

ABSTRACT 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.

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.

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

Abstract 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).

Retrospective investigation and evolutionary analysis of a novel porcine deltacoronavirus strain detected in Thailand from 2008 to 2015

Porcine deltacoronavirus (PDCoV) in Thailand was first detected in 2015. We performed a retrospective investigation of the presence of PDCoV in intestinal samples collected from piglets with diarrhea in Thailand from 2008 to 2015 using RT-PCR. PDCoV was found to be present as early as February 2013. Phylogenetic analysis demonstrated that all PDCoV variants from Thailand differ from those from other countries and belong to a novel group of PDCoV that is separate from the US and Chinese PDCoV variants. Evolutionary analysis suggested that the Thai PDCoV isolates probably diverged from a different ancestor from that of the Chinese and US PDCoV isolates and that this separation occurred after 1994.

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

Abstract 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 (56GENQ59) and 1 (140N) amino acid positions 56–59 and 140, and 2 deletions of 2 (160DG161) and 1 (1199Y) 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.

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.

A new cell-to-cell interaction model for epithelial microfold cell formation and the enhancing effect of epidermal growth factor

Abstract The formation of epithelial microfold (M) cells is mediated through cell‐to‐cell interactions between enterocytes and lymphocytes. Based on this concept, we developed a cell‐to‐cell model by encouraging interactions between enterocyte C2BBe1 and Raji B cells through a preincubation approach. Raji B cells and C2BBe1 cells were allowed to interact in detached condition for 2 h at ratios of 1:1, 1:2 and 1:4 and then plated in culture plates. Monocultured C2BBe1 cells were used as the control. Flow cytometric analysis of the M cell‐specific marker clusterin revealed that the optimum ratio of Raji B to C2BBe1 cells to obtain the maximum number of M cells was 1:1. Scanning electron micrographs exhibiting the lack of microvilli with complete tight junctions and Western blot analysis showing intense expression of clusterin confirmed the unique phenotypes of the formed M cells. Fluosphere® transport studies showed a 7‐fold increase in the cell‐to‐cell model compared to the monoculture control. Importantly, we found that the induction of M cells could be enhanced by the effect of epithelial growth factor (EGF). C2BBe1 cells were pretreated with EGF at 10, 25 and 50 ng/mL before co‐culturing with Raji B cells. Flow cytometric analysis of clusterin revealed that EGF significantly increased the formation of M cells. From mechanistic studies, we found an increase in the number of M cells involved the induction of stemness by EGF indicated by a dramatic increase in &bgr;‐catenin, Nanog, and Oct‐4, which in turn up‐regulated the cell‐to‐cell interacting protein Integrin &bgr;‐1. Furthermore, we confirmed the transport functions of the conventional, cell‐to‐cell, and cell‐to‐cell with EGF models using a Fluosphere® transport assay. Overall, we demonstrated an effective novel protocol for the formation of M cells as well as the effect of EGF on enhancing cell‐to‐cell interaction, which may benefit transport studies in M cells and promote better understanding of the biology of M cells. Graphical abstract Figure. No Caption available.