Establishment and application of a quadruplex real-time RT-qPCR assay for differentiation of TGEV, PEDV, PDCoV, and PoRVA.

Porcine viral diarrhea is a common ailment in clinical settings, causing significant economic losses to the swine industry. Notable culprits behind porcine viral diarrhea encompass transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA). Co-infections involving the viruses are a common occurrence in clinical settings, thereby amplifying the complexities associated with differential diagnosis. As a consequence, it is therefore necessary to develop a method that can detect and differentiate all four porcine diarrhea viruses (TGEV, PEDV, PDCoV, and PoRVA) with a high sensitivity and specificity. Presently, polymerase chain reaction (PCR) is the go-to method for pathogen detection. In comparison to conventional PCR, TaqMan real-time PCR offers heightened sensitivity, superior specificity, and enhanced accuracy. This study aimed to develop a quadruplex real-time RT-qPCR assay, utilizing TaqMan probes, for the distinctive detection of TGEV, PEDV, PDCoV, and PoRVA. The quadruplex real-time RT-qPCR assay, as devised in this study, exhibited the capacity to avoid the detection of unrelated pathogens and demonstrated commendable specificity, sensitivity, repeatability, and reproducibility, boasting a limit of detection (LOD) of 27 copies/µL. In a comparative analysis involving 5483 clinical samples, the results from the commercial RT-qPCR kit and the quadruplex RT-qPCR for TGEV, PEDV, PDCoV, and PoRVA detection were entirely consistent. Following sample collection from October to March in Guangxi Zhuang Autonomous Region, we assessed the prevalence of TGEV, PEDV, PDCoV, and PoRVA in piglet diarrhea samples, revealing positive detection rates of 0.2 % (11/5483), 8.82 % (485/5483), 1.22 % (67/5483), and 4.94 % (271/5483), respectively. The co-infection rates of PEDV/PoRVA, PEDV/PDCoV, TGEV/PED/PoRVA, and PDCoV/PoRVA were 0.39 %, 0.11 %, 0.01 %, and 0.03 %, respectively, with no detection of other co-infections, as determined by the quadruplex real-time RT-qPCR. This research not only established a valuable tool for the simultaneous differentiation of TGEV, PEDV, PDCoV, and PoRVA in practical applications but also provided crucial insights into the prevalence of these viral pathogens causing diarrhea in Guangxi.

Isolation and pathogenicity of porcine circovirus type 2 in mice from Guangxi province, China.

BACKGROUND: Porcine circovirus type 2 (PCV2), a member of the genus Circovirus and family Circoviridae, is a closed, small, circular, and single-stranded DNA virus, and it is a crucial swine pathogen of porcine circovirus-associated diseases (PCVADs). PCV2 was first detected in PK-15(ATCC-CCL) cells in 1974, which has caused significant economic loss to the swine industry throughout the world. And the first case of PCV2 was reported in China in 2000. At present, PCV2d is the main genotype circulating widely in China. METHODS: Lymph samples were obtained from piglets with emaciation and respiratory disease in Guangxi province, China. The main pathogens were detected via PCR from lymph samples, and then PCV2-single positive samples were used to inoculate with PK-15 cells. After successive generations, the isolate was subsequently identified by polymerase chain reaction (PCR), immunofluorescence assay (IFA), Western blot (WB), and transmission electron microscopic (TEM). The full-length genome and genetic characterization of isolates were analyzed by Sanger sequencing. The TCID50 of the PCV2-GX-6 was determined by IFA, and the pathogenicity of PCV2 in BALB/c mice was analyzed via the mouse model. RESULTS: The isolates were successfully isolated from clinical samples. The complete genome of PCV2-GX-4, PCV2-GX-6, PCV2-GX-7, PCV2-GX-11 and PCV2-GX-16 have been amplified, sequenced, and deposited in GenBank (accession no.: OR133747, OQ803314, OR133748, OR133749, OR133750). Homology and phylogenetic analysis with reference strains showed that the isolates belonged to the PCV2d genotype. The PCV2-GX-6 could be stably passaged more than 30 times in PK-15 cells. PCV2-GX-6 was identified by PCR, IFA, WB and TEM. The results of homology showed that PCV2-GX-6 was closely related to the reference strains PCV2-JS17-8 (GenBank accession no.: MH211363). Pathogenicity studies in mice have shown that PCV2-GX-6 can lead to growth inhibition of mice. Meanwhile PCV2-GX-6 caused the typical lesions of spleen, lung and kidney. The results of qPCR showed that PCV2 can effectively proliferate in the liver, spleen, lung, and kidney. CONCLUSION: PCV2-GX-6 can successfully infect BLAB/c mice, effectively proliferate in major organs, and possessed high pathogenicity. In conclusion, combined with the genotype and pathogenicity of PCV2d currently prevalent, PCV2-GX-6 can be used as a candidate vaccine strain.

