Association of THBS3 with Glycoprotein D Promotes Pseudorabies Virus Attachment, Fusion, and Entry.

Pseudorabies virus (PRV) is a neurotropic virus causing obvious neurological disorders and reproductive failure in pigs. PRV entry into target cells is a complex multistep process initiated by interacting viral envelope glycoproteins with cellular receptors. In the current study, we found that thrombospondin 3 (THBS3) plays an important role in PRV entry into target cells, indicating that THBS3 is a new PRV coreceptor. To confirm this hypothesis, the knockdown of THBS3 in several permissive cells inhibited PRV primary infection, and overexpression of THBS3 in PK15 cells promoted PRV infection. CRISPR-Cas9 knockout markedly reduced PRV infection in PK15 cells. Antibodies against THBS3 blocked PRV infection in naturally permissive target cells. Moreover, soluble THBS3 protein neutralized the infectivity of PRV. Mechanistically, THBS3 interacted with the PRV gD via its N and C termini to facilitate PRV binding in permissive and nonpermissive cells. Also, in the absence of Nectin-1, THBS3 promoted cell-to-cell fusion mediated by virus glycoproteins. While THBS3 alone could not increase virus entry, overexpression of it in the presence of Nectin-1 promoted virus entry into CHO-K1 cells. Our results have identified THBS3 as a critical player in PRV binding and subsequent membrane fusion and entry. IMPORTANCE Herpesvirus entry occurs through a cascade of virus-cell interactions, and multiple surface glycoproteins play a role in virus binding and entry during the virus invasion process. Early studies showed that attachment to cells by PRV, as well as other alphaherpesviruses, is mediated by interactions between the viral glycoprotein gC and cell membrane proteoglycans carrying heparan sulfate chains (HSPGs). However, gD may also be involved in virus binding in an HSPG-independent manner. To date, the respective cellular receptors are still unknown. In this report, we identified a host molecule, THBS3, involved in gD-mediated PRV binding and subsequent membrane fusion and entry, which increases our understanding of the initial events in alpha herpesvirus infections.

Pseudorabies virus kinase UL13 phosphorylates H2AX to foster viral replication.

The DNA damage response (DDR) pathway is critical for maintaining genomic integrity and sustaining organismal development. Viruses can either utilize or circumvent the DDR to facilitate their replication. Pseudorabies virus (PRV) infection was shown to induce apoptosis via stimulating DDR. However, the underlying mechanisms have not been fully explored to date. This study showed that PRV infection robustly activates the ATM and DNA-PK signaling pathways shortly after infection. However, inhibition of ATM, but not DNA-PK, could dampen PRV replication in cells. Importantly, we found that PRV-encoded serine/threonine kinase UL13 interacts with and subsequently phosphorylates H2AX. Furthermore, we found that UL13 deletion largely attenuates PRV neuroinvasiveness and virulence in vivo. In addtion, we showed that UL13 contributes to H2AX phosphorylation upon PRV infection both in vitro and in vivo, but does not affect ATM phosphorylation. Finally, we showed that knockdown of H2AX reduces PRV replication, while this reduction can be further enhanced by deletion of UL13. Taken together, we conclude that PRV-encoded kinase UL13 regulates DNA damage marker γH2AX and UL13-mediated H2AX phosphorylation plays a pivotal role in efficient PRV replication and progeny production.

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.

ATP-dependent activation of NLRP3 inflammasome in primary murine macrophages infected by pseudorabies virus.

Pseudorabies virus (PRV), an alphaherpesvirus, causes respiratory and reproductive diseases in pigs and severe nervous symptom in other susceptible hosts. Previous studies showed that PRV infection induced a systemic inflammatory response in mice, indicating that pro-inflammatory cytokines participated in viral neuropathy in mice. The pro-inflammatory cytokine IL-1ß is a key mediator of the inflammatory response and plays an important role in host-response to pathogens. However, the secretion of IL-1ß and its relationship with inflammasome activation during PRV infection remains poorly understood. In this study, we found that PRV infection caused significant secretion of several pro-inflammatory cytokines in macrophages and promoted IL-1ß secretion in an ATP-dependent manner. Furthermore, the expression of IL-1ß can be induced by only PRV infection and depended on NF-κB pathway activation, while the subsequent secretion of IL-1ß was mediated by ATP-induced P2 × 7R activation, loss of intracellular K+, and the subsequent NLRP3 inflammasome activation. By using a mouse infection model, we also found that ATP exacerbated clinical signs and death of mice infected by PRV in a NLRP3-dependent manner. These results indicate that ATP facilitates activation of NLRP3 inflammasome and enhances the pathogenicity of PRV in mice during its acute infection.

