Guinea pig herpes like virus is a gamma herpesvirus.

Guinea Pig Herpes-Like Virus (GPHLV) is a virus isolated from leukemic guinea pigs with herpes virus-like morphology described by Hsiung and Kaplow in 1969. GPHLV transformed embryonic cells from Syrian hamsters or rats, which were tumorigenic in adult animals. Herein, we present the genomic sequence of GPHLV strain LK40 as a reference for future molecular analysis. GPHLV has a broad host tropism and replicates efficiently in Guinea pig, Cat, and Green African Monkey-derived cell lines. GPHLV has a GC content of 35.45%. The genome is predicted to encode at least 75 open-reading frames (ORFs) with 84% (63 ORFs) sharing homology to human Kaposi Sarcoma Associated Herpes Virus (KSHV). Importantly, GPHLV encodes homologues of the KSHV oncogenes, vBCL2 (ORF16), vPK (ORF36), viral cyclin (v-cyclin, ORF72), the latency associated nuclear antigen (LANA, ORF73), and vGPCR (ORF74). GPHLV is a Rhadinovirus of Cavia porcellus, and we propose the formal name of Caviid gamma herpesvirus 1 (CaGHV-1). GPHLV can be a novel small animal model of Rhadinovirus pathogenesis with broad host tropism.

The Oncolytic herpes simplex virus type-1 (HSV-1) vaccine strain VC2 causes intratumor infiltration of functionally active T cells and inhibition of tumor metastasis and pro-tumor genes VEGF and PDL1 expression in the 4T1/Balb/c mouse model of stage four breast cancer.

Introduction: Oncolytic viruses (OVs) provide new modalities for cancer therapy either alone or in combination with synergistic immunotherapies and/or chemotherapeutics. Engineered Herpes Simplex Virus Type-1 (HSV-1) has shown strong promise for the treatment of various cancers in experimental animal models as well as in human patients, with some virus strains licensed to treat human melanoma and gliomas. In the present study we evaluated the efficacy of mutant HSV-1 (VC2) in a late stage, highly metastatic 4T1 murine syngeneic. Method: VC2 was constructed VC2 using double red recombination technology. For in-vivo efficacy we utilized a late stage 4T1 syngeneic and immunocompetent BALB/cJ mouse model breast cancer model which exhibits efficient metastasis to the lung and other organs. Results: VC2 replicated efficiently in 4T1 cells and in cell culture, achieving titers similar to those in African monkey kidney (Vero) cells. Intra-tumor treatment with VC2 did not appreciably reduce average primary tumor sizes but a significant reduction of lung metastasis was noted in mice treated intratumorally with VC2, but not with ultraviolet-inactivated VC2. This reduction of metastasis was associated with increased T cell infiltration comprised of CD4+ and CD4+CD8+ double-positive T cells. Characterization of purified tumor infiltrating T cells revealed a significant improvement in their proliferation ability compared to controls. In addition, significant T cell infiltration was observed in the metastatic nodules associated with reduction of pro-tumor PD-L1 and VEGF gene transcription. Conclusion: These results show that VC2 therapy can improve anti-tumor response associated with a better control of tumor metastasis. improve T cell responses and reduce pro-tumor biomarker gene transcription. VC2 holds promise for further development as an oncolytic and immunotherapeutic approach to treat breast and other cancers.

Cross protective efficacy of the Non-Neurotropic live attenuated herpes simplex virus type 1 vaccine VC-2 is enhanced by intradermal vaccination and deletion of glycoprotein G.

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2 respectively) cause life-long latent infections resulting in recurrent orofacial and genital blisters or sores. Ensued disease can be painful and may lead to significant mental anguish of infected individuals. Currently, there are no FDA-approved vaccines for either prophylactic or therapeutic use, and recent clinical trials of subunit vaccines failed to achieve endpoints goals. Development of a safe live-attenuated herpes simplex vaccine may provide the antigenic breadth to ultimately protect individuals from acquiring HSV disease. We have previously shown that prophylactic use of the non-neurotropic live attenuated HSV-1 vaccine, VC-2, provides potent and durable protection from genital HSV-2 disease in the guinea pig model. Here, we investigated the effects of intradermal administration as well as the deletion of the viral glycoprotein G (gG) on the efficacy of prophylactic vaccination. Vaccination with either VC-2, VC-2 gG null, or gD2 MPL/Alum offered robust protection from acute disease regardless of route of vaccination. However, both the VC-2 gG-null and the ID vaccination route were more effective compared to the parent VC2 administered by the IM route. Specifically, the VC-2 gG-null administered ID, reduced HSV-2 vaginal replication on day 2 and day 4 as well as mean recurrent lesion scores more effectively than VC2 administered IM. Most importantly, only VC-2 gG null IM and VC-2 ID significantly reduced the frequency of recurrent shedding, the most likely source for virus transmission. Similarly, while all vaccinated groups demonstrated a significant reduction in the number of animals testing PCR-positive for HSV-2 in their dorsal root ganglia following challenge only VC2 ID vaccinated animals demonstrated a significant reduction in DRG viral load. All vaccinations induced neutralizing antibodies to HSV-2 MS when compared to unvaccinated guinea pigs. Therefore, further investigation of VC-2 gG null delivered ID is warranted.

