The mutations on the envelope glycoprotein D contribute to the enhanced neurotropism of the pseudorabies virus variant.

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 gDΔR278/P279 and gDV338A 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.

Genome-wide analysis of long noncoding RNA profiles in pseudorabies-virus-infected PK15 cells.

Recently, an increasing number of studies have shown that long noncoding RNAs (lncRNAs) are involved in host metabolism after infection with pseudorabies virus (PRV). In our study, via RNA sequencing analysis, a total of 418 mRNAs, 137 annotated lncRNAs, and 312 new lncRNAs were found to be differentially expressed. These lncRNAs were closely associated with metabolic regulation and immunity-related signalling pathways, including the T-cell receptor signalling pathway, chemokine signalling pathway, mitogen-activated protein kinase (MAPK) signalling pathway, TNF signalling pathway, Ras signalling pathway, calcium signalling pathway, and phosphatidylinositol signalling system. Real-time PCR indicated that several mRNAs and lncRNAs involved in the regulation of the immune effector process, T-cell receptor signalling pathway, TNF signalling pathway, MAPK signalling pathway, and chemokine signalling pathways were significantly expressed. These mRNAs and lncRNAs might play a role in PRV infection.

A monoclonal antibody neutralizes pesudorabies virus by blocking gD binding to the receptor nectin-1.

Pseudorabies virus (PRV) was isolated from some human cases recently and the infected patients manifested respiratory dysfunction and acute neurological symptoms. However, no effective drug or vaccine, preventing the progression of PRV infection, is available. Nectin-1 was the only reported receptor for PRV cell entry both swine and human origin, representing an excellent target to block PRV infection, and especially its transmission from pigs to humans. A PRV-gD specific mAbs (10B6) was isolated from hybridomas and its neutralizing activities in vitro and in vivo were determined. 10B6 exhibited effective neutralizing activities in vitro with IC50 = 2.514 µg/ml and 4.297 µg/ml in the presence and absence of complement. And in vivo, 10B6 provided 100% protection against PRV lethal challenge with a dose of 15 mg/kg. Further, 10B6 could bind to a conserved epitope, 316QPAEPFP322, locating in gD pro-fusion domain, and finally blocks the binding of PRV-gD to nectin-1. Moreover, 10B6 showed an effective inhibition on PRV cell-attachment in a cell type-independent manner and could also block the virus spreading among cells. 10B6 exhibited effectively neutralizing activities to Chinese PRV variant strain in vitro and in vivo by blocking gD binding to nectin-1, implied both prophylactic and therapeutic interventions against PRV infections.

Deletion of the Pseudorabies Virus gE/gI-US9p complex disrupts kinesin KIF1A and KIF5C recruitment during egress, and alters the properties of microtubule-dependent transport in vitro.

During infection of neurons by alphaherpesviruses including Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) viral nucleocapsids assemble in the cell nucleus, become enveloped in the cell body then traffic into and down axons to nerve termini for spread to adjacent epithelia. The viral membrane protein US9p and the membrane glycoprotein heterodimer gE/gI play critical roles in anterograde spread of both HSV-1 and PRV, and several models exist to explain their function. Biochemical studies suggest that PRV US9p associates with the kinesin-3 motor KIF1A in a gE/gI-stimulated manner, and the gE/gI-US9p complex has been proposed to recruit KIF1A to PRV for microtubule-mediated anterograde trafficking into or along the axon. However, as loss of gE/gI-US9p essentially abolishes delivery of alphaherpesviruses to the axon it is difficult to determine the microtubule-dependent trafficking properties and motor-composition of Δ(gE/gI-US9p) particles. Alternatively, studies in HSV-1 have suggested that gE/gI and US9p are required for the appearance of virions in the axon because they act upstream, to help assemble enveloped virions in the cell body. We prepared Δ(gE/gI-US9p) mutant, and control parental PRV particles from differentiated cultured neuronal or porcine kidney epithelial cells and quantitated the efficiency of virion assembly, the properties of microtubule-dependent transport and the ability of viral particles to recruit kinesin motors. We find that loss of gE/gI-US9p has no significant effect upon PRV particle assembly but leads to greatly diminished plus end-directed traffic, and enhanced minus end-directed and bidirectional movement along microtubules. PRV particles prepared from infected differentiated mouse CAD neurons were found to be associated with either kinesin KIF1A or kinesin KIF5C, but not both. Loss of gE/gI-US9p resulted in failure to recruit KIF1A and KF5C, but did not affect dynein binding. Unexpectedly, while KIF5C was expressed in undifferentiated and differentiated CAD neurons it was only found associated with PRV particles prepared from differentiated cells.