Swine Promyelocytic Leukemia Isoform II Inhibits Pseudorabies Virus Infection by Suppressing Viral Gene Transcription in Promyelocytic Leukemia Nuclear Bodies.

Promyelocytic leukemia nuclear bodies (PML-NBs) possess an important intrinsic antiviral activity against alphaherpesvirus infection. PML is the structural backbone of NBs, comprising different isoforms. However, the contribution of each isoform to alphaherpesvirus restriction is not well understood. Here, we report the role of PML-NBs and swine PML (sPML) isoforms in pseudorabies virus (PRV) infection in its natural host swine cells. We found that sPML-NBs exhibit an anti-PRV activity in the context of increasing the expression level of endogenous sPML. Of four sPML isoforms cloned and examined, only isoforms sPML-II and -IIa, not sPML-I and -IVa, expressed in a sPML knockout cells inhibit PRV infection. Both the unique 7b region of sPML-II and the sumoylation-dependent normal formation of PML-NBs are required. 7b possesses a transcriptional repression activity and suppresses viral gene transcription during PRV infection with the cysteine residues 589 and 599 being critically involved. We conclude that sPML-NBs inhibit PRV infection partly by repressing viral gene transcription through the 7b region of sPML-II.IMPORTANCE PML-NBs are nuclear sites that mediate the antiviral restriction of alphaherpesvirus gene expression and replication. However, the contribution of each PML isoform to this activity of PML-NBs is not well characterized. Using PRV and its natural host swine cells as a system, we have discovered that the unique C terminus of sPML isoform II is required for PML-NBs to inhibit PRV infection by directly engaging in repression of viral gene transcription. Our study not only confirms in swine cells that PML-NBs have an antiviral function but also presents a mechanism to suggest that PML-NBs inhibit viral infection in an isoform specific manner.

Detection of a gE-deleted Pseudorabies virus strain in an Italian red fox.

This study describes an Aujeszky's disease case in an adult male red fox found in an urban area in Central Italy, that exhibited a fatal infection with neurological lesions, but neither itching nor skin lesions. Diagnostic examinations included histology, and parasitological, bacteriological and virological analyses. Detection of parasitic enteric pathogens, bacteria, E. coli, Leptospira spp., rabies, canine distemper virus, parvovirus, hepatitis E virus and pseudorabies virus (PrV) was performed. Results showed the presence of a gE-deleted PrVthat was closely related to the NIA-3 strain but differed from the PrV strains currently circulating in wild boars and domestic pigs in Italy. All the results led to the conclusion that the fox suffered from Aujeszky's disease caused by a gE-deleted PrV strain closely related to a vaccine strain. The epidemiological link between the PrV vaccine strain and fox infection remains unclear. It could involve vaccinated pigs as a primary source of infection by direct or indirect contact with the red fox or less likely it could be related to improper use of the vaccine in the fox.

Pseudorabies Virus Infection of Epithelial Cells Leads to Persistent but Aberrant Activation of the NF-κB Pathway, Inhibiting Hallmark NF-κB-Induced Proinflammatory Gene Expression.

The nuclear factor kappa B (NF-κB) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-κB, illustrated by proteasome-dependent degradation of the inhibitory NF-κB regulator IκB and nuclear translocation and phosphorylation of the NF-κB subunit p65. PRV-induced persistent activation of NF-κB does not result in expression of negative feedback loop genes, like the gene for IκBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-κB activation. Hence, PRV infection triggers persistent NF-κB activation in an unorthodox way and dramatically modulates the NF-κB signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-κB signaling, which may aid the virus in modulating early proinflammatory responses in the infected host.IMPORTANCE The NF-κB transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-κB, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-κB activation shares some mechanistic features with canonical NF-κB activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IκB kinase (IKK) and even renders infected cells resistant to canonical NF-κB activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-κB activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-κB activation.

Construction of a US7/US8/UL23/US3-deleted recombinant pseudorabies virus and evaluation of its pathogenicity in dogs.

