Emergence of a Pseudorabies virus variant with increased virulence to piglets

doi:10.1016/j.vetmic.2015.09.021

Veterinary Microbiology

Volume 181, Issues 3–4, 31 December 2015, Pages 236-240

https://doi.org/10.1016/j.vetmic.2015.09.021 Get rights and content

Highlights

•
PRV variant JS-2012 was more pathogenic to pigs as compared with classical SC strain.
•
Bartha-K61 vaccination provided partial protection against challenge with PRV JS-2012
•
Bartha-K61 vaccination provided complete protection against challenge with PRV SC.

Abstract

Pseudorabies virus (PRV) causes Pseudorabies (PR), an economically important disease in domestic swine. PR outbreaks on pig farms caused by PRV variant strains in Bartha-K61-vaccinated pigs have resulted in considerable economic losses in China since 2011. In this study, the pathogenicity of the PRV variant JS-2012 strain to pigs was investigated by experimentally inoculating piglets of different ages in comparison with a classic virulent PRV SC strain. The JS-2012 strain caused an earlier onset of clinical signs and higher mortality in 15, 30, and 60-day-old pigs, as compared with a classic virulent PRV SC strain. The Bartha-K61 vaccination provided complete protection against challenge with classical virulent PRV, but only partial protection against challenge with the JS-2012 strain in piglets. In conclusion, the increased virulence of the PRV variant may have partly contributed to the PR outbreak in China.

Graphical abstract

Introduction

Pseudorabies (PR) or Aujeszky’s disease (AD) caused by Pseudorabies virus (PRV), is a serious viral disease in pigs and other animals. This virus can cause nervous system disorders, respiratory disorders, reproductive failure, fever and itching, and causes huge economic losses to the swine industry in many countries (Lee and Wilson, 1979, Marcaccini et al., 2008, Mettenleiter, 1996, Pomeranz et al., 2005). PRV-infected pigs carry the virus for life in the peripheral nervous system and can infect other animals (Lee and Wilson, 1979).

Attenuated live or inactivated vaccines are widely used to control PR (Kooij, 1994, Mengeling et al., 1997, Pensaert et al., 2004, Stegeman, 1997, Visser, 1997). The Bartha-K61 strain is a safe and effective vaccine against PRV and has played an important role in the control and eradication of PR. PRV has been eradicated from domestic swine in the United States, several parts of Europe, and New Zealand (Hahn et al., 2010, Müller et al., 2011, Pannett et al., 1999). In the 1970s, the Bartha-K61 vaccine was imported from Hungary to China and used to effectively control PR (Tong and Chen, 1999). However, since late 2011, PR has occurred in Bartha-K61-vaccinated pigs on many swine farms in China (Peng et al., 2013, An et al., 2013, Wu et al., 2013, Wang et al., 2014, Yu et al., 2014, Luo et al., 2014). The symptoms of the disease were stillbirth or the birth of weak piglets with nervous system disorders, diarrhea, tremble, and opisthotonos (An et al., 2013). Since the initial outbreak in late 2011, PR has occurred in more than 15 provinces in China and has caused more than 50% mortality in infected neonatal piglets (An et al., 2013, Wang et al., 2014, Yu et al., 2014). A PRV variant was considered to be the cause of emerging PR, since the Bartha-K61 vaccine did not provide full protection against the prevalent PRV variant in sheep (An et al., 2013, Luo et al., 2014).

We isolated the PRV variant JS-2012 strain from piglets with suspected PR that had been vaccinated with Bartha-K61-vaccine (Tong et al., 2013). Phylogenetic analysis showed that JS-2012 with other emergent variants in China such as HeN1 and TJ strains formed a novel branch and relatively distant from previously isolated strains of PRV (Tong et al., 2013, Luo et al., 2014, Ye et al., 2015). Compared with Bartha strain, the emergent variants had unique insertion/deletion polymorphisms and microsatellites (Ye et al., 2015). JS-2012 strain was more pathogenic than classical virulent PRV SC strain in BALB/c mice (Tong et al., 2013). In this report, we compared the differences in pathogenicity between PRV variant JS-2012 strain and classical virulent PRV SC stain by vaccination-challenge experiment in piglets and artificial infection experiments in pigs of different age.