Effects of gE/gI deletions on the miRNA expression of PRV-infected PK-15 cells.

Pseudorabies virus (PRV) belongs to the Alphaherpesvirinae subfamily of Herpesviridae. PRV-induced pseudorabies is a highly contagious disease that has caused huge economic losses to the global swine industry. The PRV gE/gI gene deletion vaccine strain (Fa ΔgE/gI strain) constructed from the PRV Fa wild-type strain was shown to have a protective effect against infection. However, the interaction between PRV gE/gI genes and host miRNA needs further exploration, and little is known about the regulatory mechanisms of non-coding RNAs during PRV infection. miRNAs play a key regulatory role in viral infection and immune responses, so we analyzed the differential expression of miRNAs induced by the PRV Fa ΔgE/gI strain and Fa wild-type strain in the PK15 cell line. High-throughput sequencing reads were aligned to known Sus scrofa pre-miRNAs in the miRBase database. Target genes of differentially expressed miRNAs were predicted using the miRGen 3.0 database, then filtered miRNA target genes were subjected to Gene Ontology (GO) analysis and Search Tool for the Retrieval of Interacting Genes/ Proteins (STRING) analysis. Stem-loop quantitative real-time PCR was performed to confirm the accuracy of high-throughput sequencing data. In total, 387, 472, and 490 annotated and novel mature miRNAs were identified from PRV Fa ΔgE/gI strain-infected, Fa wild-type strain-infected, and non-infected PK-15 cells, respectively. Five PRV-encoded miRNAs were also identified. GO analysis showed that target genes of differentially expressed miRNAs in PRV Fa ΔgE/gI strain-infected and Fa wild-type strain-infected PK-15 cells were mainly involved in biological regulation and metabolic processes. STRING analysis showed that immune-related target genes of differentially expressed miRNAs in the Toll-like receptor signaling pathway, B cell receptor signaling pathway, T cell receptor signaling pathway, nuclear factor-κB signaling pathway, and transforming growth factor-ß signaling pathway were interrelated. This is the first report of the small RNA transcriptome in PRV mutant wild-type strain-infected and Fa ΔgE/gI strain-infected porcine cell lines. Our findings will contribute to the prevention and treatment of PRV mutant strains.

An improved animal model for herpesvirus encephalitis in humans.

Herpesviral encephalitis caused by Herpes Simplex Virus 1 (HSV-1) is one of the most devastating diseases in humans. Patients present with fever, mental status changes or seizures and when untreated, sequelae can be fatal. Herpes Simplex Encephalitis (HSE) is characterized by mainly unilateral necrotizing inflammation effacing the frontal and mesiotemporal lobes with rare involvement of the brainstem. HSV-1 is hypothesized to invade the CNS via the trigeminal or olfactory nerve, but viral tropism and the exact route of infection remain unclear. Several mouse models for HSE have been developed, but they mimic natural infection only inadequately. The porcine alphaherpesvirus Pseudorabies virus (PrV) is closely related to HSV-1 and Varicella Zoster Virus (VZV). While pigs can control productive infection, it is lethal in other susceptible animals associated with severe pruritus leading to automutilation. Here, we describe the first mutant PrV establishing productive infection in mice that the animals are able to control. After intranasal inoculation with a PrV mutant lacking tegument protein pUL21 and pUS3 kinase activity (PrV-ΔUL21/US3Δkin), nearly all mice survived despite extensive infection of the central nervous system. Neuroinvasion mainly occurred along the trigeminal pathway. Whereas trigeminal first and second order neurons and autonomic ganglia were positive early after intranasal infection, PrV-specific antigen was mainly detectable in the frontal, mesiotemporal and parietal lobes at later times, accompanied by a long lasting lymphohistiocytic meningoencephalitis. Despite this extensive infection, mice showed only mild to moderate clinical signs, developed alopecic skin lesions, or remained asymptomatic. Interestingly, most mice exhibited abnormalities in behavior and activity levels including slow movements, akinesia and stargazing. In summary, clinical signs, distribution of viral antigen and inflammatory pattern show striking analogies to human encephalitis caused by HSV-1 or VZV not observed in other animal models of disease.