Since 2011, to control the spread of pseudorabies (PR), US7/US8/UL23-deleted recombinant PRV (rPRV) vaccines based on current variants have been developed. The vaccines can provide effective immune protection to pigs, but fur-bearing animals, such as dogs, foxes, and minks, are increasingly infected by PRV due to consuming contaminated raw meat or offal from immunized pigs. It is suspected that the attenuated PRV vaccine strain is not safe for these fur-bearing animals. To confirm this, we construct a US7/US8/UL23-deleted and a US7/US8/UL23/US3-deleted rPRV based on PRV GL isolated from fox using the CRISPR/Cas9 method. Growth kinetics in vitro and pathogenicity in dogs were compared between the wild type and both rPRVs. The results showed that the growth kinetics of wild-type PRV and US7/US8/UL23-deleted rPRV were faster than those of US7/US8/UL23/US3-deleted recombinant PRV from 24 h to 48 h post infection. Moreover, PRV GL- and rPRVdelUS7/US8/UL23-infected cells formed cell-cell fusion, but the rPRVdelUS7/US8/UL23/US3-infected cells did not. Dogs challenged with wild-type PRV or US7/US8/UL23-deleted rPRV showed obvious nervous symptoms, and all the dogs died, but the group challenged with the US7/US8/UL23/US3-deleted rPRV did not show any nervous symptoms, and all the dogs survived for the duration of the experiment. Tissue viral load analyses also showed that the virulence of the US7/US8/UL23/US3-deleted rPRV was significantly reduced in dogs. This study provides evidence that the US7/US8/UL23-deleted rPRV variant still exhibits high virulence for dogs and also highlights the role of the US3 gene in the pathogenicity of PRV in dogs and provides a strategy for developing a safer vaccine.

Recombinant pseudorabies virus expressing E2 of classical swine fever virus (CSFV) protects against both virulent pseudorabies virus and CSFV.

Both classical swine fever (CSF) and pseudorabies are highly contagious, economically significant diseases of swine in China. Although vaccination with the C-strain against classical swine fever virus (CSFV) is widely carried out and severe outbreaks of CSF seldom occur in China, CSF is sporadic in many pig herds and novel sub-subgenotypes of CSFV endlessly emerge. Thus, new measures are needed to eradicate CSFV from Chinese farms. The emergence of a pseudorabies virus (PRV) variant also posed a new challenge for the control of swine pseudorabies. Here, the recombinant PRV strain JS-2012-ΔgE/gI-E2 expressing E2 protein of CSFV was developed by inserting the E2 expression cassette into the intergenic region between the gG and gD genes of the gE/gI-deletion PRV variant strain JS-2012-ΔgE/gI. The recombinant virus was stable when passaged in vitro. A single vaccination of JS-2012-ΔgE/gI-E2 via intramuscular injection fully protected against lethal challenges of PRV and CSFV. Vaccination of piglets with the recombinant JS-2012-ΔgE/gI-E2 in the presence of high levels of maternally derived antibodies (Abs) to PRV can provide partial protection against lethal challenge of CSFV. Vaccination of the recombinant PRV JS-2012-ΔgE/gI-E2 strain did not induce the production of Abs to the gE protein of PRV or to the CSFV proteins other than E2. Thus, JS-2012-ΔgE/gI-E2 appears to be a promising recombinant marker vaccine candidate against PRV and CSFV for the control and eradication of the PRV variant and CSFV.

Fusion of pseudorabies virus glycoproteins to IgG Fc enhances protective immunity against pseudorabies virus.

Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate.

Role of p53 in pseudorabies virus replication, pathogenicity, and host immune responses.

As a key cellular transcription factor that plays a central role in cellular responses to a broad range of stress factors, p53 has generally been considered as a host cell restriction factor for various viral infections. However, the defined roles of p53 in pseudorabies virus (PRV) replication, pathogenesis, and host responses remain unclear. In the present study, we initially constructed a p53 overexpressing a porcine kidney epithelial cell line (PK-15) to detect the effect of p53 on PRV replication in vitro. The results show that viral glycoprotein B (gB) gene copies and the titers of virus were significantly higher in p53 overexpressing PK-15 cells than in PK-15 and p53 inhibitor treated p53 overexpressing PK-15 cells. A similar result was also found in the p53 inhibitor PFT-α-treated PK-15 cells. We then examined the effects of p53 on PRV infection in vivo by using p53-knockout (p53-/-) mice. The results show that p53 knockout not only led to significantly reduced rates of mortality but also to reduced viral replication and development of viral encephalitis in the brains of mice following intracranial inoculation. Furthermore, we examined the effect of p53 knockout on the expression of the reported host cell regulators of PRV replication in the brains of mice by using RNA sequencing. The results show that p53 knockout downregulated the interferon (IFN) regulator genes, chemokine genes, and antiviral genes after PRV infection. This finding suggests that p53 positively regulates viral replication and pathogenesis both in vitro and in vivo. These findings offer novel targets of intrinsic host cell immunity for PRV infection.