Section snippets

Cells and viruses

Vero cells were grown and maintained in Dulbecco’s modified Eagle’s high glucose medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, USA) at 37 °C in 5% CO₂. The PRV SC strain isolated in China in 1980 was highly pathogenic to pigs and sheep (Yuan et al., 1987). The PRV variant JS-2012 strain was isolated from brain tissues of dead piglets in a farm in Jiangsu Province of China in which an outbreak of an emerging infectious disease characterized by newborn piglet deaths

The PRV SC- or JS-2012-specific neutralizing antibodies induced by Bartha-K61 vaccine

For the Bartha-K61-vaccinated piglets (group 1 and group 2), the PRV SC-specific NAbs titers (4.87 ± 0.36) were significantly higher than JS-2012-specific NAbs titers (2.87 ± 0.36, P < 0.01). Similarly, the PRV SC-specific NAbs titers of group 1 (4.79 ± 0.36) were significantly higher than JS-2012-specific NAbs titers of group 2 (2.92 ± 0.42, Table 1, P < 0.01). No PRV SC- or JS-2012-specific NAbs were detected in the unvaccinated control groups (group 3 and group 4, <2, Table 1).

Bartha-K61 vaccine provided complete protection against SC but not against JS-2012

In addition to a slight

Discussion

In late 2011, a PR-like infection disease characterized by a large number of newborn piglet deaths with neurologic symptoms occurred in Bartha-K61-vaccinated pigs on many farms in China (An et al., 2013, Yu et al., 2014). The PRV variants isolated in the Bartha-K61-vaccinated piglets with suspected PR were considered to be the cause of the disease (An et al., 2013, Yu et al., 2014). The Bartha-K61 vaccine did not provide complete protection against the emerging PRV variants in sheep (An et al.,

Acknowledgments

This work was supported by Shanghai Municipal Natural Science Foundation (No. 14ZR1448900), Shanghai city agriculture science and technology key project (2015,1-7), and the National Nonprofit Institute Research Grant of CATAS-ITBB (No. 2014JB02).

Wu Tong is a research associate of Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. His research focus is on molecular epidemiology and vaccinology of Pseudorabies virus.

References (25)

T.Q. An et al.
Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China, 2012
Emerg. Infect. Dis.
(2013)
V. Gerdts et al.
Potency of an experimental DNA vaccine against Aujeszky’s disease in pigs
Vet. Microbiol.
(1999)
Z. Gu et al.
A novel inactivated gE/gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerged variant PRV challenge
Virus Res.
(2015)
E.C. Hahn et al.
Variation of Aujeszky’s disease viruses in wild swine in USA
Vet. Microbiol.
(2010)
D. Kooij
Economic aspects of the control of Aujeszky’s disease in the European Community
Acta Vet. Hung.
(1994)
J.Y. Lee et al.
A review of pseudorabies (Aujeszky’s disease) in pigs
Can. Vet. J
(1979)
Y. Luo et al.
Pathogenicity and genomic characterization of a pseudorabies virus variant isolated from Bartha-K61-vaccinated swine population in China
Vet. Microbiol.
(2014)
A. Marcaccini et al.
Pseudorabies virus infection in mink: a host-specific pathogenesis
Vet. Immunol. Immunopathol.
(2008)
W.L. Mengeling et al.
The role of biotechnologically engineered vaccines and diagnostics in pseudorabies (Aujeszky’s disease) eradication strategies
Vet. Microbiol.
(1997)
T.C. Mettenleiter
Immunobiology of pseudorabies (Aujeszky’s disease)
Vet. Immunol. Immunopathol.
(1996)

T. Müller et al.

Pseudorabies virus in wild swine: a global perspective

Arch. Virol.

(2011)

G.R. Pannett et al.

Eradication of Aujeszky’s disease from New Zealand pig herds 1976–1997

Vet. Rec.

(1999)

Cited by (96)