A kinesin-3 recruitment complex facilitates axonal sorting of enveloped alpha herpesvirus capsids.

Axonal sorting, the controlled passage of specific cargoes from the cell soma into the axon compartment, is critical for establishing and maintaining the polarity of mature neurons. To delineate axonal sorting events, we took advantage of two neuroinvasive alpha-herpesviruses. Human herpes simplex virus 1 (HSV-1) and pseudorabies virus of swine (PRV; suid herpesvirus 1) have evolved as robust cargo of axonal sorting and transport mechanisms. For efficient axonal sorting and subsequent egress from axons and presynaptic termini, progeny capsids depend on three viral membrane proteins (Us7 (gI), Us8 (gE), and Us9), which engage axon-directed kinesin motors. We present evidence that Us7-9 of the veterinary pathogen pseudorabies virus (PRV) form a tripartite complex to recruit Kif1a, a kinesin-3 motor. Based on multi-channel super-resolution and live TIRF microscopy, complex formation and motor recruitment occurs at the trans-Golgi network. Subsequently, progeny virus particles enter axons as enveloped capsids in a transport vesicle. Artificial recruitment of Kif1a using a drug-inducible heterodimerization system was sufficient to rescue axonal sorting and anterograde spread of PRV mutants devoid of Us7-9. Importantly, biophysical evidence suggests that Us9 is able to increase the velocity of Kif1a, a previously undescribed phenomenon. In addition to elucidating mechanisms governing axonal sorting, our results provide further insight into the composition of neuronal transport systems used by alpha-herpesviruses, which will be critical for both inhibiting the spread of infection and the safety of herpesvirus-based oncolytic therapies.

Porcine IFITM1 is a host restriction factor that inhibits pseudorabies virus infection.

Interferon-inducible transmembrane proteins (IFITMs) restrict infection by several viruses, such as influenza A virus, West Nile virus and dengue virus. It has not been determined whether porcine IFITMs (pIFITMs) inhibit infection by pseudorabies virus (PRV), an enveloped, double-stranded DNA virus, which is the etiological agent of Aujeszky's disease in pigs. Here, we report that PRV infection elicited pIFITM1 expression in PK15 porcine kidney epithelial cells and 3D4/21 alveolar macrophages. pIFITM2 and pIFITM3 expression was only elevated in PK15 cells during PRV infection. Depletion of pIFITM1 using RNA interference, either in PK15 or in 3D4/21 cells, enhanced PRV infection while overexpression of pIFITM1 had the opposite effect. Knockdown of pIFITM2 and pIFITM3 did not influence PRV infection, suggesting that pIFITM2 and pIFITM3 are independent of PRV infection. PRV-induced pIFITM1 expression was dependent on the cGAS/STING/TBK1/IRF3 innate immune pathway and interferon-alpha receptor-1, suggesting that pIFITM1 is up-regulated by the type I interferon signaling pathway. The anti-PRV role of pIFITM1 was inhibited upon PRV entry. Our data demonstrate that pIFITM1 is a host restriction factor that inhibits PRV entry that may shed light on a strategy for prevention of PRV infection.

Exploring the tissue tropism of pseudorabies virus based on miRNA level analysis.

BACKGROUND: Pseudorabies virus (PRV, or suid herpesvirus, SuHV-1), a member of the herpesvirus family, has an extremely broad host range and threatens the pig industry in China. PRV can evade host innate immunity and infect the kidney, lung, brain and other tissues. At the same time, many studies have reported that microRNA (miRNA) can affect the replication of viruses by regulating gene expression levels. RESULTS: Here, to identify changes in miRNA expression and post-transcriptional regulation associated with PRV infection in the lung, spleen, and olfactory bulb, we sequenced small RNAs in tissues of rats infected or uninfected with PRV strain XJ (PRV-XJ). Sixty-one, 199 and 29 differentially-expressed miRNAs were identified in the lung, spleen, and olfactory bulb, respectively, of infected compared with uninfected rats. Among the miRNAs differentially-expressed in PRV-infected rats, 36, 171, and 15 miRNAs showed tissue-selective expression in the olfactory bulb, lung and spleen, respectively. All differentially-expressed miRNAs were analyzed for their GO functional annotations and KEGG pathway associations . CONCLUSIONS: In PRV-XJ-infected rats, miRNAs were differentially expressed in the lung, spleen and olfactory bulb. These miRNAs were involved in regulating various pathways of the nervous, respiratory and immune systems, and may affect the tissue tropism of the virus and play pivotal roles in viral infection and proliferation.

