Analysis of the spike, ORF3, and nucleocapsid genes of porcine epidemic diarrhea virus circulating on Thai swine farms, 2011–2016

Porcine epidemic diarrhea virus (PEDV) outbreaks on pig farms have caused significant economic loss in the swine industry since it was first reported in Thailand a decade ago. Anecdotal evidence suggests that PEDV is now endemic in this region, therefore genome information of circulating PEDV is important for molecular surveillance and evaluation of potential benefits of field vaccination. Here, we characterized PEDV infection on commercial Thai swine farms by screening 769 samples of feces and small intestinal contents from pigs with diarrhea between 2011 and 2016. Using reverse-transcription polymerase chain reaction targeting the spike (S) gene, 153 PEDV-positive samples were further subjected to analysis of the open reading frame 3 and nucleocapsid (N) genes. Comparison of 95 samples in which nucleotide sequencing was successfully obtained for all three genes revealed evolutionary diversity among the Thai PEDV strains. Phylogenetic analyses suggest that although some Thai strains changed little from years past, others resembled more closely to the recent strains reported in China. Interestingly, eight Thai PEDV strains possessed amino acid deletions in the N protein. The PEDV sequence divergence may be responsible for driving periodic outbreaks and continued persistence of PEDV on commercial swine farms. Our findings provide important insight into regional PEDV strains in circulation, which may assist future inclusions of suitable strains for future PEDV vaccines

Porcine rotavirus C in pigs with gastroenteritis on Thai swine farms, 2011–2016

Swine are economically important food animals, but highly contagious porcine epidemic diarrhea virus (PEDV) and rotavirus can afflict pig herds and contribute significantly to piglet morbidity and mortality. While there have been studies on rotavirus group A (RVA) in Thailand, reports of rotavirus group C (RVC) are limited. Here, we aimed to identify the prevalence of RVC circulating on Thai commercial swine farms. We analyzed 769 feces and intestine mucosal contents of pigs affected with diarrhea between 2011 and 2016 using RT-PCR specific for the PEDV spike (S), rotavirus glycoprotein (G) VP7, and protease-sensitive protein (P) VP4 genes. We found that 6.6% (51/769) of samples tested positive for RVC, of which 11 samples were co-infected with RVA and four samples were co-infected with PEDV. Three samples tested positive for all three viruses. Phylogenetic analysis of the VP7 gene showed that the most frequent RVC genotype was G1, which grouped with the prototypic RVC Cowden strain. While G6 and G9 were also common, G3 was relatively rare. Analysis of the VP4 gene revealed that the most common P type was P[5], followed by P[4], P[7], and P[1]. In all, there were six G/P combinations (G6P[5], G1P[1], G1P[4], G1P[5], G9P[4], and G9P[7]), of which G6P[5] was the most predominant

Human enteroviruses associated with and without diarrhea in Thailand between 2010 and 2016

<div><p>Non-bacterial acute gastroenteritis (AGE) associated with virus infection affects individuals living in developing countries, especially children. To investigate whether shedding of certain human enterovirus (EV) is more frequently detected in the stool of individuals with AGE of unknown etiology than individuals without AGE symptoms, we tested fecal samples collected from 2,692 individuals with diarrhea between January 2010 and December 2016. Samples were tested for rotavirus, norovirus, and EV by reverse-transcription polymerase chain reaction (RT-PCR) and adenovirus by PCR. EV-positive samples were subjected to sequencing and phylogenetic analysis to identify EV species and types. Findings were compared to EV found in 1,310 fecal samples from individuals without AGE who were diagnosed with hand, foot, and mouth disease (HFMD). While the majority of viruses identified in AGE consisted of human rotavirus (22.7%), norovirus (11.4%) and adenovirus (9.3%), we identified EV (6.2%) belonging mainly to species B, C, and rhinovirus. In contrast, >92% of EV found without AGE symptoms belonged to species A. Although AGE symptoms are not often attributed to EV infection, EV was associated with diarrhea of unknown etiology at least in 3.4% of AGE cases. While CV-A6 was most likely to be found in stools of HFMD patients, rhinovirus A and C were the two most common EV species associated with AGE. Elucidating group-specific EV infection in diseases with and without AGE will be useful in assisting identification, clinical management, and the surveillance of EV infection in the community.</p></div

Phylogenetic analysis of the nucleotide sequences of the VP4-VP2 region from EV-positive AGE samples.

<p>Phylogenetic tree was constructed using the neighbor-joining method implemented in MEGA (version 5). Bootstrap resampling values >70 are indicated at the nodes. The scale bar indicates the number of substitutions per site. Black dots denote EV obtained from samples with multiple viruses. Blue, EV-A; red, EV-B; green, EV-C; purple, EV-D; yellow, rhinovirus.</p

Enteric viruses found in AGE samples from 2010 to 2016.

<p>Proportion of EV, ADV, NV, and RV identified by year (A) and by age group (B). The number of virus-positive samples are indicated above the bar graphs. Colors are blue for RV, green for NV, yellow for ADV, and pink, EV.</p

Characteristics of the cohorts with (AGE) or without (HFMD) diarrhea in this study.

<p>Characteristics of the cohorts with (AGE) or without (HFMD) diarrhea in this study.</p

Distribution of EV species and types found in 168 EV-positive AGE samples.

<p>(A) Pie chart of EV-A to -D and rhinovirus found in the fecal samples of AGE patients. (B) Genotypes of EV and their percentages (denoted by numbers above the bar graphs). Blue, EV-A; red, EV-B; green, EV-C; purple, EV-D; yellow, rhinovirus.</p

AGE samples tested positive for diverse genotypes of RV, NV, and ADV.

<p>The genotype distribution of (A) RV based on the VP7 and VP4 genes, (B) NV based on the RdRp/VP1 region, and (C) ADV based on the hexon gene.</p

Distribution of EV species and types found in 817 EV-positive HFMD samples.

<p>(A) Pie chart of EV-A to -C and rhinovirus found in the fecal samples of HFMD patients. (B) Genotypes of EV and their percentages (denoted by numbers above the bar graphs). Blue, EV-A; red, EV-B; green, EV-C; yellow, rhinovirus.</p