Recent Advances in the Study of Alphaherpesvirus Latency and Reactivation: Novel Guidance for the Design of Herpesvirus Live Vector Vaccines.

Alphaherpesviruses, including herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV), infect a diverse array of hosts, spanning both humans and animals. Alphaherpesviruses have developed a well-adapted relationship with their hosts through long-term evolution. Some alphaherpesviruses exhibit a typical neurotropic characteristic, which has garnered widespread attention and in-depth research. Virus latency involves the retention of viral genomes without producing infectious viruses. However, under stress, this can be reversed, resulting in lytic infection. Such reactivation events can lead to recurrent infections, manifesting as diseases like herpes labialis, genital herpes, and herpes zoster. Reactivation is a complex process influenced by both viral and host factors, and identifying how latency and reactivation work is vital to developing new antiviral therapies. Recent research highlights a complex interaction among the virus, neurons, and the immune system in regulating alphaherpesvirus latency and reactivation. Neurotropic alphaherpesviruses can breach host barriers to infect neurons, proliferate extensively within their cell bodies, and establish latent infections or spread further. Whether infecting neurons or spreading further, the virus undergoes transmission along axons or dendrites, making this process an indispensable part of the viral life cycle and a critical factor influencing the virus's invasion of the nervous system. Research on the transmission process of neurotropic alphaherpesviruses within neurons can not only deepen our understanding of the virus but can also facilitate the targeted development of corresponding vaccines. This review concentrates on the relationship between the transmission, latency, and activation of alphaherpesviruses within neurons, summarizes recent advancements in the field, and discusses how these findings can inform the design of live virus vaccines for alphaherpesviruses.

Advances in the immunoescape mechanisms exploited by alphaherpesviruses.

Alphaherpesviruses, categorized as viruses with linear DNA composed of two complementary strands, can potentially to induce diseases in both humans and animals as pathogens. Mature viral particles comprise of a core, capsid, tegument, and envelope. While herpesvirus infection can elicit robust immune and inflammatory reactions in the host, its persistence stems from its prolonged interaction with the host, fostering a diverse array of immunoescape mechanisms. In recent years, significant advancements have been achieved in comprehending the immunoescape tactics employed by alphaherpesviruses, including pseudorabies virus (PRV), herpes simplex virus (HSV), varicella-zoster virus (VZV), feline herpesvirus (FeHV), equine herpesvirus (EHV), and caprine herpesvirus type I (CpHV-1). Researchers have unveiled the intricate adaptive mechanisms existing between viruses and their natural hosts. This review endeavors to illuminate the research advancements concerning the immunoescape mechanisms of alphaherpesviruses by delineating the pertinent proteins and genes involved in virus immunity. It aims to furnish valuable insights for further research on related mechanisms and vaccine development, ultimately contributing to virus control and containment efforts.

Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

Characterization of the Effects of Host p53 and Fos on Gallid Alpha Herpesvirus 1 Replication.

Treatment options for herpesvirus infections that target the interactions between the virus and the host have been identified as promising. Our previous studies have shown that transcription factors p53 and Fos are essential host determinants of gallid alpha herpesvirus 1 (ILTV) infection. The impact of p53 and Fos on ILTV replication has 'not been fully understood yet. Using the sole ILTV-permissive chicken cell line LMH as a model, we examined the effects of hosts p53 and Fos on all phases of ILTV replication, including viral gene transcription, viral genome replication, and infectious virion generation. We achieved this by manipulating the expression of p53 and Fos in LMH cells. Our results demonstrate that the overexpression of either p53 or Fos can promote viral gene transcription at all stages of the temporal cascade of ILTV gene expression, viral genome replication, and infectious virion production, as assessed through absolute quantitative real-time PCR, ILTV-specific RT-qPCR assays, and TCID50 assays. These findings are consistent with our previous analyses of the effects of Fos and p53 knockdowns on virus production and also suggest that both p53 and Fos may be dispensable for ILTV replication. Based on the synergistic effect of regulating ILTV, we further found that there is an interaction between p53 and Fos. Interestingly, we found that p53 also has targeted sites upstream of ICP4, and these sites are very close to the Fos sites. In conclusion, our research offers an in-depth understanding of how hosts p53 and Fos affect ILTV replication. Understanding the processes by which p53 and Fos regulate ILTV infection will be improved by this knowledge, potentially paving the way for the development of novel therapeutics targeting virus-host interactions as a means of treating herpesvirus infections.