Isolation and molecular characterization of a variant of Chinese gC-genotype II pseudorabies virus from a hunting dog infected by biting a wild boar in Japan and its pathogenicity in a mouse model.

We isolated a variant of Chinese pseudorabies virus from a hunting dog with symptoms similar to Aujeszky's disease and designated the isolate MY-1 strain. The dog developed symptoms 6 days after hunting and biting a wild boar and died the day after onset. The Bam HI restriction profile of MY-1 DNA was different from those of the Japanese reference strain Yamagata-S81 and two vaccine strains, Bartha and Begonia, and resembled Bam HI-RFLP (restriction fragment length polymorphism) type IV. Complete nucleotide sequences were determined, and phylogenetic analyses revealed that MY-1 belonged to the same cluster of old Chinese strains and variant strains isolated recently in China, but most of the open reading frames of MY-1 were located on a different branch from those of these Chinese strains. Based on a gC phylogenetic analysis, MY-1 belonged to gC-genotype II composed of those Chinese strains. In mice, the 50% lethal dose (LD50) of MY-1 (103.0 TCID50) was almost the same as those of Yamagata-S81 and Bartha. The LD50 value of Begonia was 10≥4.5 TCID50. The mean survival periods of mice after infection with 104 TCID50 of MY-1, Yamagata-S81 and Bartha were 3.9 days, 2.3 days, and 8.0 days, respectively. The results suggested that the variant of Chinese PRV with slightly weaker pathogenicity than that of wild virulent viruses might be maintained in wild boars in Japan. Furthermore, we would like to propose that old Chinese strains, recent Chinese variant strains, and MY-1 should be grouped as an Asian type PRV.

Acyloxyacyl hydrolase modulates pelvic pain severity.

Chronic pelvic pain causes significant patient morbidity and is a challenge to clinicians. Using a murine neurogenic cystitis model that recapitulates key aspects of interstitial cystitis/bladder pain syndrome (IC), we recently showed that pseudorabies virus (PRV) induces severe pelvic allodynia in BALB/c mice relative to C57BL/6 mice. Here, we report that a quantitative trait locus (QTL) analysis of PRV-induced allodynia in F2CxB progeny identified a polymorphism on chromosome 13, rs6314295 , significantly associated with allodynia (logarithm of odds = 3.11). The nearby gene encoding acyloxyacyl hydrolase ( Aoah) was induced in the sacral spinal cord of PRV-infected mice. AOAH-deficient mice exhibited increased vesicomotor reflex in response to bladder distension, consistent with spontaneous bladder hypersensitivity, and increased pelvic allodynia in neurogenic cystitis and postbacterial chronic pain models. AOAH deficiency resulted in greater bladder pathology and tumor necrosis factor production in PRV neurogenic cystitis, markers of increased bladder mast cell activation. AOAH immunoreactivity was detectable along the bladder-brain axis, including in brain sites previously correlated with human chronic pelvic pain. Finally, AOAH-deficient mice had significantly higher levels of bladder vascular endothelial growth factor, an emerging marker of chronic pelvic pain in humans. These findings indicate that AOAH modulates pelvic pain severity, suggesting that allelic variation in Aoah influences pelvic pain in IC.

Dunaliella salina alga extract inhibits the production of interleukin-6, nitric oxide, and reactive oxygen species by regulating nuclear factor-κB/Janus kinase/signal transducer and activator of transcription in virus-infected RAW264.7 cells.

