Emergence of highly virulent pseudorabies virus in southern China

Abstract

Pseudorabies has been controlled efficiently in China for many years by vaccination. However, it suddenly broke out in many pig farms in 2012-2013 in southern China. In this study, a systematic investigation that included virus isolation, genetic and pathological studies, and immunogenicity analysis was carried out with the aim of understanding the pathogenetic and antigenic features of novel isolates of pseudorabies virus (PRV). Of 38 tissue samples collected from pigs with clinical signs of pseudorabies on 13 farms in 4 provinces in southern China in 2012-2013, 29 showed wild-type PRV infection by polymerase chain reaction. Sequence analysis of 5 isolates from the 4 provinces showed that they belonged to a relatively independent cluster that shared 2 insertions of a single amino acid in the gE gene and 1 insertion of 7 amino acids in the gC gene. In experiments, isolate ZJ01 caused death in 100% of pigs that were either 14 or 80 days old. The serum antibodies to the commercial PRV vaccines had significantly lower neutralizing activity against the ZJ01 isolate than against the vaccine strains. The antigenic relatedness between ZJ01 and the vaccine strains was 0.378 to 0.455. These findings indicated that a novel, highly virulent PRV strain with antigenic variance had spread widely in southern China.

Figure 1

Clinical signs and gross lesions observed in the pigs with pseudorabies. A — Abortion. B and C — Severe neurologic disorders. D — Diarrhea. E — Cerebral edema and hemorrhage. F, G, and H — Lung, tonsil, and spleen with multiple small focal areas of necrosis.

Figure 2

Amino acid sequence alignment of the gE and gC genes of pseudorabies virus (PRV). A — New PRV strains ZJ01, ZJ02, GD01, GX01, and JX01 have an insertion of 1 amino acid at positions 48 and 496 in gE. B — An insertion of 7 amino acids between positions 63 and 69 in gC and several mutations between positions 180 and 200 are shared by the 5 new strains and some strains isolated previously in China but not others.

Figure 3

Phylogenetic trees of PRV based on the gE (A) and gC (B) genes. The 5 new strains are all in an independent cluster that is closely related to the traditional PRV isolates in China and other Asian countries.

Figure 4

Survival and viral DNA load, as determined by real-time polymerase chain reaction, after challenge with PRV strain ZJ01 in 14-day-old pigs (A and B) and 80-day-old pigs (C and D); for the latter, 2 different inoculation doses were used. In each experiment a control group was inoculated with phosphate-buffered saline (PBS). TCID₅₀— median tissue culture infective dose. Different letters (a to c) indicate a significant difference (P < 0.05) between the tissues.

Figure 5

Gross lesions and histopathological features of tissues stained with hematoxylin and eosin from piglets challenged with ZJ01. A — Cerebral edema and hemorrhage. B — Lung with many blood spots. C — Liver with multiple small focal areas of necrosis. D — Nonsuppurative meningoencephalitis; original magnification ×100. E — Lung tissue with severe hemorrhage and congestion; original magnification ×100. F — Liver tissue with hemorrhage, hepatocyte necrosis, and inflammatory cell infiltration; original magnification ×200.