An indirect ELISA to detect antibodies to the gC of bovine alphaherpesvirus 1 (BoAHV1) displaying no crossreactivity with antibodies induced by bovine alphaherpesvirus 5 (BoAHV5).

Seroprevalence of bovine alphaherpesvirus type 1 (BoAHV1) infections may be contaminated by crossreactive antibodies to bovine alphaherpesvirus type 5 (BoAHV5). To avoid such crossreactivity, an indirect enzyme-linked immunosorbent assay prepared with a recombinant glycoprotein C (gC) antigen (ELISA-gC1) was developed, aiming the detection of antibodies to BoAHV1, with no crossreactivity with BoAHV5 antibodies. The antigen for the ELISA-gC1 was the product of the expression of 219 bp from the N-terminal portion of the BoAHV1 gC gene, which bears low homology between the two virus types. The test was validated on 131 bovine serum samples, including 26 sera from BoAHV1-experimentally immunized, 38 sera from BoAHV5-experimentally infected or immunized calves, and 67 sera from calves seronegative for both BoAHV1 and BoAHV5, as determined by serum neutralization (SN). When compared to SN for BoAHV1, the ELISA-gC1 presented 100% sensitivity, 95.5 % specificity, 100 % negative predictive value, 89.6 % positive predictive value, 98.8 % precision, and a kappa correlation coefficient (κ) 0.95. None of the 38 BoAHV5-seropositive calves was detected by the ELISA-gC1. The ELISA-gC1 proved highly effective for the identification of BoAHV1-positive sera, with no crossreactivity with anti-BoAHV5 antibodies, thus able to distinguish serological responses from BoAHV1- and BoAHV5-seropositive cattle. Its capacity to detect BoAHV1-specific antibodies should allow the determination of the actual BoAHV1 prevalence in herds, which cannot be serologically determined in countries where BoAHV5 is also prevalent due to antibody crossreactivity. Apart from recognizing exclusively BoAHV1-infected cattle, the ELISA-gC1 may also be used in support of BoAHV5 epidemiological studies by allowing the exclusion of BoAHV1-seropositive animals.

N-Linked Glycosylation and Expression of Duck Plague Virus pUL10 Promoted by pUL49.5.

Duck plague virus (DPV) is a member of the alphaherpesvirus subfamily, and its genome encodes a conserved envelope protein, protein UL10 (pUL10). pUL10 plays complex roles in viral fusion, assembly, cell-to-cell spread, and immune evasion, which are closely related to its protein characteristics and partners. Few studies have been conducted on DPV pUL10. In this study, we identified the characteristics of pUL10, such as the type of glycosylation modification and subcellular localization. The characteristic differences in pUL10 in transfection and infection suggest that there are other viral proteins that participate in pUL10 modification and localization. Therefore, pUL49.5, the interaction partner of pUL10, was explored. We found that pUL10 interacts with pUL49.5 during transfection and infection. Their interaction entailed multiple interaction sites, including noncovalent forces in the pUL49.5 N-terminal domains and C-terminal domains and a covalent disulfide bond between two conserved cysteines. pUL49.5 promoted pUL10 expression and mature N-linked glycosylation modification. Moreover, deletion of UL49.5 in DPV caused the molecular mass of pUL10 to decrease by approximately3 to 10 kDa, which suggested that pUL49.5 was the main factor affecting the N-linked glycosylation of DPV pUL10 during infection. This study provides a basis for future exploration of the effect of pUL10 glycosylation on virus proliferation. IMPORTANCE Duck plague is a disease with high morbidity and mortality rates, and it causes great losses for the duck breeding industry. Duck plague virus (DPV) is the causative agent of duck plague, and DPV UL10 protein (pUL10) is a homolog of glycoprotein M (gM), which is conserved in herpesviruses. pUL10 plays complex roles in viral fusion, assembly, cell-to-cell spread, and immune evasion, which are closely related to its protein characteristics and partners. In this study, we systematically explored whether pUL49.5 (a partner of pUL10) plays roles in the localization, modification, and expression of pUL10.

Crystal structures of glycoprotein D of equine alphaherpesviruses reveal potential binding sites to the entry receptor MHC-I.

Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus type 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) to initiate entry into equine cells. We have characterized the gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein-protein docking simulations, surface plasmon resonance (SPR) analysis, and biological assays. The structures of gD1 and gD4 revealed the existence of a common V-set immunoglobulin-like (IgV-like) core comparable to those of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Altering the key residues resulted in impaired virus growth in cells, which highlights the important role of these residues in the gD-MHC-I interaction. Taken together, our results add to our understanding of the initial herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development.

Neutralizing antibodies to bovine and bubaline alphaherpesviruses in water buffaloes (Bubalus bubalis).

Water buffaloes (Bubalus bubalis) have been introduced in many regions of the world as a source of animal protein. In many instances, bubaline cattle are reared close to or mixed with bovine or zebuine cattle. However, little is known about infectious diseases of bubaline and the interactions that may arise involving the microbiota of those species. Alphaherpesviruses of ruminants (bovine alphaherpesviruses types 1 and 5, BoHV-1, BoHV-5; bubaline alphaherpesvirus 1, BuHV-1) are highly cross-reactive in serological assays performed with bovine or zebuine sera. However, the profile of reactivity of bubaline cattle sera to alphaherpesviruses remains unknown. As such, it is not known which virus strain (or strains) would be most appropriate to be used as the challenge virus in the laboratory in search for alphaherpesvirus-neutralizing antibodies. In this study, the profile of neutralizing antibodies to alphaherpesviruses in bubaline sera was determined against different types/subtypes of bovine and bubaline alphaherpesviruses. Sera (n=339) were screened in a 24-h serum neutralization test (SN) against 100 TCID50 of each of the challenge viruses. From those, 159 (46.9 %) neutralized at least one of the viruses assayed; 131 (38.6%) sera neutralized the three viral strains used for screening. The viral strain that was neutralized by the largest number of sera was BoHV-5b A663 (149/159; 93.7%). A few sera neutralized only one of the challenge viruses: four sera neutralized BoHV-1 LA only; another neutralized BoHV-5 A663 only and four others neutralized BuHV-1 b6 only. SN testing with two additional strains gave rise to similar results, where maximum sensitivity (defined here as the largest number of sera that neutralized the challenge viruses) was obtained by adding positive results attained with three of the challenge strains. Differences in neutralizing antibody titers were not significant to allow inferences on which would be the most likely virus that induced the antibody responses detected here.

An ELISA to Detect Antibodies to Bovine Alphaherpesviruses 1 and 5 and Bubaline Alphaherpesvirus 1 in Cattle Sera.

Bovine alphaherpesvirus 1 (subtypes 1.1, 1.2a, and 1.2b), type 5 (subtypes 5a, 5b, and 5c), and bubaline herpesvirus 1 (BuHV-1) induce highly, though not fully cross-reactive serological responses. Most types and subtypes of these viruses circulate particularly in countries of the southern hemisphere, notably Brazil and Argentina. Therefore, the detection of infected animals is important in defining prevention and control strategies, particularly when flocks are destined for international trade. Identification of infected herds is most often achieved by assays that detect antibodies, such as enzyme immunoassays (ELISAs). However, to date, no ELISA has been evaluated in its capacity to detect antibodies to these alphaherpesviruses. Here, an ELISA was developed to detect antibodies to all currently recognized BoAHV-1, BoAHV-5, and BuAHV-1 types/subtypes, and its sensitivity and specificity were determined. Six hundred bovine sera were screened in serum neutralization tests (SN) against the seven viruses. ELISAs prepared with each of the viruses were compared to SN. Subsequently, a combined assay with multiple antigens LISA was prepared by mixing five viral antigens, chosen for their highest sensitivity in the preparative assays. In comparison to SN, the mAgELISA sensitivity was 96.5% with 96.1% specificity (κ = 0.93; PPV = 95.0%; NPV = 97.3%). The findings reveal that the mAgELISA developed here is highly suitable for the detection of antibodies, comparable in sensitivity and specificity to that of SN when performed with all known types and subtypes of bovine and bubaline alphaherpesviruses.

Microscopic lesions and modulation of gene expression in cervical medulla during BoAHV-1and BoAHV-5 infection: A mini-review.