Recent investigations have demonstrated that carotenoid extract of Dunaliella salina alga (Alga) contains abundant ß-carotene and has good anti-inflammatory activities. Murine macrophage (RAW264.7 cells) was used to establish as an in vitro model of pseudorabies virus-induced reactive oxygen species (ROS) response. In this study, antioxidant activities of Alga were measured based on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, trolox equivalent antioxidant capacity assays, reducing power, and virus-induced ROS formation in RAW264.7 cells. Anti-inflammatory activities of Alga were assessed by its ability to inhibit the production of interleukin-6 and nitric oxide (NO) using enzyme-linked immunosorbent assay, then the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway was investigated by measuring the inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear factor-κB (p50 and p65), JAK, STAT-1/3, and suppressor of cytokine signaling 3 (SOCS3) by Western blotting. In addition, Alga inhibited virus replication by plaque assay. Our results showed that the Alga had high antioxidant activity, significantly reduced the virus-induced accumulation of ROS, and inhibited the levels of nitric oxide and interleukin-6. Further studies revealed that Alga also downregulated the gene and protein expressions of iNOS, COX-2, nuclear factor-κB (p50 and p65), and the JAK/STAT pathway. The inhibitory effects of Alga were similar to pretreatment with specific inhibitors of JAK and STAT-3 in pseudorabies virus -infected RAW264.7 cells. Alga enhanced the expression of SOCS3 to suppress the activity of the JAK/STAT signaling pathway in pseudorabies virus-infected RAW264.7 cells. In addition, Alga has decreased viral replication (p < 0.005) at an early stage. Therefore, our results demonstrate that Alga inhibits ROS, interleukin6, and nitric oxide production via suppression of the JAK/STAT pathways and enhanced the expression of SOCS3 in virus-infected RAW264.7 cells.

Structural basis of nectin-1 recognition by pseudorabies virus glycoprotein D.

An early and yet indispensable step in the alphaherpesvirus infection is the engagement of host receptors by the viral envelope glycoprotein D (gD). Of the thus-far identified gD receptors, nectin-1 is likely the most effective in terms of its wide usage by multiple alphaherpesviruses for cell entry. The molecular basis of nectin-1 recognition by the gD protein is therefore an interesting scientific question in the alphaherpesvirus field. Previous studies focused on the herpes simplex virus (HSV) of the Simplexvirus genus, for which both the free gD structure and the gD/nectin-1 complex structure were reported at high resolutions. The structural and functional features of other alphaherpesviral gDs, however, remain poorly characterized. In the current study, we systematically studied the characteristics of nectin-1 binding by the gD of a Varicellovirus genus member, the pseudorabies virus (PRV). We first showed that PRV infects host cells via both human and swine nectin-1, and that its gD exhibits similar binding affinities for nectin-1 of the two species. Furthermore, we demonstrated that removal of the PRV gD membrane-proximal residues could significantly increase its affinity for the receptor binding. The structures of PRV gD in the free and the nectin-1-bound states were then solved, revealing a similar overall 3D fold as well as a homologous nectin-1 binding mode to its HSV counterpart. However, several unique features were observed at the binding interface of PRV gD, enabling the viral ligand to utilize different gD residues (from those of HSV) for nectin-1 engagement. These observed binding characteristics were further verified by the mutagenesis study using the key-residue mutants of nectin-1. The structural and functional data obtained in this study, therefore, provide the basis of receptor recognition by PRV gD.

Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus.

Recombinase polymerase amplification assays using real-time fluorescent detection (real-time RPA assay) and lateral flow dipstick (RPA LFD assay) were developed targeting the gD gene of pseudorabies virus (PRV). Both assays were performed at 39 °C within 20 min. The sensitivity of the real-time RPA assay and the RPA LFD assay was 100 copies per reaction and 160 copies per reaction, respectively. Both assays did not detect DNAs from other virus or PRV negative samples. Therefore, the developed RPA assays provide a rapid, simple, sensitive and specific alternative tool for detection of PRV.

Age-Dependent Differences in Pseudorabies Virus Neuropathogenesis and Associated Cytokine Expression.

