Isolation and Characterization of Yunnan Variants of the Pseudorabies Virus and Their Pathogenicity in Rats

Abstract

Porcine pseudorabies has long existed in China and is a serious threat to the Chinese farming industry. To understand the prevalence and genetic variation of the porcine pseudorabies virus (PRV) and its pathogenicity in Yunnan Province, China, we collected 560 serum samples across seven Yunnan Province regions from 2020 to 2021 and detected anti-gE antibodies in these samples. Sixty-one clinical tissue samples were also collected from pigs with suspected PRV that were vaccinated with Bartha-K61. PRV-gE antibodies were found in 29.6% (166/560) of the serum samples. The PRV positivity rate in clinical tissue samples was 13.1% (8/61). Two isolates, PRV-KM and PRV-QJ, were obtained. The identity of the gB, gD, and gE genes between these isolates and the Chinese mutants exceeded 99.5%. These isolates and the classical Fa strain were used to infect 4-week-old rats intranasally to assess their pathogenicity. All infected rats showed the typical clinical and pathological features of PRV two days post-infection. The viral loads in the organs differed significantly among the infected groups. Viruses were detected in the saliva and feces at 12 h. Significant dynamic changes in total white blood cell counts (WBC), lymphocyte counts (Lym), and neutrophil counts (Gran) occurred in the blood of the infected groups at 24 and 48 h. These results show that mutant PRV strains are prevalent in Bartha-K61-vaccinated pigs in Yunnan Province, China. Moreover, rats shed PRV in their saliva and feces during early infection, indicating the need for rodent control in combatting PRV infections in Yunnan Province, China.

Keywords: Yunnan; characterization; isolation; mutant strain; rat pathogenicity; swine pseudorabies.

Figure 1

Pseudorabies (PR) epidemiological survey and pseudorabies virus (PRV) isolation: (A) serum gE-antibody-positivity rate of different pig herds in seven regions of Yunnan Province during 2020–2021.; (B) cytopathogenic effect (CPE) of PRV-KM and PRV-QJ on BHK-21 cells; (C) amplification of PRV gE fragments (810 bp) from PRV-KM- and PRV-QJ-inoculated BHK-21 cells. PCR amplification was performed with PCR+ as the positive control and PCR- as the negative control; (D) nucleotide phylogenetic tree of the gB gene in PRV isolates; (E) nucleotide phylogenetic tree of the gD gene in PRV isolates; (F) nucleotide phylogenetic tree of the gE gene in PRV isolates. The black circles indicate the PRV-KM and PRV-QJ strains obtained from this study.

Figure 2

Results of the pathogenicity tests of the pseudorabies virus (PRV) strains Fa, KM, and QJ in rats: (A) plot of the results of the uninfected control group (UC) compared with the Fa-, KM-, and QJ-positive organ indices (n = 3 in each group); (B) pathological changes in mice that died after experimental infection with the Fa, KM, and QJ strains (hematoxylin and eosin staining, 400×); (C) plots of the viral load in the brain, heart, liver, spleen, lungs, kidneys, and testes of the Fa, KM, and QJ groups (n = 3 in each group). ns means no significant difference, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001; (D–F) WBC, Lym, and Gran counts in the UC, Fa, KM, and QJ groups at 0, 24, and 48 h post-infection (n = 5 in each group).

Figure 3

Salivary and fecal viral load results of the PRV-KM and PRV-QJ groups: (A) the 0–60-h salivary viral load values of the PRV-KM and PRV-QJ groups (n = 3 in each group); (B) the 0–60-h fecal viral load values of the PRV-KM and PRV-QJ groups (n = 3 in each group).