A novel duplex TaqMan probe-based real-time RT-qPCR for detecting and differentiating classical and variant porcine epidemic diarrhea viruses.

Two different genotypes of porcine epidemic diarrhea virus (PEDV), the classical and variant strains, are classified by multiple insertions and deletions in their S genes. It is critical to detect and differentiate two genotypes in the pork industry to prevent PEDV outbreaks. In the present study, a novel duplex TaqMan RT-PCR was developed for detecting and differentiating PEDV strains in China. There was no cross-amplification between the two probes when using standard recombinant plasmids, and the specificity was further confirmed by using other seven non-PEDV swine pathogens. The minimum copies required for the detection of both classical and variant PEDV were 4.8 × 102 DNA copies/reaction. The repeatability of TaqMan RT-PCR was evaluated using standard recombinant plasmids and gave coefficients of variation 0.19-4.93. In recent 5 years, 79 clinical samples were collected from piglets with severe diarrhea in the Central China. Among these clinical samples, 51 were confirmed as PEDV positive by conventional RT-PCR, whereas 63 variant PEDV, 3 co-infections and 1 classical PEDV were confirmed by this duplex TaqMan RT-PCR, with viral loads of 102-108, 102-103, and 104 copies/reaction, respectively. Therefore, the duplex TaqMan RT-PCR could be a useful method for detecting and differentiating variant and classical PEDV strains. The results showed that variant PEDV was prevalent in clinical samples in central China. Moreover, in this study, co-infection by PEDV strains was detected for the first time and might help explain the emergence of the novel recombinant PEDV in recent years.

Molecular epidemiology of porcine reproductive and respiratory syndrome virus in Central China since 2014: The prevalence of NADC30-like PRRSVs.

Porcine reproductive and respiratory syndrome (PRRS), characterized by respiratory disorders in piglets and reproductive failure in sows, is still the great threat of swine industry. Recently, Emergence of the novel NADC30-like PRRS viruses (PRRSVs) has caused widespread outbreaks of PRRS. To investigate the epidemic characteristics of PRRSVs in Central China since 2014, 6372 clinical serum samples were tested by ELISA, 250 tissue samples were tested by RT-PCR, and among these, 30 ORF5 and 17 Nsp2 genes sequences were analyzed. Phylogenetic tree based on ORF5 revealed that, 17 isolates were clustered into subgroup 1, represented by the NADC30. And for the Nsp2, The strains which had a discontinuous 131-amino-acid deletion in Nsp2, called NADC30-like strains, were clustered into subgroup 2. Our data suggested that the NADC30-like PRRSV strains spread quickly and are now circulating and prevalent in Central China as well as the classical HP-PRRSV strains. In addition, amino acid variation analysis of GP5 revealed that the amino acid sequences of NADC30-like PRRSV strains underwent rapid evolution and contained extensive amino acid substitutions in important motifs, such as potential neutralization epitope and the N-glycosylation sites. In summary, our data would provide a large amount of detailed information on molecular variation and genetic diversity of PRRSV in central China.

Molecular epidemiology of outbreak-associated pseudorabies virus (PRV) strains in central China.

In several parts of China, there have been a large number of pseudorabies (PR) outbreaks which have devastated many swine farms even though the herds had been previously immunized with gE-deleted vaccines (Bartha-K61). The emergence of these outbreak-associated PRV strains might indicate that Bartha-K61 vaccine could not provide effective protection and poses challenges for current serologic diagnostics of anti-PRV antibodies. Here, we performed phylogenetic analyses based on partial gE, gB, and gC genes to provide information about the molecular epidemiology, diagnostics, and immune protection in these outbreak-associated PRV strains. Our results indicated that the maximal nucleotide sequence divergence for gE, gB, and gC genes are 1.7, 0.4, and 2.7 % within the cluster where outbreak-associated PRV strains were located, and are 2.3, 2.7, and 7.6 % with other clusters in the phylogenetic trees, respectively. Phylogenetic analyses revealed that gE, gB, and gC genes of the twelve outbreak-associated PRV strains clustered to a relatively independent branch of the tree, and evolved from the same ancestor with strains Ea-China-1999, Fa-China-2001, and BJ-China-2008. The genetic relationship between these outbreak-associated PRV strains and strain Bartha is not close which may genetically explain the emergence of PR outbreaks in Bartha-K61-vaccinated swine farms. We suggest that these outbreak-associated PRV strains originate from earlier strains in local regions in China.