Intramuscular Vaccination With the HSV-1(VC2) Live-Attenuated Vaccine Strain Confers Protection Against Viral Ocular Immunopathogenesis Associated With γδT Cell Intracorneal Infiltration.

Herpes simplex virus type-1 (HSV-1) ocular infection is one of the leading causes of infectious blindness in developed countries. The resultant herpetic keratitis (HK) is caused by an exacerbated reaction of the adaptive immune response that persists beyond virus clearance causing substantial damage to the cornea. Intramuscular immunization of mice with the HSV-1(VC2) live-attenuated vaccine strain has been shown to protect mice against lethal ocular challenge. Herein, we show that following ocular challenge, VC2 vaccinated animals control ocular immunopathogenesis in the absence of neutralizing antibodies on ocular surfaces. Ocular protection is associated with enhanced intracorneal infiltration of γδ T cells compared to mock-vaccinated animals. The observed γδ T cellular infiltration was inversely proportional to the infiltration of neutrophils, the latter associated with exacerbated tissue damage. Inhibition of T cell migration into ocular tissues by the S1P receptors agonist FTY720 produced significant ocular disease in vaccinated mice and marked increase in neutrophil infiltration. These results indicate that ocular challenge of mice immunized with the VC2 vaccine induce a unique ocular mucosal response that leads into the infiltration of γδ T cells resulting in the amelioration of infection-associated immunopathogenesis.

Two Sides to Every Story: Herpes Simplex Type-1 Viral Glycoproteins gB, gD, gH/gL, gK, and Cellular Receptors Function as Key Players in Membrane Fusion.

Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) are prototypical alphaherpesviruses that are characterized by their unique properties to infect trigeminal and dorsal root ganglionic neurons, respectively, and establish life-long latent infections. These viruses initially infect mucosal epithelial tissues and subsequently spread to neurons. They are associated with a significant disease spectrum, including orofacial and ocular infections for HSV-1 and genital and neonatal infections for HSV-2. Viral glycoproteins within the virion envelope bind to specific cellular receptors to mediate virus entry into cells. This is achieved by the fusion of the viral envelope with the plasma membrane. Similarly, viral glycoproteins expressed on cell surfaces mediate cell-to-cell fusion and facilitate virus spread. An interactive complex of viral glycoproteins gB, gD/gH/gL, and gK and other proteins mediate these membrane fusion phenomena with glycoprotein B (gB), the principal membrane fusogen. The requirement for the virion to enter neuronal axons suggests that the heterodimeric protein complex of gK and membrane protein UL20, found only in alphaherpesviruses, constitute a critical determinant for neuronal entry. This hypothesis was substantiated by the observation that a small deletion in the amino terminus of gK prevents entry into neuronal axons while allowing entry into other cells via endocytosis. Cellular receptors and receptor-mediated signaling synergize with the viral membrane fusion machinery to facilitate virus entry and intercellular spread. Unraveling the underlying interactions among viral glycoproteins, envelope proteins, and cellular receptors will provide new innovative approaches for antiviral therapy against herpesviruses and other neurotropic viruses.

Intramuscular vaccination of mice with the human herpes simplex virus type-1(HSV-1) VC2 vaccine, but not its parental strain HSV-1(F) confers full protection against lethal ocular HSV-1 (McKrae) pathogenesis.

