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. IMPORTANCEHerpes 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.

ABSTRACTWe have shown that the herpes simplex virus 1 (HSV-1) gK gene is essential for efficient replication and spread in the corneal epithelium and trigeminal ganglion neuroinvasion in mice (A. T. David, A. Baghian, T. P. Foster, V. N. Chouljenko, and K. G. Kousoulas, Curr. Eye Res. 33:455–467, 2008). To further investigate the role of gK in neuronal infection, we utilized a microfluidic chamber system separating neuronal cell bodies and axonal termini. HSV-1 (McKrae) engineered virus constitutively expressing enhanced green fluorescence protein (GFP) was efficiently transmitted in both a retrograde and an anterograde manner. These results were corroborated by expression of virion structural proteins in either chamber, as well as detection of viral genomes and infectious viruses. In contrast, efficient infection of either chamber with a gK-null virus did not result in infection of the apposed chamber. These results show that gK is an important determinant in virion axonal infection. Moreover, the inability of the gK-null virus to be transmitted in an anterograde manner suggests that virions acquire cytoplasmic envelopes prior to entering axons.IMPORTANCEHerpes simplex virus 1 (HSV-1) enters mucosal epithelial cells and neurons via fusion of the viral envelope with cellular membranes, mediated by viral glycoprotein B (gB) in cooperation with other viral glycoproteins. Retrograde transport of virions to neuronal cell bodies (somata) establishes lifelong latent infection in ganglionic neurons. We have previously reported that gK binds gB and is required for gB-mediated membrane fusion (Jambunatathan et al., J. Virol. 85:12910–12918, 2011; V. N. Chouljenko, A. V. Iyer, S. Chowdhury, J. Kim, and K. G. Kousoulas, J. Virol. 84:8596–8606, 2010). In the current study, we constructed a recombinant virus with the gK gene deleted in the highly virulent ocular HSV-1 strain McKrae. This recombinant virus failed to infect rat ganglionic neuronal axons alone or cocultured with Vero cells in microfluidic chambers. In addition, lack of gK expression prevented anterograde transmission of virions. These results suggest that gK is a critical determinant for neuronal infection and transmission.

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.

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 (gK⌬31-68 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 gK⌬31-68 mutation spans a well-defined ␤-sheet structure within the amino terminus of gK, which is conserved among alphaherpesviruses. The HSV-1(McKrae) gK⌬31-68 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) gK⌬31-68 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. IMPORTANCEAlphaherpesviruses, 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 ␤-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. Herpes simplex virus 1 (HSV-1) encodes at least 26 tegument proteins and 11 virally encoded glycoproteins, as well as several nonglycosylated membrane-associated proteins. Viral glycoproteins gD, gB, gH, and gL serve critical roles in virion entry (1-5). Virion entry is initiated by the binding of glycoproteins gB and gC to glycosaminoglycan (GAG) moieties of cell surface proteoglycans (6). This initial attachment causes the interaction of gD with one or more of its specific receptors, including the herpesvirus entry mediator (HVEM) (HveA), nectin-1 (HVEC), and 3-Osulfated HS. In addition, gB binds to PILR-␣, NMHC-IIA, and myelin-associated glycoprotein (MAG) receptors (7). HSV-1 enters into neurons strictly via a pH-independent fusion of the viral envelope with neuronal plasma membranes (8-10), while it can enter a wide range of nonneuronal cells via either pH-independent or pH-dependent endocytosis (11). Fusion of the vira...

Introduction: Zika virus (ZIKV) has emerged in dengue (DENV) endemic areas, where these two related flaviviruses continue to co-circulate. DENV is a complex of four serotypes and infections can progress to severe disease. It is thought that this is mediated by antibody dependent enhancement (ADE) whereby antibodies from a primary DENV infection are incapable of neutralizing heterologous DENV infections with another serotype. ADE has been demonstrated among other members of the Flavivirus group.Methods: We utilize an in vitro ADE assay developed for DENV to determine whether ZIKV is enhanced by a commonly available DENV serotype 2-derived monoclonal antibody (4G2).Results: We show that ZIKV infection in vitro is enhanced in the presence of the 4G2 mAb.Discussion: Our results demonstrate that ADE between ZIKV and DENV is possible and that the 4G2 antibody is a useful tool for the effects of pre-existing anti-DENV antibodies during ZIKV infections.

Salmonella Enteritidis is a major foodborne pathogen for which chickens serve as reservoir hosts. Reducing Salmonella Enteritidis carriage in chickens would reduce contamination of poultry meat and eggs with this pathogen. We investigated the prophylactic efficacy of feed supplemented with caprylic acid (CA), a natural, generally recognized as safe eight-carbon fatty acid, for reducing Salmonella Enteritidis colonization in chicks. One hundred commercial day-old chicks were randomly divided into five groups of 20 birds each: CA control (no Salmonella Enteritidis, CA), positive control (Salmonella Enteritidis, no CA), negative control (no Salmonella Enteritidis, no CA), and 0.7 or 1% CA. Water and feed were provided ad libitum. On day 8, birds were inoculated with 5.0 log CFU of Salmonella Enteritidis by crop gavage. Six birds from each group were euthanized on days 1, 7, and 10 after challenge, and Salmonella Enteritidis populations in the cecum, small intestine, cloaca, crop, liver, and spleen were enumerated. The study was replicated three times. CA supplementation at 0.7 and 1% consistently decreased Salmonella Enteritidis populations recovered from the treated birds. Salmonella Enteritidis counts in the tissue samples of CA-treated chicks were significantly lower (P < 0.05) than those of control birds on days 7 and 10 after challenge. Feed intake and body weight did not differ between the groups. Histological examination revealed no pathological changes in the cecum and liver of CA-supplemented birds. The results suggest that prophylactic CA supplementation through feed can reduce Salmonella Enteritidis colonization in day-old chicks and may be a useful treatment for reducing Salmonella Enteritidis carriage in chickens.

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Cattle serve as a major reservoir of E. coli O157:H7 and excrete the pathogen in feces. Environmental persistence of E. coli O157:H7 plays a vital role in its epidemiology on farms, and cattle water troughs are a demonstrated long-term reservoir of E. coli O157:H7 for animals. The objective of this study was to investigate the potential of low concentrations of trans-cinnamaldehyde for killing E. coli O157:H7 in cattle drinking-water. A five-strain mixture of E. coli O157:H7 was inoculated (at approximately 8.0 log colony-forming units [CFU]/mL) into 100 mL samples of well water containing 0, 0.03, 0.05, 0.07, or 0.1% trans-cinnamaldehyde. Additionally, water samples containing (1% w/v) bovine feces or feed were also included. The samples were incubated at 21 degrees , 8 degrees , or 4 degrees C for 7 days and tested for viable E. coli O157:H7 on days 0, 1, 3, 5, and 7. Triplicate samples of each treatment and control were included and the study was replicated twice. All concentrations of trans-cinnamaldehyde were effective in killing E. coli O157:H7 in water, but the magnitude of killing significantly increased with increase in trans-cinnamaldehyde concentration and storage temperature (p < 0.05). The presence of feed or feces in water decreased the antibacterial effect of trans-cinnamaldehyde on E. coli O157:H7 (p < 0.05). This study indicated that trans-cinnamaldehyde is effective in killing E. coli O157:H7 in cattle drinking-water, but detailed palatability studies on cattle intake of water containing the antimicrobial are needed.