Bovine herpesvirus (BoAHV) types 1 and 5 are closely-related neurotropic alpha-herpesviruses. BoAHV-1 generally causes respiratory and genital disease but can occasionally cause encephalitis. BoAHV-5 is the causative agent of non suppurative meningoencephalitis in calves. During neuroinvasion, both viruses reach the central and peripheral nervous system. While brain alterations are well-described, the changes that occur in the medulla have not been fully detailed. In this work, we integrated and analyzed the virological findings, the microscopic lesions and the changes that occur in the expression of genes related to the innate immunity, cell cycle and apoptosis in the cervical medulla of calves experimentally-infected with BoAHV-1 and BoAHV-5. This will contribute to the understanding of the differential neuropathogenesis of these alpha-herpesviruses of cattle.

P53 maintains gallid alpha herpesvirus 1 replication by direct regulation of nucleotide metabolism and ATP synthesis through its target genes.

P53, a well-known tumor suppressor, has been confirmed to regulate the infection of various viruses, including chicken viruses. Our previous study observed antiviral effect of p53 inhibitor Pifithrin-α (PFT-α) on the infection of avian infectious laryngotracheitis virus (ILTV), one of the major avian viruses economically significant to the poultry industry globally. However, the potential link between this antiviral effect of PFT-α and p53 remains unclear. Using chicken LMH cell line which is permissive for ILTV infection as model, we explore the effects of p53 on ILTV replication and its underlying molecular mechanism based on genome-wide transcriptome analysis of genes with p53 binding sites. The putative p53 target genes were validated by ChIP-qPCR and RT-qPCR. Results demonstrated that, consistent with the effects of PFT-α on ILTV replication we previously reported, knockdown of p53 repressed viral gene transcription and the genome replication of ILTV effectively. The production of infectious virions was also suppressed significantly by p53 knockdown. Further bioinformatic analysis of genes with p53 binding sites revealed extensive repression of these putative p53 target genes enriched in the metabolic processes, especially nucleotide metabolism and ATP synthesis, upon p53 repression by PFT-α in ILTV infected LMH cells. Among these genes, eighteen were involved in nucleotide metabolism and ATP synthesis. Then eight of the 18 genes were selected randomly for validations, all of which were successfully identified as p53 target genes. Our findings shed light on the mechanisms through which p53 controls ILTV infection, meanwhile expand our knowledge of chicken p53 target genes.

PFT-α inhibits gallid alpha herpesvirus 1 replication by repressing host nucleotide metabolism and ATP synthesis.

Therapeutics targeting virus-host interactions have been considered promising strategies for treating herpesvirus infection. Our previous study on avian infectious laryngotracheitis virus (ILTV), an avian herpesvirus economically important to the poultry industry worldwide, identified the small molecule Pifithrin-α (PFT-α) as a potential therapeutic agent. However, the underlying mechanisms of its antiviral function remain largely unknown. Using the ILTV-permissive chicken cell line LMH as the model, we found that PFT-α effectively suppressed the transcription and genome replication of ILTV and greatly reduced the level of infectious virions. Genome-wide transcriptome analysis revealed extensive repression of the metabolic processes of infected cells by PFT-α administration. Further metabolome assays of ILTV-infected cells using liquid chromatography coupled with mass spectrometry suggest host nucleotide metabolism and ATP synthesis as the key targets of PFT-α treatment during its repression of ILTV replication, which was experimentally supported by the reduced transcription of many key enzymes essential to nucleotide metabolism and ATP synthesis. The present study provides insights into the mechanisms by which PFT-α inhibits ILTV infection, which may increase the probability of successful clinical application of this molecule.

Replication and Spread of Oncolytic Herpes Simplex Virus in Solid Tumors.

Oncolytic herpes simplex virus (oHSV) is a highly promising treatment for solid tumors. Intense research and development efforts have led to first-in-class approval for an oHSV for melanoma, but barriers to this promising therapy still exist that limit efficacy. The process of infection, replication and transmission of oHSV in solid tumors is key to obtaining a good lytic destruction of infected cancer cells to kill tumor cells and release tumor antigens that can prime anti-tumor efficacy. Intracellular tumor cell signaling and tumor stromal cells present multiple barriers that resist oHSV activity. Here, we provide a review focused on oncolytic HSV and the essential viral genes that allow for virus replication and spread in order to gain insight into how manipulation of these pathways can be exploited to potentiate oHSV infection and replication among tumor cells.

ICP22/IE63 Mediated Transcriptional Regulation and Immune Evasion: Two Important Survival Strategies for Alphaherpesviruses.