The Carboxyl Terminus of Tegument Protein pUL21 Contributes to Pseudorabies Virus Neuroinvasion.

Following its entry into cells, pseudorabies virus (PRV) utilizes microtubules to deliver its nucleocapsid to the nucleus. Previous studies have shown that PRV VP1/2 is an effector of dynein-mediated capsid transport. However, the mechanism of PRV for recruiting microtubule motor proteins for successful neuroinvasion and neurovirulence is not well understood. Here, we provide evidence that PRV pUL21 is an inner tegument protein. We tested its interaction with the cytoplasmic light chains using a bimolecular fluorescence complementation (BiFC) assay and observed that PRV pUL21 interacts with Roadblock-1. This interaction was confirmed by coimmunoprecipitation (co-IP) assays. We also determined the efficiency of retrograde and anterograde axonal transport of PRV strains in explanted neurons using a microfluidic chamber system and investigated pUL21's contribution to PRV neuroinvasion in vivo Further data showed that the carboxyl terminus of pUL21 is essential for its interaction with Roadblock-1, and this domain contributes to PRV retrograde axonal transport in vitro and in vivo Our findings suggest that the carboxyl terminus of pUL21 contributes to PRV neuroinvasion.IMPORTANCE Herpesviruses are a group of DNA viruses that infect both humans and animals. Alphaherpesviruses are distinguished by their ability to establish latent infection in peripheral neurons. After entering neurons, the herpesvirus capsid interacts with cellular motor proteins and undergoes retrograde transport on axon microtubules. This elaborate process is vital to the herpesvirus lifecycle, but the underlying mechanism remains poorly understood. Here, we determined that pUL21 is an inner tegument protein of pseudorabies virus (PRV) and that it interacts with the cytoplasmic dynein light chain Roadblock-1. We also observed that pUL21 promotes retrograde transport of PRV in neuronal cells. Furthermore, our findings confirm that pUL21 contributes to PRV neuroinvasion in vivo Importantly, the carboxyl terminus of pUL21 is responsible for interaction with Roadblock-1, and this domain contributes to PRV neuroinvasion. This study offers fresh insights into alphaherpesvirus neuroinvasion and the interaction between virus and host during PRV infection.

Virulent Pseudorabies Virus Infection Induces a Specific and Lethal Systemic Inflammatory Response in Mice.

Pseudorabies virus (PRV) is an alphaherpesvirus that infects the peripheral nervous system (PNS). The natural host of PRV is the swine, but it can infect most mammals, including cattle, rodents, and dogs. In these nonnatural hosts, PRV always causes a severe acute and lethal neuropathy called the "mad itch," which is uncommon in swine. Thus far, the pathophysiological and immunological processes leading to the development of the neuropathic itch and the death of the animal are unclear. Using a footpad inoculation model, we established that mice inoculated with PRV-Becker (virulent strain) develop a severe pruritus in the foot and become moribund at 82 h postinoculation (hpi). We found necrosis and inflammation with a massive neutrophil infiltration only in the footpad and dorsal root ganglia (DRGs) by hematoxylin and eosin staining. PRV load was detected in the foot, PNS, and central nervous system tissues by quantitative reverse transcription-PCR. Infected mice had elevated plasma levels of proinflammatory cytokines (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) and chemokines (Gro-1 and monocyte chemoattractant protein 1). Significant IL-6 and G-CSF levels were detected in several tissues at 82 hpi. High plasma levels of C-reactive protein confirmed the acute inflammatory response to PRV-Becker infection. Moreover, mice inoculated with PRV-Bartha (attenuated, live vaccine strain) did not develop pruritus at 82 hpi. PRV-Bartha also replicated in the PNS, and the infection spread further in the brain than PRV-Becker. PRV-Bartha infection did not induce the specific and lethal systemic inflammatory response seen with PRV-Becker. Overall, we demonstrated the importance of inflammation in the clinical outcome of PRV infection in mice and provide new insights into the process of PRV-induced neuroinflammation.IMPORTANCE Pseudorabies virus (PRV) is an alphaherpesvirus related to human pathogens such as herpes simplex virus 1 and varicella-zoster virus (VZV). The natural host of PRV is the swine, but it can infect most mammals. In susceptible animals other than pigs, PRV infection always causes a characteristic lethal pruritus known as the "mad itch." The role of the immune response in the clinical outcome of PRV infection is still poorly understood. Here, we show that a systemic host inflammatory response is responsible for the severe pruritus and acute death of mice infected with virulent PRV-Becker but not mice infected with attenuated strain PRV-Bartha. In addition, we identified IL-6 and G-CSF as two main cytokines that play crucial roles in the regulation of this process. Our findings give new insights into neuroinflammatory diseases and strengthen further the similarities between VZV and PRV infections at the level of innate immunity.