Herpes simplex virus type-1 (HSV-1) can cause severe ocular infection and blindness. We have previously shown that the HSV-1 VC2 vaccine strain is protective in mice and guinea pigs against genital herpes infection following vaginal challenge with HSV-1 or HSV-2. In this study, we evaluated the efficacy of VC2 intramuscular vaccination in mice against herpetic keratitis following ocular challenge with lethal human clinical strain HSV-1(McKrae). VC2 vaccination in mice produced superior protection and morbidity control in comparison to its parental strain HSV-1(F). Specifically, after HSV-1(McKrae) ocular challenge, all VC2 vaccinated- mice survived, while 30% of the HSV-1(F)- vaccinated and 100% of the mock-vaccinated mice died post challenge. VC2-vaccinated mice did not exhibit any symptoms of ocular infection and completely recovered from initial conjunctivitis. In contrast, HSV-1(F)-vaccinated mice developed time-dependent progressive keratitis characterized by corneal opacification, while mock-vaccinated animals exhibited more severe stromal keratitis characterized by immune cell infiltration and neovascularization in corneal stroma with corneal opacification. Cornea in VC2-immunized mice exhibited significantly increased infiltration of CD3+ T lymphocytes and decreased infiltration of Iba1+ macrophages in comparison to mock- or HSV-1(F)-vaccinated groups. VC2 immunization produced higher virus neutralization titers than HSV-1(F) post challenge. Furthermore, VC-vaccination significantly increased the CD4 T central memory (TCM) subsets and CD8 T effector memory (TEM) subsets in the draining lymph nodes following ocular HSV-1 (McKrae) challenge, then mock- or HSV-1(F)-vaccination. These results indicate that VC2 vaccination produces a protective immune response at the site of challenge to protect against HSV-1-induced ocular pathogenesis.

The Functional and Molecular Effects of Doxycycline Treatment on Borrelia burgdorferi Phenotype.

Recent studies have shown that Borrelia burgdorferi can form antibiotic-tolerant persisters in the presence of microbiostatic drugs such as doxycycline. Precisely how this occurs is yet unknown. Our goal was to examine gene transcription by B. burgdorferi following doxycycline treatment in an effort to identify both persister-associated genes and possible targets for antimicrobial intervention. To do so, we performed next-generation RNA sequencing on doxycycline-treated spirochetes and treated spirochetes following regrowth, comparing them to untreated B. burgdorferi. A number of genes were perturbed and most of those which were statistically significant were down-regulated in the treated versus the untreated or treated/re-grown. Genes upregulated in the treated B. burgdorferi included a number of Erp genes and rplU, a 50S ribosomal protein. Among those genes associated with post-treatment regrowth were bba74 (Oms28), bba03, several peptide ABC transporters, ospA, ospB, ospC, dbpA and bba62. Studies are underway to determine if these same genes are perturbed in B. burgdorferi treated with doxycycline in a host environment.

Cysteines and N-Glycosylation Sites Conserved among All Alphaherpesviruses Regulate Membrane Fusion in Herpes Simplex Virus 1 Infection.

Neurotropism is a defining characteristic of alphaherpesvirus pathogenicity. Glycoprotein K (gK) is a conserved virion glycoprotein of all alphaherpesviruses that is not found in other herpesvirus subfamilies. The extracellular amino terminus of gK has been shown to be important to the ability of the prototypic alphaherpesvirus herpes simplex virus 1 (HSV-1) to enter neurons via axonal termini. Here, we determined the role of the two conserved N-linked glycosylation (N48 and N58) sites of gK in virus-induced cell fusion and replication. We found that N-linked glycosylation is important to the regulation of HSV-1-induced membrane fusion since mutating N58 to alanine (N58A) caused extensive virus-induced cell fusion. Due to the known contributions of N-linked glycosylation to protein processing and correct disulfide bond formation, we investigated whether the conserved extracellular cysteine residues within the amino terminus of gK contributed to the regulation of HSV-1-induced membrane fusion. We found that mutation of C37 and C114 residues led to a gK-null phenotype characterized by very small plaque formation and drastic reduction in infectious virus production, while mutation of C82 and C243 caused extensive virus-induced cell fusion. Comparison of N-linked glycosylation and cysteine mutant replication kinetics identified disparate effects on infectious virion egress from infected cells. Specifically, cysteine mutations caused defects in the accumulation of infectious virus in both the cellular and supernatant fractions, while glycosylation site mutants did not adversely affect virion egress from infected cells. These results demonstrate a critical role for the N glycosylation sites and cysteines for the structure and function of the amino terminus of gK.IMPORTANCE We have previously identified important entry and neurotropic determinants in the amino terminus of HSV-1 glycoprotein K (gK). Alphaherpesvirus-mediated membrane fusion is a complex and highly regulated process that is not clearly understood. gK and UL20, which are highly conserved across all alphaherpesviruses, play important roles in the regulation of HSV-1 fusion in the context of infection. A greater understanding of mechanisms governing alphaherpesvirus membrane fusion is expected to inform the rational design of therapeutic and prevention strategies to combat herpesviral infection and pathogenesis. This work adds to the growing reports regarding the importance of gK to alphaherpesvirus pathogenesis and details important structural features of gK that are involved in gK-mediated regulation of virus-induced membrane fusion.