Inhibition of pseudorabies virus replication via upregulated interferon response by targeting 7-dehydrocholesterol reductase
2024, Veterinary Microbiology
Pseudorabies virus (PRV) is an alpha-herpesvirus capable of infecting a range of animal species, particularly its natural host, pigs, resulting in substantial economic losses for the swine industry. Recent research has shed light on the significant role of cholesterol metabolism in the replication of various viruses. However, the specific role of cholesterol metabolism in PRV infection remains unknown. Here, we demonstrated that the expression of 7-dehydrocholesterol reductase (DHCR7) is upregulated following PRV infection, as evidenced by the proteomic analysis. Subsequently, we showed that DHCR7 plays a crucial role in promoting PRV replication by converting 7-dehydrocholesterol (7-DHC) into cholesterol, leading to increased cellular cholesterol levels. Importantly, DHCR7 inhibits the phosphorylation of interferon regulatory factor 3 (IRF3), resulting in reduced levels of interferon-beta (IFN-β) and interferon-stimulated genes (ISGs). Finally, we revealed that the DHCR7 inhibitor, trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride (AY9944), significantly suppresses PRV replication both in vitro and in vivo. Taken together, the study has established a connection between cholesterol metabolism and PRV replication, offering novel insights that may guide future approaches to the prevention and treatment of PRV infections.
Intradermal vaccination with Porcilis® Begonia can clinically protect against fatal PRV challenge with the highly virulent ZJ01 field strain
2024, Microbial Pathogenesis
Since pseudorabies (PR) re-emerged and rapidly spread in China at the end of 2011, researchers have focused on effective vaccine strategies to prevent and control pseudorabies virus (PRV) infection in pig herds. Due to the extensive application of an attenuated vaccine based on the Bartha-K61 strain isolated in Hungary in 1961 and the variation of the PRV strain, it has been suggested that traditional vaccines based on the Bartha-K61 strain offer only partial protection against variant strains. It was therefore evaluated whether the Porcilis® Begonia vaccine, which is based on the NIA-3 strain with deletions in the gE and TK genes, is efficacious against experimental infection with the virulent, contemporary Chinese PRV strain ZJ01.
In this study, piglets were vaccinated with Porcilis® Begonia through either the intradermal (ID) route or the intramuscular (IM) route and subsequently challenged intranasally with strain ZJ01 at 4 weeks post-vaccination. An unvaccinated challenge group and an unvaccinated/nonchallenged group were also included in the study. All animals were monitored for 14 days after challenge. Vaccinated and negative control pigs stayed healthy during the study, while the unvaccinated control animals developed lesions associated with PRV ZJ01 challenge, and 44% of these pigs died before the end of the experiment. This study demonstrated that ID or IM vaccination of pigs with a vaccine based on the NIA-3 strain Porcilis® Begonia clinically protects against fatal PRV challenge with the ZJ01 strain.
Pathogenicity and immunogenicity of a quadruple gene-deleted pseudorabies virus variant as a vaccine candidate
2024, Veterinary Microbiology
Since late 2011, the PRV variants have emerged in China, characterized by the increased virulence. The traditional attenuated vaccines have proven insufficient in providing complete protection, resulting in substantial economic losses to swine industry. In this study, a vaccine candidate strain, ZJ01-ΔgI/gE/TK/UL21, carrying the quadruple gene deletion was derived from the previously generated three gene-deleted virus ZJ01-ΔgI/gE/TK. As anticipated, piglets inoculated with ZJ01-ΔgI/gE/TK/UL21 exhibited normal body temperatures and showed no viral shedding, consistent with the observations from piglets treated with ZJ01-ΔgI/gE/TK. Importantly, a significant higher level of interferon induction was observed among piglets in the ZJ01-ΔgI/gE/TK/UL21 group compared to those in the ZJ01-ΔgI/gE/TK group. Upon challenge with the PRV variant ZJ01, piglets immunized with ZJ01-ΔgI/gE/TK/UL21 exhibited reduced viral shedding compared to the ZJ01-ΔgI/gE/TK group. Furthermore, piglets vaccinated with ZJ01-ΔgI/gE/TK/UL21 exhibited minimal pathological lesions in brain tissues, similar to those in the ZJ01-ΔgI/gE/TK group. These results underscore the potential of ZJ01-ΔgI/gE/TK/UL21 as a promising vaccine for controlling PRV infection.
The mutations on the envelope glycoprotein D contribute to the enhanced neurotropism of the pseudorabies virus variant
2023, Journal of Biological Chemistry
The pseudorabies virus (PRV) TJ strain, a variant of PRV, induces more severe neurological symptoms and higher mortality in piglets and mice than the PRV SC strain isolated in 1980. However, the mechanism underlying responsible for the discrepancy in virulence between these strains remains unclear. Our study investigated the differences in neurotropism between PRV TJ and PRV SC using both in vitro and in vivo models. We discovered that PRV TJ enters neural cells more efficiently than PRV SC. Furthermore, we found that PRV TJ has indistinguishable genomic DNA replication capability and axonal retrograde transport dynamics compared to the PRV SC. To gain deeper insights into the mechanisms underlying these differences, we constructed gene-interchanged chimeric virus constructs and assessed the affinity between envelope glycoprotein B, C, and D (gD) and corresponding receptors. Our findings confirmed that mutations in these envelope proteins, particularly gD, significantly contributed to the heightened attachment and penetration capabilities of PRV TJ. Our study revealed the critical importance of the gD^ΔR278/P279 and gD^V338A in facilitating viral invasion. Furthermore, our observations indicated that mutations in envelope proteins have a more significant impact on viral invasion than on virulence in the mouse model. Our findings provide valuable insights into the roles of natural mutations on the PRV envelope glycoproteins in cell tropism, which sheds light on the relationship between cell tropism and clinical symptoms and offers clues about viral evolution.
Isolation and identification of two novel pseudorabies viruses with natural recombination or TK gene deletion in China
2023, Veterinary Microbiology
Pseudorabies virus (PRV), the causative agent of Aujeszky's disease, has gained increased attention in China in recent years due to outbreaks of emergent pseudorabies. However, there is limited information about the evolution and pathogenicity of emergent PRV field strains in China. In this study, two PRV field strains were isolated from an intensive pig farm with suspected PRV infection. These were named the GXLB-2015 and GXGG-2016 strains and their growth characteristics together with their genome sequences and pathogenicity were determined. Nucleotide homology and phylogenetic analysis revealed the GXLB-2015 stain was relatively close to the foreign PRV isolated strains with respect to the whole genome sequence. However, it formed an independent branch between the foreign PRV isolates and the previous PRV variants isolated in China. Further recombination and genetic evolution analysis showed that the GXLB-2015 strain was a natural recombinant between the Bartha strain and PRV variants. The GXGG-2016 strain was highly homologous with the Chinese classical strains, but it has a natural deletion of 69 aa in the thymidine kinase (TK) gene. Pathogenicity analysis showed that, the GXLB-2015 strain had the strongest pathogenicity to mice with an LD₅₀ of 10^3.5, while the GXGG-2016 strain with the TK gene deletion was not pathogenic to mice. Taken together, our data provide direct evidence for the genomic recombination and natural TK gene deletion of PRVs, which may provide a reference for a better understanding of PRV evolution in China and contribute to the clinical control of PRV infection in pig farms.
Recombinant porcine Interferon-α and Interleukin-2 fusion protein (rPoIFNα+IL-2) shows potent anti-pseudorabies virus activity in vitro and in vivo
2023, Veterinary Microbiology
Pseudorabies virus (PRV) variants have been widely prevalent since 2011, leading to substantial losses to the swine industry. Although PRV can cause cross-species transmission and induce human infection, no drugs can currently prevent PRV infection. Interferons (IFNs) and interleukin-2 (IL-2) are important cytokines that mediate several biological functions including antiviral activity and immune regulation. In this study, we expressed and purified a recombinant porcine IFN-α and IL-2 fusion protein (rPoIFNα+IL-2), which did not show a cytotoxic effect on PK-15 cells. The antiviral activity was evaluated in PK-15 cells using the cytopathic effect inhibition method, and the results indicated that rPoIFNα+IL-2 can inhibit the replication of PRV, with an antiviral activity of approximately 10⁴ U/mL. Moreover, the proliferation of peripheral blood mononuclear cells was enhanced by rPoIFNα+IL-2. Additionally, rPoIFNα+IL-2 substantially increased the expression of IFN-stimulated genes, including IFIT1, ISG15, MX1, and OAS, which are critical for antiviral activity. Furthermore, rPoIFNα+IL-2 alleviated the clinical symptoms and reduced mortality in mice infected with PRV. Simultaneously, rPoIFNα+IL-2 increased the expression levels of IFN-γ and IL-10 and inhibited the expression of IL-1β and IL-6. Additionally, the viral DNA copies in different tissues in the rPoIFNα+IL-2-treated group were lower than those in the untreated group. These findings indicate that rPoIFNα+IL-2 may serve as an antiviral agent for the prevention and treatment of PRV infection and may expand the potential function of IFN antiviral drugs in the future.