Antiviral Effect of Resveratrol in Piglets Infected with Virulent Pseudorabies Virus.

Pseudorabies virus (PRV) is one of the most important pathogens of swine, resulting in devastating disease and economic losses worldwide. Nevertheless, there are currently no antiviral drugs available for PRV infection. Resveratrol (Res) was identified to exert its antiviral activity by inhibiting the PRV replication in preliminary investigations. In our previous study, we found that Res has anti-PRV activity in vitro. Here, we show that Res can effectively reduce the mortality and increase the growth performance of PRV-infected piglets. After Res treatment, the viral loads significantly (p < 0.001) decreased. Pathological symptoms, particularly inflammation in the brain caused by PRV infection, were significantly (p < 0.001) relieved by the effects of Res. In Res-treated groups, higher levels of cytokines in serum, including interferon gama, interleukin 12, tumor necrosis factor-alpha and interferon alpha were observed at 7 days post infection. These results indicated that Res possesses potent inhibitory activity against PRV-infection through inhibiting viral reproduction, alleviating PRV-induced inflammation and enhancing animal immunity, suggesting that Res is expected to be a new alternative control measure for PRV infection.

Polymorphisms affecting the gE and gI proteins partly contribute to the virulence of a newly-emergent highly virulent Chinese pseudorabies virus.

An outbreak of a highly virulent pseudorabies virus strain, ZJ01, occurred in PRV-vaccinated pigs in China in 2011. In this study, ZJ01 caused fatal diseases, while the Chinese prototypic PRV strain LA caused mild respiratory disorders. Full-genome sequencing results indicate the two viruses can be classified into two sub-clusters that distinct from traditional European and US strains. To examine the potential role of the gE and gI proteins in ZJ01 virulence, we generated several recombinant viruses. In two chimeric viruses (rZJ01-LA/gEI and rLA-ZJ01/gEI), the gE and gI genes were swapped using corresponding genes from ZJ01 and LA. rZJ01-LA/gEI and the parental virus rZJ01 retained high virulence in piglets, although the survival time for rZJ01-LA/gEI infected piglets was obviously prolonged. In contrast, rLA-ZJ01/gEI exhibited higher virulence than its parental virus rLA. We conclude that changes in gE and gI proteins partly contribute to the enhanced virulence of ZJ01 strain.

Reduced virulence of a pseudorabies virus isolate from wild boar origin in domestic pigs correlates with hampered visceral spread and age-dependent reduced neuroinvasive capacity.

Morbidity and mortality associated with pseudorabies virus (PRV) infection are dependent on the age of the pig and the virulence of the strain. PRV strains circulating in wild boar are considered to be low virulent, but no mechanistic explanation for their reduced virulence is available. Here infection of 2- and 15-week-old domestic pigs with the PRV wild boar strain BEL24043 did not induce clinical symptoms in 15-week-old pigs, but resulted in important neurological and respiratory disease in 2-week-old piglets. A detailed study of the (neuro) pathogenesis and associated cytokine mRNA expression showed that the reduced virulence of the wild boar strain, compared to what was previously reported for the virulent domestic NIA3 strain, is due to a severely hampered spread to visceral organs in pigs of both age categories and to an efficient suppression of viral replication at primary replication sites of 15-week-old pigs and to a lesser extent in those of 2-week-old piglets. The age-dependent difference in induced symptoms seems to be due to an immature development state of the immune and/or nervous system in 2-week-old pigs. An extensive viral replication associated with a robust expression of cytokine-related mRNA was found in the olfactory bulb of 2-week-old piglets, correlating with observed neurological disease. Neuroinvasion also occurred via the trigeminal route in 2-week-old pigs, but viral replication was efficiently suppressed in the trigeminal ganglion in the presence of a moderate induction of cytokine-related mRNA. Viral replication in the peripheral and central nervous system of 15-week-old pigs was limited and efficiently suppressed.