Intramuscular Immunization of Mice with the Live-Attenuated Herpes Simplex Virus 1 Vaccine Strain VC2 Expressing Equine Herpesvirus 1 (EHV-1) Glycoprotein D Generates Anti-EHV-1 Immune Responses in Mice.

Vaccination remains the best option to combat equine herpesvirus 1 (EHV-1) infection, and several different strategies of vaccination have been investigated and developed over the past few decades. Herein, we report that the live-attenuated herpes simplex virus 1 (HSV-1) VC2 vaccine strain, which has been shown to be unable to enter into neurons and establish latency in mice, can be utilized as a vector for the heterologous expression of EHV-1 glycoprotein D (gD) and that the intramuscular immunization of mice results in strong antiviral humoral and cellular immune responses. The VC2-EHV-1-gD recombinant virus was constructed by inserting an EHV-1 gD expression cassette under the control of the cytomegalovirus immediate early promoter into the VC2 vector in place of the HSV-1 thymidine kinase (UL23) gene. The vaccines were introduced into mice through intramuscular injection. Vaccination with both the VC2-EHV-1-gD vaccine and the commercially available vaccine Vetera EHVXP 1/4 (Vetera; Boehringer Ingelheim Vetmedica) resulted in the production of neutralizing antibodies, the levels of which were significantly higher in comparison to those in VC2- and mock-vaccinated animals (P < 0.01 or P < 0.001). Analysis of EHV-1-reactive IgG subtypes demonstrated that vaccination with the VC2-EHV-1-gD vaccine stimulated robust IgG1 and IgG2a antibodies after three vaccinations (P < 0.001). Interestingly, Vetera-vaccinated mice produced significantly higher levels of IgM than mice in the other groups before and after challenge (P < 0.01 or P < 0.05). Vaccination with VC2-EHV-1-gD stimulated strong cellular immune responses, characterized by the upregulation of both interferon- and tumor necrosis factor-positive CD4+ T cells and CD8+ T cells. Overall, the data suggest that the HSV-1 VC2 vaccine strain may be used as a viral vector for the vaccination of horses as well as, potentially, for the vaccination of other economically important animals.IMPORTANCE A novel virus-vectored VC2-EHV-1-gD vaccine was constructed using the live-attenuated HSV-1 VC2 vaccine strain. This vaccine stimulated strong humoral and cellular immune responses in mice, suggesting that it could protect horses against EHV-1 infection.

Vaccination of rhesus macaques with the live-attenuated HSV-1 vaccine VC2 stimulates the proliferation of mucosal T cells and germinal center responses resulting in sustained production of highly neutralizing antibodies.

We have shown that the live-attenuated HSV-1 VC2 vaccine strain with mutations in glycoprotein K (gK) and the membrane protein UL20 is unable to establish latency in vaccinated animals and produces a robust immune response capable of completely protecting mice against lethal vaginal HSV-1 or HSV-2 infections. To better understand the immune response generated by vaccination with VC2, we tested its ability to elicit immune responses in rhesus macaques. Vaccinated animals showed no signs of disease and developed increasing HSV-1 and HSV-2 reactive IgG1 after two booster vaccinations, while IgG subtypes IgG2 and IgG3 remained at low to undetectable levels. All vaccinated animals produced high levels of cross protective neutralizing antibodies. Flow cytometry analysis of cells isolated from draining lymph nodes showed that VC2 vaccination stimulated significant increases in plasmablast (CD27highCD38high) and mature memory (CD21-IgM-) B cells. T cell analysis on cells isolated from draining lymph node biopsies demonstrated a statistically significant increase in proliferating (Ki67+) follicular T helper cells and regulatory CXCR5+ CD8+ cytotoxic T cells. Analysis of plasma isolated two weeks post vaccination showed significant increases in circulating CXCL13 indicating increased germinal center activity. Cells isolated from vaginal biopsy samples collected over the course of the study exhibited vaccination-dependent increases in proliferating (Ki67+) CD4+ and CD8+ T cell populations. These results suggest that intramuscular vaccination with the live-attenuated HSV-1 VC2 vaccine strain can stimulate robust IgG1 antibody responses that persist for >250days post vaccination. In addition, vaccination lead to the maturation of B cells into plasmablast and mature memory B cells, the expansion of follicular T helper cells, and affects in the mucosal immune responses. These data suggest that the HSV VC2 vaccine induces potent immune responses that could help define correlates of protection towards developing an efficacious HSV-1/HSV-2 vaccine in humans.