In the process of infecting the host, alphaherpesviruses have derived a series of adaptation and survival strategies, such as latent infection, autophagy and immune evasion, to survive in the host environment. Infected cell protein 22 (ICP22) or its homologue immediate early protein 63 (IE63) is a posttranslationally modified multifunctional viral regulatory protein encoded by all alphaherpesviruses. In addition to playing an important role in the efficient use of host cell RNA polymerase II, it also plays an important role in the defense process of the virus overcoming the host immune system. These two effects of ICP22/IE63 are important survival strategies for alphaherpesviruses. In this review, we summarize the complex mechanism by which the ICP22 protein regulates the transcription of alphaherpesviruses and their host genes and the mechanism by which ICP22/IE63 participates in immune escape. Reviewing these mechanisms will also help us understand the pathogenesis of alphaherpesvirus infections and provide new strategies to combat these viral infections.

The Roles of Envelope Glycoprotein M in the Life Cycle of Some Alphaherpesviruses.

The envelope glycoprotein M (gM), a surface virion component conserved among alphaherpesviruses, is a multiple-transmembrane domain-containing glycoprotein with a complex N-linked oligosaccharide. The gM mediates a diverse range of functions during the viral life cycle. In this review, we summarize the biological features of gM, including its characterization and function in some specicial alphaherpesviruses. gM modulates the virus-induced membrane fusion during virus invasion, transports other proteins to the appropriate intracellular membranes for primary and secondary envelopment during virion assembly, and promotes egress of the virus. The gM can interact with various viral and cellular components, and the focus of recent research has also been on interactions related to gM. And we will discuss how gM participates in the life cycle of alphaherpesviruses.

Viral Load and Cell Tropism During Early Latent Equid Herpesvirus 1 Infection Differ Over Time in Lymphoid and Neural Tissue Samples From Experimentally Infected Horses.

Upper respiratory tract infections with Equid Herpesvirus 1 (EHV-1) typically result in a peripheral blood mononuclear cell-associated viremia, which can lead to vasculopathy in the central nervous system. Primary EHV-1 infection also likely establishes latency in trigeminal ganglia (TG) via retrograde axonal transport and in respiratory tract-associated lymphatic tissue. However, latency establishment and reactivation are poorly understood. To characterize the pathogenesis of EHV-1 latency establishment and maintenance, two separate groups of yearling horses were experimentally infected intranasally with EHV-1, strain Ab4, and euthanized 30 days post infection (dpi), (n = 9) and 70 dpi (n = 6). During necropsy, TG, sympathetic trunk (ST), retropharyngeal and mesenteric lymph nodes (RLn, MesLn) and kidney samples were collected. Viral DNA was detected by quantitative PCR (qPCR) in TG, ST, RLn, and MesLn samples in horses 30 and 70 dpi. The number of positive TG, RLn and MesLn samples was reduced when comparing horses 30 and 70 dpi and the viral copy number in TG and RLn significantly declined from 30 to 70 dpi. EHV-1 late gene glycoprotein B reverse transcriptase PCR and IHC results for viral protein were consistently negative, thus lytic replication was excluded in the present study. Mild inflammation could be detected in all neural tissue samples and inflammatory infiltrates mainly consisted of CD3+ T-lymphocytes (T-cells), frequently localized in close proximity to neuronal cell bodies. To identify latently infected cell types, in situ hybridization (ISH, RNAScope®) detecting viral DNA was used on selected qPCR- positive neural tissue sections. In ganglia 30 dpi, EHV-1 ISH signal was located in the neurons of TG and ST, but also in non-neuronal support or interstitial cells surrounding the neuron. In contrast, distinct EHV-1 signal could only be observed in neurons of TG 70 dpi. Overall, detection of latent EHV-1 in abdominal tissue samples and non-neuronal cell localization suggests, that EHV-1 uses T-cells during viremia as alternative route toward latency locations in addition to retrograde neuronal transport. We therefore hypothesize that EHV-1 follows the same latency pathways as its close relative human pathogen Varicella Zoster Virus.

PRV-encoded UL13 protein kinase acts as an antagonist of innate immunity by targeting IRF3-signaling pathways.