Two classes of protective antibodies against Pseudorabies virus variant glycoprotein B: Implications for vaccine design.

Pseudorabies virus (PRV) belongs to the Herpesviridae family, and is an important veterinary pathogen. Highly pathogenic PRV variants have caused severe epidemics in China since 2011, causing huge economic losses. To tackle the epidemics, we identified a panel of mouse monoclonal antibodies (mAbs) against PRV glycoprotein B (gB) that effectively block PRV infection. Among these 15 mAbs, fourteen of them block PRV entry in a complement-dependent manner. The remaining one, 1H1 mAb, however can directly neutralize the virus independent of complement and displays broad-spectrum neutralizing activities. We further determined the crystal structure of PRV gB and mapped the epitopes of these antibodies on the structure. Interestingly, all the complement-dependent neutralizing antibodies bind gB at the crown region (domain IV). In contrast, the epitope of 1H1 mAb is located at the bottom of domain I, which includes the fusion loops, indicating 1H1 mAb might neutralize the virus by interfering with the membrane fusion process. Our studies demonstrate that gB contains multiple B-cell epitopes in its crown and base regions and that antibodies targeting different epitopes block virus infection through different mechanisms. These findings would provide important clues for antiviral drug design and vaccine development.

The pUL37 tegument protein guides alpha-herpesvirus retrograde axonal transport to promote neuroinvasion.

A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses.

Comparison of Pathogenicity-Related Genes in the Current Pseudorabies Virus Outbreak in China.

There is currently a pandemic of pseudorabies virus (PRV) variant strains in China. Despite extensive research on PRV variant strains in the past two years, few studies have investigated PRV pathogenicity-related genes. To determine which gene(s) is/are linked to PRV virulence, ten putative virulence genes were knocked out using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 technology. The pathogenicity of these mutants was evaluated in a mouse model. Our results demonstrated that of the ten tested genes, the thymidine kinase (TK) and glycoprotein M (gM) knockout mutants displayed significantly reduced virulence. However, mutants of other putative virulence genes, such as glycoprotein E (gE), glycoprotein I (gI), Us2, Us9, Us3, glycoprotein G (gG), glycoprotein N (gN) and early protein 0 (EP0), did not exhibit significantly reduced virulence compared to that of the wild-type PRV. To our knowledge, this study is the first to compare virulence genes from the current pandemic PRV variant strain. This study will provide a valuable reference for scientists to design effective live attenuated vaccines in the future.

Subclinical Herpes Simplex Virus Type 1 Infections Provide Site-Specific Resistance to an Unrelated Pathogen.

HSV-1 infections of the cornea range in severity from minor transient discomfort to the blinding disease herpes stromal keratitis, yet most patients experience a single episode of epithelial keratitis followed by re-establishment of a clear cornea. We asked whether a single transient episode of HSV-1 epithelial keratitis causes long-term changes in the corneal microenvironment that influence immune responses to subsequent corneal infection or trauma. We showed that C57BL/6 mouse corneas infected with HSV-1 KOS, which induces transient herpes epithelial keratitis without herpes stromal keratitis sequelae, possessed a significant leukocytic infiltrate composed primarily of CD4+ T cells and macrophages along with elevated chemokines and cytokines that persisted without loss of corneal clarity (subclinical inflammation). Chemokine and cytokine expression was CD4+ T cell dependent, in that their production was significantly reduced by systemic CD4+ T cell depletion starting before infection, although short-term (3-d) local CD4+ T cell depletion postinfection did not influence chemokine levels in cornea. Corneas with subclinical inflammation developed significantly greater trauma-induced inflammation when they were recipients of syngeneic corneal transplants but also exhibited significantly increased resistance to infections by unrelated pathogens, such as pseudorabies virus. The resistance to pseudorabies virus was CD4+ T cell dependent, because it was eliminated by local CD4+ T cell depletion from the cornea. We conclude that transient HSV-1 corneal infections cause long-term alterations of the corneal microenvironment that provide CD4-dependent innate resistance to subsequent infections by antigenically unrelated pathogens.