The severity of clinical symptoms induced by pseudorabies virus (PRV) infection of its natural host is inversely related to the age of the pig. During this study, 2- and 15-week-old pigs were inoculated with PRV strain NIA3. This resulted in important clinical disease, although the associated morbidity and mortality were lower in older pigs. Quantitative PCR analysis of viral DNA in different organs confirmed the general knowledge on PRV pathogenesis. Several new findings and potential explanations for the observed age-dependent differences in virulence, however, were determined from the study of viral and cytokine mRNA expression at important sites of neuropathogenesis. First, only limited viral and cytokine mRNA expression was detected in the nasal mucosa, suggesting that other sites may serve as the primary replication site. Second, PRV reached the trigeminal ganglion (TG) and brain stem rapidly upon infection but, compared to 2-week-old pigs, viral replication was less pronounced in 15-week-old pigs, and the decrease in viral mRNA expression was not preceded by or associated with an increased cytokine expression. Third, extensive viral replication associated with a robust expression of cytokine mRNA was detected in the olfactory bulbs of pigs from both age categories and correlated with the observed neurological disease. Our results suggest that age-dependent differences in PRV-induced clinical signs are probably due to enhanced viral replication and associated immunopathology in immature TG and the central nervous system neurons of 2-week-old pigs and that neurological disease is related with extensive viral replication and an associated immune response in the olfactory bulb. IMPORTANCE: It is well known that alphaherpesvirus infections of humans and animals result in more severe clinical disease in newborns than in older individuals and that this is probably related to differences in neuropathogenesis. The underlying mechanisms, however, remain unclear. Pseudorabies virus infection of its natural host, the pig, provides a suitable infection model to study this more profoundly. We show here that the severe neurological disease observed in 2-week-old pigs does not appear to be related to a hampered innate immune response but is more likely to reflect the immature development state of the trigeminal ganglia (TG) and central nervous system (CNS) neurons, resulting in an inefficient suppression of viral replication. In 15-week-old pigs, viral replication was efficiently suppressed in the TG and CNS without induction of an extensive immune response. Furthermore, our results provide evidence that neurological disease could, at least in part, be related to viral replication and associated immunopathology in the olfactory bulb.

Identification and Analysis of Novel Viral and Host Dysregulated MicroRNAs in Variant Pseudorabies Virus-Infected PK15 Cells.

Pseudorabies (PR) is one of the most devastating diseases in the pig industry. To identify changes in microRNA (miRNA) expression and post-transcriptional regulatory responses to PRV infection in porcine kidney epithelial (PK15) cells, we sequenced a small RNA (sRNA) library prepared from infected PK15 cells and compared it to a library prepared from uninfected cells using Illumina deep sequencing. Here we found 25 novel viral miRNAs by high-throughput sequencing and 20 of these miRNAs were confirmed through stem-loop RT-qPCR. Intriguingly, unlike the usual miRNAs encoded by the α-herpesviruses, which are found clustered in the large latency transcript (LLT), these novel viral miRNAs are throughout the PRV genome like ß-herpesviruses. Viral miRNAs are predicted to target multiple genes and form a complex regulatory network. GO analysis on host targets of viral miRNAs were involved in complex cellular processes, including the metabolic pathway, biological regulation, stimulus response, signaling process and immune response. Moreover, 13 host miRNAs were expressed with significant difference after infection with PRV: 8 miRNAs were up-regulated and 5 miRNAs were down-regulated, which may affect viral replication in host cell. Our results provided new insight into the characteristic of miRNAs in response to PRV infection, which is significant for further study of these miRNAs function.

Pathogenicity and immunogenicity of a gE/gI/TK gene-deleted pseudorabies virus variant in susceptible animals.

A pseudorabies virus (PRV) variant with enhanced pathogenicity has emerged in many vaccinated swine herds in China since 2011. rPRVTJ-delgE/gI, a previously described gE/gI-deleted PRV based on the PRV variant TJ strain, has been shown to be avirulent to pigs yet virulent to sheep. To ensure desirable biosafety, we further deleted the thymidine kinase (TK) gene of rPRVTJ-delgE/gI to generate a gE/gI/TK-deleted mutant rPRVTJ-delgE/gI/TK, and evaluated its pathogenicity and immunogenicity in susceptible animals. Groups of mice (n=5), sheep (n=3), and pigs (n=4) were inoculated with different doses of rPRVTJ-delgE/gI/TK or rPRVTJ-delgE/gI, and clinical signs, viral shedding, pathological changes, and serum antibodies were examined following inoculation. The results showed that rPRVTJ-delgE/gI/TK displayed higher safety than rPRVTJ-delgE/gI for mice (10(3)-10(6) TCID50) and sheep (10(5) TCID50), and pigs inoculated with rPRVTJ-delgE/gI/TK (10(5) TCID50) induced PRV-specific antibodies and protection against lethal PRV challenge comparable to those immunized with rPRVTJ-delgE/gI. In conclusion, rPRVTJ-delgE/gI/TK has the potential to be developed as a vaccine for controlling the currently prevalent PR in China.