A nanoparticle vaccine based on the VP121-26 and VP2 structural proteins of Senecavirus A induces robust protective immune responses.

Senecavirus A (SVA) is a causative agent that can cause vesicular disease in swine, which causes a great threat to the swine husbandry in the world. Therefore, it is necessary to develop a vaccine that can effectively prevent the spread of SVA. In this study, we developed a 24-polymeric nano-scaffold using ß-annulus peptide from tomato bushy effect virus (TBSV) by coupling this antigen to SVA B cell epitope VP121-26 and VP2 proteins via linkers, respectively. The SVA-based nanoparticle protein of the VP1(B)-ß-VP2 was expressed and purified by low-cost prokaryotic system to prepare a SVA nanoparticle vaccine. The immunological protective effect of SVA nanoparticle vaccine was evaluated in mouse and swine models, respectively. The results suggested that both mice and swine could induce high levels SVA neutralizing antibodies and IgG antibodies after two doses immunization. In addition, the swine challenge protection experiment showed that the protection rate of immune SVA nanoparticle vaccine and SVA inactivated vaccine both were 80 %, while the negative control had no protection effect. It demonstrated that SVA nanoparticle vaccine effectively prevented SVA infection in swine. In summary, the preparation of SVA vaccine by using ß-annulus peptide is a promising candidate vaccine for prevent SVA transmission, and provides a new idea for the development of novel SVA vaccines.

Pathogenicity Studies of NADC34-like Porcine Reproductive and Respiratory Syndrome Virus LNSY-GY and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus GXGG-8011 in Piglets.

The porcine reproductive and respiratory syndrome virus (PRRSV) has caused significant economic losses to the swine industry. The U.S., China, and Peru have reported NADC30-like or NADC34-like PRRSV-infected piglets, which have been identified as the cause of a significant number of abortions in clinics. Although the pathogenicity of NADC30-like PRRSV and NADC34-like PRRSV in piglets exhibits significant variability globally, studies on their pathogenicity in China are limited. In this study, the animal experiments showed that within 8-14 days post-infection, both piglets infected with NADC30-like PRRSV GXGG-8011 and those infected with NADC34-like PRRSV LNSY-GY exhibited significant weight loss compared to the control piglets. Additionally, the viremia of the LNSY-GY persisted for 28 days, while the viremia of piglets infected with the GXGG-8011 lasted for 17 days. Similarly, the duration of viral shedding through the fecal-oral route after the LNSY-GY infection was longer than that observed after the GXGG-8011 infection. Furthermore, post-infection, both the LNSY-GY and GXGG-8011 led to pronounced histopathological lesions in the lungs of piglets, including interstitial pneumonia and notable viral colonization. However, the antibody production in the LNSY-GY-infected group occurred earlier than that in the GXGG-8011-infected group. Our research findings indicate that LNSY-GY is a mildly pathogenic strain in piglets, whereas we speculate that the GXGG-8011 might be a highly pathogenic strain.