Utility of a Dengue-Derived Monoclonal Antibody to Enhance Zika Infection In Vitro.

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.

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.

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.

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.

Enhancing the thermal destruction of Escherichia coli O157:H7 in ground beef patties by trans-cinnamaldehyde.

The effect of trans-cinnamaldehyde (TC) on the inactivation of Escherichia coli O157:H7 in undercooked ground beef patties was investigated. A five-strain mixture of E. coli O157:H7 was inoculated into ground beef (7.0log CFU/g), followed by addition of TC (0, 0.15, and 0.3%). The meat was formed into patties and stored at 4 degrees C for 5 days or at -18 degrees C for 7 days. The patties were cooked to an internal temperature of 60 or 65 degrees C, and E. coli O157:H7 was enumerated. The numbers of E. coli O157:H7 did not decline during storage of patties. However, cooking of patties containing TC significantly reduced (P<0.05) E. coli O157:H7 counts, by >5.0log CFU/g, relative to the reduction in controls cooked to the same temperatures. The D-values at 60 and 65 degrees C of E. coli O157:H7 in TC-treated patties (1.85 and 0.08min, respectively) were significantly lower (P<0.05) than the corresponding D-values for the organism in control patties (2.70 and 0.29min, respectively). TC-treated patties were more color stable and showed significantly lower lipid oxidation (P<0.05) than control samples. TC enhanced the heat sensitivity of E. coli O157:H7 and could potentially be used as an antimicrobial for ensuring pathogen inactivation in undercooked patties. However detailed sensory studies will be necessary to determine the acceptability to consumers of TC in ground beef patties.

Prophylactic supplementation of caprylic acid in feed reduces Salmonella enteritidis colonization in commercial broiler chicks.

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.

Reduction of Escherichia coli O157:H7 in cattle drinking-water by trans-cinnamaldehyde.

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.