Herpes Simplex Virus 1 UL37 Protein Tyrosine Residues Conserved among All Alphaherpesviruses Are Required for Interactions with Glycoprotein K, Cytoplasmic Virion Envelopment, and Infectious Virus Production.

The herpes simplex virus 1 (HSV-1) UL37 protein functions in virion envelopment at trans-Golgi membranes, as well as in retrograde and anterograde transport of virion capsids. Recently, we reported that UL37 interacts with glycoprotein K (gK) and its interacting partner protein UL20 (N. Jambunathan, D. Chouljenko, P. Desai, A. S. Charles, R. Subramanian, V. N. Chouljenko, and K. G. Kousoulas, J Virol 88:5927-5935, 2014, http://dx.doi.org/10.1128/JVI.00278-14), facilitating cytoplasmic virion envelopment. Alignment of UL37 homologs encoded by alphaherpesviruses revealed the presence of highly conserved residues in the central portion of the UL37 protein. A cadre of nine UL37 site-specific mutations were produced and tested for their ability to inhibit virion envelopment and infectious virus production. Complementation analysis revealed that replacement of tyrosines 474 and 480 with alanine failed to complement the UL37-null virus, while all other mutated UL37 genes complemented the virus efficiently. The recombinant virus DC474-480 constructed with tyrosines 474, 476, 477, and 480 mutated to alanine residues produced a gK-null-like phenotype characterized by the production of very small plaques and accumulation of capsids in the cytoplasm of infected cells. Recombinant viruses having either tyrosine 476 or 477 replaced with alanine produced a wild-type phenotype. Immunoprecipitation assays revealed that replacement of all four tyrosines with alanines substantially reduced the ability of gK to interact with UL37. Alignment of HSV UL37 with the human cytomegalovirus and Epstein-Barr virus UL37 homologs revealed that Y480 was conserved only for alphaherpesviruses. Collectively, these results suggest that the UL37 conserved tyrosine 480 residue plays a crucial role in interactions with gK to facilitate cytoplasmic virion envelopment and infectious virus production. IMPORTANCE: The HSV-1 UL37 protein is conserved among all herpesviruses, functions in both retrograde and anterograde transport of virion capsids, and plays critical roles in cytoplasmic virion envelopment by interacting with gK. We show here that UL37 tyrosine residues conserved among all alphaherpesviruses serve critical roles in cytoplasmic virion envelopment and interactions with gK.

Methods for Efficient Elimination of Mitochondrial DNA from Cultured Cells.

Here, we document that persistent mitochondria DNA (mtDNA) damage due to mitochondrial overexpression of the Y147A mutant uracil-N-glycosylase as well as mitochondrial overexpression of bacterial Exonuclease III or Herpes Simplex Virus protein UL12.5M185 can induce a complete loss of mtDNA (ρ0 phenotype) without compromising the viability of cells cultured in media supplemented with uridine and pyruvate. Furthermore, we use these observations to develop rapid, sequence-independent methods for the elimination of mtDNA, and demonstrate utility of these methods for generating ρ0 cells of human, mouse and rat origin. We also demonstrate that ρ0 cells generated by each of these three methods can serve as recipients of mtDNA in fusions with enucleated cells.

Deletion of a Predicted ß-Sheet Domain within the Amino Terminus of Herpes Simplex Virus Glycoprotein K Conserved among Alphaherpesviruses Prevents Virus Entry into Neuronal Axons.