Roles of nucleic acid substrates and cofactors in the vhs protein activity of pseudorabies virus.

Pseudorabies virus (PrV) belongs to the α-herpesvirinae of which human simplex virus (HSV) is the prototype virus. One of the hallmarks of HSV infection is shutoff of protein synthesis that is mediated by various viral proteins including vhs (virion host shutoff), which is encoded by the UL41 gene. However, the function of PrV vhs is poorly understood. Due to the low sequence similarity (39.3%) between the HSV and PrV UL41 proteins, vhs might not share the same biochemistry characteristics. The purpose of this study was to characterize the nuclease activity of the PrV vhs protein with respect to substrate specificity, its requirements in terms of cofactors, and the protein regions, as well as key amino acids, which contribute to vhs activity. Our results indicated that, similar to HSV vhs, PrV vhs is able to degrade ssRNA and mRNA. However, PrV vhs also targeted rRNA for degradation, which is novel compared to the HSV-1 vhs. Activity assays indicated that Mg(2+) alone enhances RNA degradation mediated by PrV vhs, while K(+) and ATP are not sufficient to induce activity. Finally, we demonstrated that each of the four highly conserved functional boxes of PrV vhs contributes to RNA degradation and that, in particular, residues 152, 169, 171, 172, 173 343, 345, 352 and 356, which are conserved among α-herpesviruses, are key amino acids needed for PrV vhs ribonuclease activity.

Construction of a gE-Deleted Pseudorabies Virus and Its Efficacy to the New-Emerging Variant PRV Challenge in the Form of Killed Vaccine.

The new-emerging PRV variants plague the vaccinated pigs and caused huge economic loss to local pig industry in China since 2011. The current commercial PRV vaccines cannot provide complete protection as the new-emerging PRV variants are antigenically different from the classical viruses. It is urgent to develop more safe and effective PRV vaccines based on the current circulating field isolates. In this study, a gE gene-deleted PRV based on the PRV HN1201, a representative PRV variant, was generated and the efficacy was tested on 3-week-old pigs in the form of killed vaccine. After fatal PRV HN1201 challenge, all vaccinated pigs survived without showing any clinical symptoms, but all unvaccinated pigs exhibited pseudorabies-specific respiratory and neurological signs with 100% mortality rate within 6 days after infection. The vaccinated pigs developed high level of gB and neutralizing antibodies after vaccination which may correlate to the protection provided by vaccine. Therefore, this gE gene-deleted PRV could be a promising vaccine candidate for the control of currently epidemic pseudorabies in China.

Exposure of extensively farmed wild boars (Sus scrofa scrofa) to selected pig pathogens in Greece.

BACKGROUND: Increased density and distribution of wild boar populations are likely to promote interactions and transmission of certain pathogens, not only among wild boar but also from wild boar to livestock or humans and vice versa. OBJECTIVE: The purpose of this study was to determine seroprevalence against seven selected pathogens in wild boar living in four different areas in Greece. ANIMALS AND METHODS: In total, 359 serum samples were collected from extensively farmed wild boar (Sus scrofa scrofa) originating from four distinct geographical areas throughout Greece from April 2012 to August 2013. Samples were tested for antibodies to Actinobacillus pleuropneumoniae, African swine fever virus (ASFV), Aujeszky's disease virus (ADV), classical swine fever virus (CSFV), Erysipelothrix rhusiopathiae, Mycoplasma hyopneumoniae and porcine reproductive and respiratory syndrome virus (PRRSV). Prevalence was compared among the four regions using Fisher's exact test. RESULTS: Low overall seropositivities of 2.4% and 5.6% were detected for E. rhusiopathiae and PRRSV, respectively, higher ones for ADV (32.0%) and the highest (72.5% and 90.5%) for M. hyopneumoniae and A. pleuropneumoniae, respectively. All sera tested were found negative for antibodies directed against CSFV and ASFV. CONCLUSIONS: This is the first report of exposure of wild boars to selected pig pathogens in Greece. These results are indicative of the circulation of these pathogens in Greece with the exception of CSFV and ASFV and suggestive of the potential role of wild boars on their maintenance and transmission to their domestic counterparts and vice versa.