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The pseudorabies virus (PRV) gene encoding thymidine kinase (tk) is an important virulence-associated factor. Attenuation of PRV in susceptible animals is a frequent result of tk deletion. The aim of the study was to assess the pathogenicity of tk-deleted PRV in rats.

Background: Pseudorabies virus (PRV) of the family Herpesviridae is the causative agent of Aujeszky's disease. Attenuation of PRV by serial passaging in vitro is a well-established method; however, the dynamic variations occurring on viral genome during this process have not been characterized. Methods: Genome sequencing and comparative genomic analyses of a virulent pseudorabies virus and a series of its plaque-purified strains via serial passaging in vitro were performed, and the properties in vitro and in vivo of which were further characterized. Results: Compared to the parental virus, replication in vitro was enhanced in the highly passaged F50, F91, and F120. In contrast, lethality in mice decreased gradually with passage number. Genome sequencing of F50, F91, and F120 showed deletion of a large fragment containing gE, which is likely related to their attenuation. In addition, single nucleotide variations were identified in many genes of F50, F91, and F120. In-frame and frameshift indels were also detected in specific genes of passaged strains. Particularly frameshift mutations were observed in highly passaged strains, resulting in a truncated but overexpressed pUL46. Conclusion: During attenuation of PRV by serial passaging in Vero cells, dynamic variation patterns including a large deletion, single nucleotide variations, small in-frame indels, and also frameshifts mutations successively emerged, contributing to evolution of the viral population and enabling the gradual attenuation of the virus. These data provide clues to better understand PRV attenuation during passaging. [ABSTRACT FROM AUTHOR]

Since late 2011, a pseudorabies virus (PRV) variant with increased virulence in old pigs had caused major disease outbreaks and great economic losses to the pig industry in China. The gene mutations that contributed to the increased virulence were unclear. To study the basis of the enhanced pathogenicity, an infectious bacterial artificial chromosome (BAC) clone consisting of the complete genome of the PRV variant was developed. Using homologous recombination and Cre/LoxP recombination, the recombinant virus rJS-2012-BAC carrying a BAC insertion downstream of the open reading frame (ORF) of gG was constructed. The circular genome of rJS-2012-BAC was extracted from infected Vero cells and transformed into Escherichia coli DH10B, generating the BAC clone pBAC-JS2012. The loxP sites flanking the BAC vector were used to excise the BAC sequences using Cre recombinase. The reconstituted BAC-excision virus, vJS2012 L, from pBAC-JS2012 exhibited similar biological properties to the wild-type virulent strain JS-2012. To manipulate the BAC clone pBAC-JS2012, the galK selection system was adopted to delete the gI/gE gene from pBAC-JS2012 in E. coli and to generate the gI/gE-deleted virus vJS2012-ΔgE/gI. The BAC clone, pBAC-JS2012, retained the same level of virulence as its parent strain and was easily manipulated using a galK system which would facilitate the study of the enhanced pathogenicity of the PRV variant as well as other studies on PRV. [ABSTRACT FROM AUTHOR]

Emerging pseudorabies virus (PRV) variant have led to pseudorabies outbreaks in Chinese pig farms. The commercially available PRV vaccine provides poor protection against the PRV variant. In this study, a gE/gI deleted PRV strain JS-2012-△gE/gI was generated from a PRV variant strain using homologous DNA recombination. Compared to the parental strain JS-2012, JS-2012-△gE/gI grew slowly and showed small plaque morphology on Vero cells. The safety and immunological efficacy of JS-2012-△gE/gI was evaluated as a vaccine candidate. JS-2012-△gE/gI was avirulent to suckling piglets, but was able to provide full protection for young piglets against challenge with both the classical virulent PRV and the emerging PRV variant. After sows were vaccinated with the gE/gI-deleted strain, their suckling offspring were resistant to an otherwise lethal challenge with the classical and the variant PRVs. Piglets inoculated with JS-2012-△gE/gI did not develop PRV-specific gE-ELISA antibodies. Thus, JS-2012-△gE/gI appears to be a promising marker vaccine candidate to control PRV variant circulating in pig farms in China. [ABSTRACT FROM AUTHOR]

• ZCCHC3 overexpression inhibits PRV proliferation. • ZCCHC3 knockdown facilitates PRV proliferation. • ZCCHC3 positively regulates IFN-β expression. • PRV infection reduces endogenous ZCCHC3 expression. • PRV UL13 and UL24 proteins suppress the expression of ZCCHC3. Zinc finger CCHC-type containing protein 3 (ZCCHC3) acts as an antiviral factor that interacts with RIG-I and cGAS to modulate innate signaling against viral infections. Here, we investigated the role of porcine ZCCHC3 during pseudorabies virus (PRV) proliferation. We found that porcine ZCCHC3 plays an inhibitory role in the proliferation of PRV by regulating cellular innate immune responses. Further, overexpression of ZCCHC3 inhibited gB protein levels and viral titers, whereas knockdown of ZCCHC3 promoted viral growth. ZCCHC3 overexpression increased IFN-β expression to upregulate downstream gene expression, thus leading to the suppression of viral replication. However, PRV infection reduced the endogenous expression of ZCCHC3 in permissive cells. Importantly, PRV-encoded UL13 and UL24 proteins were identified to inhibit the expression of ZCCHC3, thus antagonizing its antiviral effect. Collectively, our data underscore the important role of ZCCHC3 against PRV infection and promote understandings of viral proteins in PRV pathogenesis. [ABSTRACT FROM AUTHOR]