UNLABELLED: We have shown previously that herpes simplex virus 1 (HSV-1) lacking expression of the entire glycoprotein K (gK) or expressing gK with a 38-amino-acid deletion (gKΔ31-68 mutation) failed to infect ganglionic neurons after ocular infection of mice. We constructed a new model for the predicted three-dimensional structure of gK, revealing that the gKΔ31-68 mutation spans a well-defined ß-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gKΔ31-68 virus was tested for the ability to enter into ganglionic neuronal axons in cell culture of explanted rat ganglia using a novel virus entry proximity ligation assay (VEPLA). In this assay, cell surface-bound virions were detected by the colocalization of gD and its cognate receptor nectin-1 on infected neuronal surfaces. Capsids that have entered into the cytoplasm were detected by the colocalization of the virion tegument protein UL37, with dynein required for loading of virion capsids onto microtubules for retrograde transport to the nucleus. HSV-1(McKrae) gKΔ31-68 attached to cell surfaces of Vero cells and ganglionic axons in cell culture as efficiently as wild-type HSV-1(McKrae). However, unlike the wild-type virus, the mutant virus failed to enter into the axoplasm of ganglionic neurons. This work suggests that the amino terminus of gK is a critical determinant for entry into neuronal axons and may serve similar conserved functions for other alphaherpesviruses. IMPORTANCE: Alphaherpesviruses, unlike beta- and gammaherpesviruses, have the unique ability to infect and establish latency in neurons. Glycoprotein K (gK) and the membrane protein UL20 are conserved among all alphaherpesviruses. We show here that a predicted ß-sheet domain, which is conserved among alphaherpesviruses, functions in HSV-1 entry into neuronal axons, suggesting that it may serve similar functions for other herpesviruses. These results are in agreement with our previous observations that deletion of this gK domain prevents the virus from successfully infecting ganglionic neurons after ocular infection of mice.

Draft Genome Sequences of Edwardsiella ictaluri Strains LADL11-100 and LADL11-194 Isolated from Zebrafish Danio rerio.

Here, we report the draft genome sequences of Edwardsiella ictaluri strains LADL11-100 and LADL11-194, two isolates from natural outbreaks of edwardsiellosis in the zebrafish Danio rerio, as well as the sequences of the plasmids carried by the zebrafish strain of E. ictaluri.

Impact and regulation of lambda interferon response in human metapneumovirus infection.

UNLABELLED: Human metapneumovirus (hMPV) is a respiratory paramyxovirus that is distributed worldwide and induces significant airway morbidity. Despite the relevance of hMPV as a pathogen, many aspects of the immune response to this virus are still largely unknown. In this report, we focus on the antiviral immune response, which is critical for viral clearance and disease resolution. Using in vitro and in vivo systems, we show that hMPV is able to induce expression of lambda interferon 1 (IFN-λ1), IFN-λ2, IFN-λ3, and IFN-λ4. The induction of IFN-λ expression by hMPV was dependent on interferon regulatory factor 7 (IRF-7) expression but not on IRF-3 expression. Treatment of hMPV-infected mice with IFN-λ reduced the disease severity, lung viral titer, and inflammatory response in the lung. Moreover, the IFN-λ response induced by the virus was regulated by the expression of the hMPV G protein. These results show that type III interferons (IFN-λs) play a critical protective role in hMPV infection. IMPORTANCE: Human metapneumovirus (hMPV) is a pathogen of worldwide importance. Despite the relevance of hMPV as a pathogen, critical aspects of the immune response induced by this virus remain unidentified. Interferons (IFNs), including IFN-λ, the newest addition to the interferon family, constitute an indispensable part of the innate immune response. Here, we demonstrated that IFN-λ exhibited a protective role in hMPV infection in vitro and in an experimental mouse model of infection.

Genomic diversification in strains of Rickettsia felis Isolated from different arthropods.

Rickettsia felis (Alphaproteobacteria: Rickettsiales) is the causative agent of an emerging flea-borne rickettsiosis with worldwide occurrence. Originally described from the cat flea, Ctenocephalides felis, recent reports have identified R. felis from other flea species, as well as other insects and ticks. This diverse host range for R. felis may indicate an underlying genetic variability associated with host-specific strains. Accordingly, to determine a potential genetic basis for host specialization, we sequenced the genome of R. felis str. LSU-Lb, which is an obligate mutualist of the parthenogenic booklouse Liposcelis bostrychophila (Insecta: Psocoptera). We also sequenced the genome of R. felis str. LSU, the second genome sequence for cat flea-associated strains (cf. R. felis str. URRWXCal2), which are presumably facultative parasites of fleas. Phylogenomics analysis revealed R. felis str. LSU-Lb diverged from the flea-associated strains. Unexpectedly, R. felis str. LSU was found to be divergent from R. felis str. URRWXCal2, despite sharing similar hosts. Although all three R. felis genomes contain the pRF plasmid, R. felis str. LSU-Lb carries an additional unique plasmid, pLbaR (plasmid of L. bostrychophila associated Rickettsia), nearly half of which encodes a unique 23-gene integrative conjugative element. Remarkably, pLbaR also encodes a repeats-in-toxin-like type I secretion system and associated toxin, heretofore unknown from other Rickettsiales genomes, which likely originated from lateral gene transfer with another obligate intracellular parasite of arthropods, Cardinium (Bacteroidetes). Collectively, our study reveals unexpected genomic diversity across three R. felis strains and identifies several diversifying factors that differentiate facultative parasites of fleas from obligate mutualists of booklice.