View all citing articles on Scopus

View full text

Emergence of a Pseudorabies virus variant with increased virulence to piglets

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Cells and viruses

The PRV SC- or JS-2012-specific neutralizing antibodies induced by Bartha-K61 vaccine

Bartha-K61 vaccine provided complete protection against SC but not against JS-2012

Discussion

Acknowledgments

Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China, 2012

Emerg. Infect. Dis.

Potency of an experimental DNA vaccine against Aujeszky’s disease in pigs

Vet. Microbiol.

A novel inactivated gE/gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerged variant PRV challenge

Virus Res.

Variation of Aujeszky’s disease viruses in wild swine in USA

Vet. Microbiol.

Economic aspects of the control of Aujeszky’s disease in the European Community

Acta Vet. Hung.

A review of pseudorabies (Aujeszky’s disease) in pigs

Can. Vet. J

Pathogenicity and genomic characterization of a pseudorabies virus variant isolated from Bartha-K61-vaccinated swine population in China

Vet. Microbiol.

Pseudorabies virus infection in mink: a host-specific pathogenesis

Vet. Immunol. Immunopathol.

The role of biotechnologically engineered vaccines and diagnostics in pseudorabies (Aujeszky’s disease) eradication strategies

Vet. Microbiol.

Immunobiology of pseudorabies (Aujeszky’s disease)

Vet. Immunol. Immunopathol.

Pseudorabies virus in wild swine: a global perspective

Arch. Virol.

Eradication of Aujeszky’s disease from New Zealand pig herds 1976–1997

Vet. Rec.