• PRV pUL16 localized to the mitochondria, nuclei and cytoplasm. • PRV pUL16 weakened the inhibition of LRPPRC for the growth of PRV. • LRPPRC influenced STING-mediated activation of NF-κB. Pseudorabies is caused by pseudorabies virus (PRV), a member of the Herpesvirus family, and has caused tremendous damage to the pig industry. Protein unique lone 16 (pUL16) is a conserved envelope protein in all herpesviruses, that is known to play an important role in several aspects, including virus diffusion in cells and virulence in mice. It has been shown that the pUL16 can interact with the virus proteins UL11, UL49, UL21, gD, and gE. However, the research to date on pUL16 has only focused on etiology, without discussing the possible cellular pathways involved in PRV infection. Leucine-rich PPR motif-containing protein (LRPPRC) is a multifunctional cellular protein that participates in various cellular processes, such as RNA processing, splicing, stabilization, editing, translation, and energy metabolism. This was the first caspase-independent apoptosis protein to be identified. In this study, immune precipitation and mass spectrometry was performed to define the function of the pUL16 in PRV infection to study the possible cellular pathways in which pUL16 may participate. It was found that LRPRRC could interact with PRV pUL16, which may indicate that UL16 is involved in a redox reaction or cellular apoptosis. This is the first study of the interaction between pUL16 and host proteins, which has positive significance to gain a further understanding of the pUL16. [ABSTRACT FROM AUTHOR]

• pUL16 interacted with VP26. • Deletion of UL16 and UL35 significantly reduced the viral characteristics. Pseudorabies virus (PRV) is related to alphaherpesvirus and varicellovirus. pUL16 is a conserved protein in all herpesviruses, and studies have shown that UL16 can interact with the viral proteins pUL11, pUL49, pUL21, gD, and gE. In this study, we found that pUL16 interacted with the viral capsid protein VP26, which could not translocate into the nucleus itself but did appear in the nucleus. We further determined whether pUL16 assists the translocation of VP26 into the nucleus. We found that pUL16 interacted with VP26 with or without viral proteins, and since VP26 itself did not contain a nuclear location signal, we concluded that pUL16 assisted the translocation of VP26 into the nucleus. Deletion of UL16 and UL35 significantly reduced the 50 % tissue culture infective dose, virulence, attachment, and internalization of PRV in cells. These results show that the interaction between pUL16 and VP26 influences the growth and virulence of pseudorabies virus. Our research is the first study to show that pUL16 interacts with VP26, which may explain the targeting site of UL16 and viral capsids. It is also the first to show that UL16 assists the transport of other viral proteins to organelles. Previous researches on pUL16 usually emphasized its interaction with pUL11, pUL21, and gE, and sometimes commented on pUL49 and gD. Our research focuses on the novel interaction between pUL16 and VP26, thereby enriching the studies on herpesviruses and possibly providing different directions for researchers. [ABSTRACT FROM AUTHOR]

• PRV infection leads to endogenous OASL downregulation. • RIG-I is required for OASL-mediated anti-PRV activity. • OASL exerts inhibitory effects on PRV proliferation. • PRV UL24 impairs RIG-I signaling. • PRV UL24 reduces OASL transcription in an IRF3-dependent manner. Oligoadenylate synthetases-like (OASL) protein exerts various effects on DNA and RNA viruses by inhibiting cGAS-mediated IFN production and by enhancing RIG-I-mediated IFN induction, respectively. In this study, we aimed to examine the role of OASL in pseudorabies virus (PRV) proliferation and investigate the function of the PRV UL24 protein in cellular innate immunity. We found that OASL regulates PRV proliferation by enhancing RIG-I signaling. PRV infection decreased the expression of OASL at both the mRNA and protein levels in PK15 and HeLa cells. OASL expression suppressed the proliferation of PRV in a RIG-I-dependent manner and boosted RIG-I-mediated IFN expression as well as IFN-stimulated gene (ISG) induction. In contrast, knockdown of OASL enhanced PRV proliferation and reduced RIG-I signaling. However, the PRV UL24 protein was found to impair RIG-I signaling, thus inhibiting transcription of IFN and ISGs. In addition, the UL24 protein reduced RIG-I-induced expression of endogenous OASL in an IRF3-dependent manner, thereby antagonizing the OASL antiviral effect. Taken together, our findings characterize the role of OASL in PRV proliferation and provide new insights into the role of UL24 in PRV pathogenesis. [ABSTRACT FROM AUTHOR]

• Pseudorabies Virus UL24 inhibited IFN-β. • UL24 antagonized cGAS-STING signal pathway. • UL24 interacted with and degraded IRF7 via the proteasome pathway. The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway plays an important role in the innate immune response by the production of type I interferon (IFN) against DNA virus infection. However, viruses have evolved a variety of strategies to antagonize the host antiviral response to facilitate infection and replication. Pseudorabies virus (PRV), a DNA virus that causes great economic losses to the swine industry, encodes approximate 70 proteins, including some that are involved in evasion of host immunity. However, the mechanism employed by PRV to regulate type I IFN remains unclear. The results of the present study showed that the transcription levels of type I IFN were significantly upregulated by a UL24-deleted PRV strain. Furthermore, IFN-β activation induced by poly(dA:dT) or stimulated by cGAS-STING was inhibited by UL24 overexpression in PK15 cells. Co-immunoprecipitation analysis demonstrated that UL24 interacts with and can degrade interferon regulatory factor 7 (IRF7) through the proteasome pathway in a dose-dependent manner. Together, these results showed that PRV UL24 interacted with IRF7 via the proteasome pathway and antagonized cGAS-STING-mediated activation of IFN-β. [ABSTRACT FROM AUTHOR]