Immunogenic response of recombinant pseudorabies virus carrying B646L and B602L genes of African swine fever virus in mice.

African swine fever (ASF) is an acute infectious disease that poses a high lethality risk to domestic pigs and wild boars, causing substantial economic losses to the global pig industry. The prevention and control of ASF remain challenging, necessitating the urgent development of a safe and effective vaccine. This study focused on the essential structural protein p72 of ASFV (encoded by the B646L gene) and its chaperone protein pB602L (encoded by the B602L gene) as the target antigenic proteins. Based on CRISPR/Cas9 gene-editing technology, we constructed a live attenuated recombinant pseudorabies virus vector expressing the p72 and pB602L proteins (designated as rPRVXJ-EGFP/B602L/B646L), and assessed its immunization effect in mice. The recombinant virus rPRVXJ-EGFP/B602L/B646L successfully proliferated and demonstrated stable expression of the p72 and pB602L proteins in BHK-21 cells. Moreover, it exhibited excellent safety when used in mice and induced specific humoral and cellular immune responses targeting p72 and pB602L. In addition, it provided complete protection (100%) against the virulent PRV strain (PRV-XJ). These results indicate that the recombinant virus rPRVXJ-EGFP/B602L/B646L possesses robust immunogenicity and safety in mice. In conclusion, PRV represents a promising viral vector for expressing ASFV gene, and our study serves as an essential reference for the development of viral vector vaccines against ASFV.

Establishment of a reverse transcription recombinase-aided amplification detection method for porcine group a rotavirus.

Porcine rotavirus type A (PoRVA) is the main cause of dehydration and diarrhea in piglets, which has a great impact on the development of the pig industry worldwide. A rapid, accurate and sensitive detection method is conducive to the monitoring, control, and removal of PoRVA. In this study, a PoRVA real-time fluorescent reverse transcription recombinase-aided amplification (RT-RAA) assay was developed. Based on the PoRVA VP6 gene, specific primers and probes were designed and synthesized. The sensitivity of RT-RAA and TaqMan probe-based RT-qPCR was 7 copies per reaction and 5 copies per reaction, respectively. The sensitivity of the RT-RAA method was close to TaqMan probe-based RT-qPCR. The detection results of RT-RAA and TaqMan probe-based quantitative real-time RT-PCR methods were completely consistent in 241 clinical samples. Therefore, we successfully established a rapid and specific RT-RAA diagnostic method for PoRVA.

Epidemiological investigation of swine Japanese encephalitis virus based on RT-RAA detection method.

JEV is one of the zoonotic pathogens that cause serious diseases in humans. JEV infection can cause abortion, mummified foetus and stillbirth in sows, orchitis and semen quality decline in boars, causing huge economic losses to pig industry. In order to investigate the epidemiology of JEV in pigs in Sichuan province, a rapid and efficient fluorescent Reverse transcription recombinase-aided amplification (RT-RAA) detection method was established. Aborted fetuses and testicular swollen boar samples were detected by RT-RAA in pigs in the mountain areas around Sichuan Basin, and the detection rate of JEV was 6.49%. The positive samples were identified as JEV GI strain and GIIIstrain by sequencing analysis. We analyzed the whole gene sequence of a positive sample for the GI virus. The Envelope Protein (E protein) phylogenetic tree analysis was far related to the Chinese vaccine strain SA14-14-2, and was most closely related to the JEV GI strains SH17M-07 and SD0810 isolated from China. The results showed that we established an efficient, accurate and sensitive method for clinical detection of JEV, and JEV GI strains were prevalent in Sichuan area. It provides reference for the prevention and control of JEV in Sichuan.

The Immunity Protection of Central Nervous System Induced by Pseudorabies Virus DelgI/gE/TK in Mice.

Based on a variant strain, we constructed a gE/gI/TK-deleted pseudorabies virus (PRV). A total of 18 female mice were randomized to a vaccination group to receive PRV XJ delgE/gI/TK, a vehicle group to receive Dulbecco's modified Eagle's medium, and a mock group to confirm the protection of PRV delgE/gI/TK on the central nervous system in mice. Subsequently, the vaccination and vehicle groups were infected with PRV XJ. The mice in the vehicle group showed more severe neurological symptoms and higher viral loads than those in the vaccination group. The exudation of Evans blue and the expression of tight junction protein showed no difference in all groups. HE staining showed vacuolar neuronal degeneration in the vehicle group brain, but no tissue lesions were observed in the vaccination group. TNF-α, IL-6, and synuclein were upregulated in the brain of mice in the vehicle group, while those were inhibited among mice in the vaccination group. IFN-ß, IFN-γ, ISG15, Mx1, and OAS1 showed no difference in the brain between the vaccination and vehicle groups. In addition, TNF-α and IL-6 were inhibited, and antiviral factors were increased in the intestine of the mice in the vaccination group compared to those in the vehicle group. Our study showed that PRV XJ delgE/gI/TK inhibited neurological damage and the inflammation of the intestine and brain induced by PRV and activated the innate immunity of the intestine.

