Isolation, Characterization and Reverse Genetic System Establishment of a Highly Virulent PEDV Strain

Isolation and assessment of characterization of porcine epidemic diarrhea virus GD1 strain from China

Porcine epidemic diarrhea virus (PEDV) is an important pathogen in swine, causing severe economic losses to the global swine industry. As a coronavirus, PEDV is prone to mutations and recombination. Therefore, characterizing circulating strains is essential for disease control. In this study, a PEDV strain (GD1) was isolated from diarrheic piglets in Guangdong Province, China, and identified by RT-PCR and immunofluorescence. Pathogenicity was evaluated in piglet challenge experiment. The results showed that GD1 strain could be stably passaged in Vero cells, forming syncytia. Phylogenetic analysis based on the complete genome and spike (S) gene both placed GD1 strain within the GIIc genotype. Amino acid sequence alignment of the S protein revealed multiple amino acid mutations in the neutralizing epitopes COE, SE16, and SS6 of GD1 strain compared with the classical vaccine strain CV777. Recombination analysis suggested that GD1 may be a novel recombination strain originating from strains SD2021 and GDS28, with the recombination breakpoint located in the ORF1a region. Pathogenicity assessment demonstrated that GD1 is highly virulent, inducing severe watery diarrhea and vomiting in newborn piglets within 24 h post-inoculation, and causing histopathological lesions characterized by intestinal villous atrophy and shedding. Immunohistochemistry confirmed that PEDV antigen was predominantly distributed in the villous epithelial cells of the small intestine. The successful isolation and characterization of the GD1 strain provide important viral resources for further research on the genetic evolution of PEDV and the development of matched vaccines.

Development of a rapid antiviral screening assay based on GFP reporter virus of bovine enterovirus

Genetic diversity and recombination of NA-PRRSV field strains in Vietnam: Implications for vaccine efficacy.

Porcine reproductive and respiratory syndrome (PRRS) causes severe reproductive losses in pregnant sows and piglets, resulting in substantial economic impact on the swine industry worldwide. However, due to the significant genetic diversity and rapid evolutionary changes of the pathogen, continuous surveillance and detailed genetic analysis of circulating strains are essential. The current study aimed to evaluate the genetic diversity of the hypervariable (HV) region of non-structural protein 2 (nsp2) among North American PRRSV strains isolated from swine farms in Vietnam. Phylogenetic analysis and multiple sequence alignment were conducted to determine subtype classification and assess genetic variability. A total of 48 field isolates were obtained, of which 12.5% belonged to classical NA-PRRSV, 16.6% to NADC30-like and 70.9% to HP-PRRSV, primarily distributed across sublineages 1.4, 5.1, 8.7 and 8.9. Amino acid comparisons found multiple insertions, deletions and substitutions at various positions within the hypervariable region of nsp2. The study revealed substantial genetic variation in the HV region of nsp2 among NA-PRRSV field strains, largely associated with recombination and immune escape. These findings highlight epidemiological risks to vaccine efficacy and underscore the need for continuous molecular surveillance to support effective PRRSV control in Vietnam.

The S32 amino acid deletion in the GP5 protein modulates the infectivity of porcine reproductive and respiratory syndrome virus

ABSTRACT The high genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant challenge to effective vaccination and infection prevention. Recently, NADC30-like PRRSV has become dominant in China. In this study, we characterized a PRRSV isolate XJ2020 from a vaccinated pig. The isolate exhibited moderate pathogenicity in piglets and clustered within the same sub-lineage as NADC30. Sequence analysis identified a unique deletion of the serine residue at position 32 (S32) in the GP5 protein. This deletion was introduced into a highly pathogenic PRRSV (HP-PRRSV) strain, JX2015, to generate the mutant JX2015-ΔS32 using a reverse genetic approach. We found that JX2015-△S32 showed reduced propagation in Marc-145 cells and decreased sensitivity to neutralization by MLV-derived anti-PRRSV positive serum, but exhibited enhance replication in primary porcine alveolar macrophages (PAMs) compared to its parental strain JX2015. Interestingly, the viral load in lung and ileum samples from JX2015-△S32-infected piglets was higher, but the lung injury was milder than in JX2015-infected piglets. Overall, this study highlights the S32 site in the PRRSV GP5 protein as an important factor influencing virus propagation, neutralization, cell tropism and pathogenicity in piglets, and suggests that targeting this residue could lead to the development of more effective PRRSV vaccines.

Development and immunological evaluation of a multi-antigen recombinant adenovirus vaccine candidate against PRRSV and PCV2

In this study, a recombinant adenovirus vaccine candidate constructed and evaluated to addressed the widespread clinical problem of co-infections of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus Type 2 (PCV2). Epidemiological analysis revealed a PRRSV-positive rate of 40.8%, with prevalent strains belonging to Lineage 1 and Lineage 8. Analysis of key amino acid sites in the GP5 protein indicated the presence of highly virulent strain characteristics and widespread wild-type strain variation. Based on these findings and previous studies, we selected multiple PRRSV proteins (GP2–GP5, M, N) from multiple strains and the validated PCV2 Cap protein fused with dominant T/B cell epitopes, and successfully constructed four recombinant adenoviruses using a 2A peptide-linked strategy. These constructs stably expressed the target antigens in HEK293 cells and demonstrated good passage stability. Animal experiments showed that piglets immunized with the recombinant adenoviruses developed high levels of PRRSV- and PCV2-specific antibodies and neutralizing antibodies, and exhibited lymphocyte proliferation and secretion of cytokines including IFN-γ, IL-2, and IL-4, indicating the vaccine simultaneously elicited both humoral and cellular immune responses. Following PRRSV challenge, immunized groups, particularly the multi-antigen combined immunization group, demonstrated significant clinical protection. This study provides a promising vaccine candidate and a theoretical foundation for the coordinated control of PRRSV and PCV2.

Isolation, Characterization, and Pathogenicity of a Novel Recombinant Fowl Adenovirus 8a Strain in China

Inclusion body hepatitis (IBH), a disease predominantly associated with fowl adenovirus serotypes 8a (FAdV-8a) and 8b (FAdV-8b), poses a significant challenge to poultry production on a global scale. This study reports a severe outbreak of IBH that occurred in 2021 at a chicken layer farm in Hunan Province, China. Liver tissues were collected from visibly affected layer chickens for viral isolation in LMH cell cultures. Subsequent phylogenetic analysis was conducted based on DNA sequencing and bioinformatics data to determine the genetic relationships of the isolated virus. Pathogenicity trials were performed on specific-pathogen-free (SPF) chickens. A novel FAdV-8, designated HuN21, with recombination between serotypes FAdV-8a/FAdV-8b, was isolated. Recombination and phylogenetic analyses revealed that this recombinant strain harbored a genomic backbone and fiber gene from FAdV-8a and FAdV-8b, respectively. HuN21’s pathogenicity differed significantly by age groups, showing high pathogenicity (100% mortality, classic IBH signs) in 1-day-old SPF chickens, compared with no mortality despite viral replication in 28-day-old chickens. These data provide robust evidence for recombination between different serotypes of FAdV-8. Notably, the unique genetic and pathogenic characteristics of the HuN21 strain indicate potential as a candidate strain for the development of a bivalent inactivated vaccine or a natural vaccine against FAdV-8a/FAdV-8b.