Lower seroprevalence of Toxoplasma gondii in swine from central China after an outbreak of African swine fever.

Toxoplasma gondii is widespread worldwide and can infect swine. This study evaluated the seroprevalence of T. gondii in swine from central China after an outbreak of African swine fever (ASF). A total of 2683 swine serum samples were collected from farms in four provinces. Of the serum samples, 1.42% (38/2683) (95% CI, 1.03-1.94) tested positive for T. gondii IgG antibody by a modified agglutination test (MAT) (cut-off: 1:25). Comparing with the results of previous studies, specifically our survey from before the outbreak, the seroprevalence of T. gondii in swine from central China was significantly decreased after the occurrence of ASF (OR = 7.679, 2015-2017 vs. 2019-2020). In general, the proportion of seropositive animals increased with the age of the swine, indicating post-natal transmission of T. gondii. Furthermore, there was a significant difference in seroprevalence between suckling pigs and weaned pigs (p < 0.05). This is the first large-scale investigation of T. gondii infection in swine after an ASF outbreak in China. The lower seroprevalence of T. gondii in swine after ASF may be due to stricter biosecurity measures on the farms, but results indicated swine exposure to zoonotic parasites despite these measures. This highlights that pigs must be considered a potential source of human T. gondii infections.

TITLE: Baisse de la séroprévalence de Toxoplasma gondii chez les porcs du centre de la Chine après une épidémie de peste porcine africaine. ABSTRACT: Toxoplasma gondii est répandu dans le monde entier et peut infecter les porcs. Cette étude a évalué la séroprévalence de T. gondii chez les porcs du centre de la Chine après une épidémie de peste porcine africaine (PPA). Au total, 2 683 échantillons de sérum de porc ont été prélevés dans des fermes de quatre provinces. Parmi les échantillons de sérum, 1,42 % (38/2683) (IC à 95 %, 1,03­1,94) étaient positifs pour les anticorps IgG de T. gondii par un test d'agglutination modifié (MAT) (seuil : 1:25). En comparaison avec les résultats d'études précédentes, en particulier notre enquête d'avant l'épidémie, la séroprévalence de T. gondii chez les porcs du centre de la Chine a été significativement diminuée après l'apparition de la PPA (OR = 7,679, 2015-2017 vs 2019-2020). En général, la proportion d'animaux séropositifs augmentait avec l'âge des porcs, indiquant une transmission postnatale de T. gondii. De plus, il y avait une différence significative entre la séroprévalence chez les cochons de lait et les cochons sevrés (p < 0,05). Il s'agit de la première enquête à grande échelle sur l'infection à T. gondii chez le porc après l'épidémie de PPA en Chine. La séroprévalence plus faible de T. gondii chez les porcs après la PPA peut être due à des mesures de biosécurité plus strictes dans les fermes, mais les résultats ont indiqué une exposition des porcs aux parasites zoonotiques malgré ces mesures. Cela souligne que les porcs doivent être constamment considérés comme une source potentielle d'infection humaine à T. gondii.

Complete Genome Sequence of the Porcine Epidemic Diarrhea Virus Variant CH/HNYF/2014.

Sow's milk is a potential route for the vertical transmission of porcine epidemic diarrhea virus (PEDV) from sow to suckling piglet. We report here the complete genome sequence of PEDV strain CH/HNYF/2014, which was isolated from milk samples : This information provides further understanding of the transmission mechanisms and genetic diversity of PEDV.