The recombinant pseudorabies virus expressing porcine deltacoronavirus spike protein is safe and effective for mice.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a new pathogenic porcine intestinal coronavirus, which has appeared in many countries since 2012. PDCoV disease caused acute diarrhea, vomiting, dehydration and death in piglets, resulted in significant economic loss to the pig industry. However, there is no commercially available vaccine for PDCoV. In this study, we constructed recombinant pseudorabies virus (rPRVXJ-delgE/gI/TK-S) expressing PDCoV spike (S) protein and evaluated its safety and immunogenicity in mice. RESULTS: The recombinant strain rPRVXJ-delgE/gI/TK-S obtained by CRISPR/Cas gE gene editing technology and homologous recombination technology has genetic stability in baby hamster syrian kidney-21 (BHK-21) cells and is safe to mice. After immunizing mice with rPRVXJ-delgE/gI/TK-S, the expression levels of IFN-γ and IL-4 in peripheral blood of mice were up-regulated, the proliferation of spleen-specific T lymphocytes and the percentage of CD4+ and CD8+ lymphocytes in mice spleen was increased. rPRVXJ-delgE/gI/TK-S showed good immunogenicity for mice. On the seventh day after booster immunity, PRV gB and PDCoV S specific antibodies were detected in mice, and the antibody level continued to increase, and the neutralizing antibody level reached the maximum at 28 days post- immunization (dpi). The recombinant strain can protect mice with 100% from the challenge of virulent strain (PRV XJ) and accelerate the detoxification of PDCoV in mice. CONCLUSION: The recombinant rPRVXJ-delgE/gI/TK-S strain is safe and effective with strong immunogenicity and is expected to be a candidate vaccine against PDCoV and PRV.

Development of a reverse transcription recombinase-aided amplification assay for detection of Getah virus.

GETV, an arbo-borne zoonotic virus of the genus Alphavirus, which causes diarrhea and reproduction disorders in swine, lead to serious economic losses to the swine industry in China. At present, the existing methods for GETV detection are time-consuming and low sensitivity, so, a rapid, accurate and sensitive GETV detection method is urgently needed. In this study, a fluorescent reverse transcription recombinase-assisted amplification method (RT-RAA) was successfully established for the rapid detection of GETV. The sensitivity of this method to GETV was 8 copies/reaction and 20 TCID50/reaction. No cross-reaction with other viruses. A total of 118 samples were prepared for GETV detection using fluorescent RT-RAA and SYBR Green I RT-qPCR, the coincidence rate of the two methods was 100%. The results suggest that the RT-RAA method is rapid, sensitive and specific for GETV detection and can be applied in the clinical.

[Immunogenicity of engineered Lactobacillus plantarum expressing porcine epidemic diarrhea virus S1 gene].

To investigate whether the engineered Lactobacillus plantarum expressing the porcine epidemic diarrhea virus (PEDV) S1 gene can protect animals against PEDV, guinea pigs were fed with recombinant L. plantarum containing plasmid PVE5523-S1, with a dose of 2×108 CFU/piece, three times a day, at 14 days intervals. Guinea pigs fed with wild type L. plantarum and the engineered L. plantarum containing empty plasmid pVE5523 were used as negative controls. For positive control, another group of guinea pigs were injected with live vaccine for porcine epidemic diarrhea and porcine infectious gastroenteritis (HB08+ZJ08) by intramuscular injection, with a dose of 0.2 mL/piece, three times a day, at 14 days intervals. Blood samples were collected from the hearts of the four groups of guinea pigs at 0 d, 7 d, 14 d, 24 d, 31 d, 41 d and 48 d, respectively, and serum samples were isolated for antibody detection and neutralization test analysis by enzyme-linked immunosorbent assay (ELISA). The spleens of guinea pigs were also aseptically collected to perform spleen cells proliferation assay. The results showed that the engineered bacteria could stimulate the production of secretory antibody sIgA and specific neutralizing antibody, and stimulate the increase of IL-4 and IFN-γ, as well as the proliferation of spleen cells. These results indicated that the engineered L. plantarum containing PEDV S1 induced specific immunity toward PEDV in guinea pigs, which laid a foundation for subsequent oral vaccine development.