Pseudorabies virus (PRV), a porcine alphaherpesvirus, causes neurological disorders and reproductive failure in swine. It is capable of avoiding host antiviral responses, resulting in viral latency in infected animals. The mechanisms by which many PRV proteins help the virus to evade immune surveillance are poorly understood. In this study, we found that the PRV protein kinase, UL13, inhibits the IFN-ß signaling pathway by targeting interferon regulatory factor 3 (IRF3) for ubiquitination and degradation. PRV with mutant of UL13 is impaired in its ability to hinder IRF3 and interferon-ß (IFN-ß) activation, and has significantly less pathogenesis in mice that wild-type PRV. Our findings reveal an as yet undescribed mechanism utilized by PRV to evade host immune responses. PRV UL13 is a potential target for attenuated vaccines and antiviral drugs.

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.

Bovine alphaherpesvirus type 5 replicates more efficiently than bovine alphaherpesvirus type 1 in undifferentiated human neural cells.

Bovine herpesvirus (BoHV) types 1 and 5 are two closely related alpha-herpesviruses of cattle with neuroinvasive potential. BoHV-5 causes non-suppurative meningoencephalitis in calve whereas encephalitis caused by BoHV-1 has been occasionally reported. As an initial step to understand the biology of both BoHV types in neural cells, undifferentiated SH-SY5Y human neuroblastoma cells were infected with BoHV-1 strains Cooper and Los Angeles (LA), BoHV-5 strain 97/613 and A663, a BoHV-5/BoHV-1 natural recombinant. Cytopathic effect (CPE) in these cells was evident earlier for BoHV-5 strain 97/613 and CPE progression was slower for BoHV-1, particularly for Cooper strain. Virus antigen was detected as early as 8 h post-infection (hpi) for all strains, with the exception of BoHV-1 Cooper for which antigen expression was detectable by 24 hpi. All strains released detectable infectious virus in the extracellular medium by 8 hpi, confirming that undifferentiated SH-SY5Y cells are fully permissive to BoHV infection. Significantly different extracellular virus titers among the different strains were detected by 24 hpi, with BoHV-5 97/613 reaching the maximal virus production. The lowest extracellular titer was recorded for BoHV-1 Cooper at all the evaluated time-points. BoHV-1 Cooper, BoHV-1 LA and BoHV-5 97/613 had a steady increase in intracellular virus production. The evaluation of lysis plaques formation revealed that BoHV-5 A663 produced the largest plaques followed by BoHV-5 97/613. Both BoHV-1 strains produced smaller plaques when compared with BoHV-5. Despite a slower replicative cycle, strain A663 is more efficient in cell to cell dissemination. Thus, it is evident that BoHV-5 strains have growth advantages in undifferentiated neural cells compared with BoHV-1. This in vitro model might be useful to analyze the neuropathogenic potential of bovine alphaherpesviruses.

Antiviral activity of Α-hydroxytropolones on caprine alphaherpesvirus 1 in vitro.

The emergence of human alphaherpesvirus strains (i.e. HHV-1 and -2) resistant to commonly used antiviral drugs has prompted the research for alternative, biologically active anti-herpetic agents. Natural-product and synthetic α-hydroxytropolones (αHTs) have been identified as lead therapeutic agents for a number of infections, including HHV-1 and -2, and several veterinary herpesviruses, i.e. bovine alphaherpesvirus 1 (BoHV-1), equine alphaherpesvirus 1 (EHV-1) and feline alphaherpesvirus 1 (FHV-1). In the present study we evaluated the activity in vitro of two natural and two synthetic α-hydroxytropolones (αHTs) against Caprine alphaherpesvirus 1 (CpHV-1) which is regarded as a useful homologous animal model for the study of HSV-2 infection, chiefly for the assessment of antiviral drugs in in vivo studies. AlphaHTs were able to decrease significantly CpHV-1 viral titres up to 4.25 log10 TCID50/50 µl and suppressed extensively CpHV-1 nucleic acids up to 8.71 log10 viral DNA copy number/10 µl. This study demonstrated the efficacy of αHTs against CpHV-1 in vitro, adding to their activity observed against the human and animal alphaherpesviruses in vitro. The activity of αHTs against CpHV-1 appeared similar but not identical to the patterns of activity observed against other alphaherpesviruses, suggesting virus-related variability in terms of response to specific αHT molecules. These findings open several perspectives in terms of future studies using the CpHV-1 homologous animal model, for the development of therapeutic tools against herpesviruses.