A single intramuscular vaccination of mice with the HSV-1 VC2 virus with mutations in the glycoprotein K and the membrane protein UL20 confers full protection against lethal intravaginal challenge with virulent HSV-1 and HSV-2 strains.

Herpes Simplex Virus type-1 (HSV-1) and type-2 (HSV-2) establish life-long infections and cause significant orofacial and genital infections in humans. HSV-1 is the leading cause of infectious blindness in the western world. Currently, there are no available vaccines to protect against herpes simplex infections. Recently, we showed that a single intramuscular immunization with an HSV-1(F) mutant virus lacking expression of the viral glycoprotein K (gK), which prevents the virus from entering into distal axons of ganglionic neurons, conferred significant protection against either virulent HSV-1(McKrae) or HSV-2(G) intravaginal challenge in mice. Specifically, 90% of the mice were protected against HSV-1(McKrae) challenge, while 70% of the mice were protected against HSV-2(G) challenge. We constructed the recombinant virus VC2 that contains specific mutations in gK and the membrane protein UL20 preventing virus entry into axonal compartments of neurons, while allowing efficient replication in cell culture, unlike the gK-null virus, which has a major defect in virus replication and spread. Intramuscular injection of mice with 107 VC2 plaque forming units did not cause any significant clinical disease in mice. A single intramuscular immunization with the VC2 virus protected 100% of mice against lethal intravaginal challenge with either HSV-1(McKrae) or HSV-2(G) viruses. Importantly, vaccination with VC2 produced robust cross protective humoral and cellular immunity that fully protected vaccinated mice against lethal disease. Quantitative PCR did not detect any viral DNA in ganglionic tissues of vaccinated mice, while unvaccinated mice contained high levels of viral DNA. The VC2 virus may serve as an efficient vaccine against both HSV-1 and HSV-2 infections, as well as a safe vector for the production of vaccines against other viral and bacterial pathogens.

Functional hierarchy of herpes simplex virus type-1 membrane proteins in corneal infection and virus transmission to ganglionic neurons.

PURPOSE: To determine the relative importance of viral glycoproteins gK, gM, gE and the membrane protein UL11 in infection of mouse corneas and ganglionic neurons. METHODS: Mouse eyes were scarified and infected with herpes simplex virus (HSV)-1(F), gE-null, gM-null, gK-null, or UL11-null viruses. Clinical signs of ocular disease were monitored daily. Virus shedding was determined at 24, 48 and 72 h post infection. Viral DNA within trigeminal ganglia (TG) was quantified by quantitative PCR at 30 d post infection. RESULTS: The gE-null virus replicated as efficiently as the parental virus and formed viral plaques approximately half-the-size in comparison with the HSV-1(F) wild-type virus. The UL11-null and gM-null viruses replicated approximately one log less efficiently than the wild-type virus, and formed plaques that were on average one-third the size and one-half the size of the wild-type virus, respectively. The gK-null virus replicated more than 3-logs less efficiently than the wild-type virus and formed very small plaques (5-10 cells). Mice infected with the wild-type virus exhibited mild clinical ocular symptoms, while mice infected with the mutant viruses did not show any significant ocular changes. The wild-type virus produced the highest virus shedding post infection followed by the gM-null, gE-null and UL11-null viruses, while no gK-null virus was detected at any time point. All TG collected from mice infected with the wild-type virus and 6-of-10 of TG retrieved from mice infected with the UL11-null virus contained high numbers of viral genomes. The gE-null and gM-null-infected ganglia contained moderate-to-low number of viral genomes in 4-of-10 and 2-of-10 mice, respectively. No viral genomes were detected in ganglionic tissues obtained from gK-null eye infections. CONCLUSIONS: The results show that gK plays the most important role among gM, gE and UL11 in corneal and ganglionic infection in the mouse eye model.

Phenylalanine residues at the carboxyl terminus of the herpes simplex virus 1 UL20 membrane protein regulate cytoplasmic virion envelopment and infectious virus production.

UNLABELLED: The herpes simplex virus type 1 (HSV-1) UL20 gene encodes a 222-amino-acid nonglycosylated envelope protein which forms a complex with viral glycoprotein K (gK) that functions in virion envelopment, egress, and virus-induced cell fusion. To investigate the role of the carboxyl terminus of the UL20 protein (UL20p) in cytoplasmic virion envelopment, a cadre of mutant viruses was constructed and characterized. The deletion of six amino acids from the carboxyl terminus of UL20p caused an approximately 1-log reduction in infectious virus production compared to that of the wild-type virus. Surprisingly, a phenylalanine-to-alanine replacement at amino acid position 210 caused a gain-of-function phenotype, increasing infectious virus production up to 1 log more than in the wild-type virus. In contrast, the replacement of two membrane-proximal phenylalanines with alanines caused drastic inhibition of infectious virion production and cytoplasmic virion envelopment. Prediction of the membrane topology of UL20p revealed that these two amino acid changes cause retraction of the carboxyl terminus of UL20p from the intracellular space. Confocal microscopy revealed that none of the engineered UL20 mutations affected intracellular transport of UL20p to trans-Golgi network membranes. In addition, a proximity ligation assay showed that none of the UL20 mutations affected UL20p colocalization and potential interactions with the UL37 protein recently found to interact with the gK/UL20 protein complex. Collectively, these studies show that phenylalanine residues within the carboxyl terminus of UL20p are involved in the regulation of cytoplasmic virion envelopment and infectious virus production. IMPORTANCE: We have shown previously that the UL20/gK protein complex serves crucial roles in cytoplasmic virion envelopment and that it interacts with the UL37 tegument protein to facilitate cytoplasmic virion envelopment. In this study, we investigated the role of phenylalanine residues within the carboxyl terminus of UL20p, since aromatic and hydrophobic amino acids are known to be involved in protein-protein interactions through stacking of their aromatic structures. Characterization of mutant viruses carrying phenylalanine (Phe)-to-alanine (Ala) mutations revealed that the two membrane-proximal Phe residues were critical for the proper UL20p membrane topology and efficient virion envelopment and infectious virus production. Surprisingly, a Phe-to-Ala change located approximately in the middle of the UL20p carboxyl terminus substantially enhanced cytoplasmic envelopment and overall production of infectious virions. This work revealed that Phe residues within the UL20p carboxyl terminus are involved in the regulation of cytoplasmic virion envelopment and infectious virus production.

Herpes simplex virus 1 protein UL37 interacts with viral glycoprotein gK and membrane protein UL20 and functions in cytoplasmic virion envelopment.

UNLABELLED: We have shown that glycoprotein K (gK) and its interacting partner, the UL20 protein, play crucial roles in virion envelopment. Specifically, virions lacking either gK or UL20 fail to acquire an envelope, thus causing accumulation of capsids in the cytoplasm of infected cells. The herpes simplex virus 1 (HSV-1) UL37 protein has also been implicated in cytoplasmic virion envelopment. To further investigate the role of UL37 in virion envelopment, the recombinant virus DC480 was constructed by insertion of a 12-amino-acid protein C (protC) epitope tag within the UL37 amino acid sequence immediately after amino acid 480. The DC480 mutant virus expressed full-size UL37 as detected by the anti-protC antibody in Western immunoblots, accumulated unenveloped capsids in the cytoplasm of infected cells, and produced very small plaques on African green monkey kidney (Vero) cells that were similar in size to those produced by the UL20-null and UL37-null viruses. The DC480 virus replicated nearly 4 log less efficiently than the parental wild-type virus when grown on Vero cells. However, DC480 mutant virus titers increased nearly 20-fold when the virus was grown on FRT cells engineered to express the UL20 gene in comparison to the titers on Vero cells, while the UL37-null virus replicated approximately 20-fold less efficiently than the DC480 virus on FRT cells. Coimmunoprecipitation experiments and proximity ligation assays showed that gK and UL20 interact with the UL37 protein in infected cells. Collectively, these results indicate that UL37 interacts with the gK-UL20 protein complex to facilitate cytoplasmic virion envelopment. IMPORTANCE: Herpes simplex viruses acquire their final envelopes by budding into cytoplasmic membranes derived from the trans-Golgi network (TGN). The tegument proteins UL36 and UL37 are known to be transported to the TGN sites of virus envelopment and to function in virion envelopment, since mutants lacking UL37 accumulate capsids in the cytoplasm that are unable to bud into TGN membranes. Viral glycoprotein K (gK) also functions in cytoplasmic envelopment, in a protein complex with the membrane-associated protein UL20 (UL20mp). This work shows for the first time that the UL37 protein functionally interacts with gK and UL20 to facilitate cytoplasmic virion envelopment. This work may lead to the design of specific drugs that can interrupt UL37 interactions with the gK-UL20 protein complex, providing new ways to combat herpesviral infections.