Sequences of the ribonucleotide reductase-encoding genes of felid herpes simplex virus 1 and molecular phylogenetic analysis.

The felid herpes simplex virus 1 (FHV-1) genes encoding the two ribonucleotide reductase (RR) subunits (RR1, large subunit and RR2, small subunit) were cloned and their nucleotide (nt) sequence determined. The RR1 open reading frame (ORF) is 2358 nts long and is predicted to encode a protein of 786 amino acids (aa). In common with herpes simplex viruses in the Varicellovirus genus of the alphaherpes virus subfamily, FHV-1 RR1 lacks the N-terminal serine threonine protein kinase region present in herpes simplex virus (HSV)-1 and -2. FHV-1 RR1 has a predicted aa identity of 47-64% with other alphaherpes virus RR1 peptides, falling to 26-29% for gammaherpes viruses. The RR2 ORF is 996 nts long, predicted to encode a protein of 332 aa and has aa identities of 64-70% with alphaherpes viruses and 38-39% with gammaherpes viruses. Molecular phylogenetic analysis groups FHV-1 with equid herpes simplex viruses 1 and 4 (EHV 1 and 4), pseudorabies virus (PRV) and bovid herpes simplex virus 1 (BHV 1) within the genus Varicellovirus.

Cunnilingus and vaginal intercourse are risk factors for herpes simplex virus type 1 acquisition in women.

OBJECTIVE: Although numerous cross-sectional studies have identified herpes simplex virus type 1 (HSV-1) as an important genital pathogen, the specific sexual activities associated with HSV-1 infection are not well delineated. Our objective was to identify demographic and behavioral variables in women associated with the prevalence and acquisition of HSV-1. STUDY: From 1998 through 2000, we enrolled 1207 nonpregnant 18- to 30-year-old women from 3 Pittsburgh, Pennsylvania, area health clinics in a prospective cohort study. Serum from the women was tested each visit for the presence of type-specific HSV-1 antibodies. RESULTS: At enrollment, HSV-1 serum antibodies were detected in only 38% of women < or =20 years of age. Black race, < or =12 years education, older age, and a history of at least 5 lifetime male sex partners were independently associated with the prevalence of HSV-1. In longitudinal analyses, women who had vaginal intercourse were more likely than sexually inactive women to acquire HSV-1 (6.8 vs. 1.2 cases per 100 woman-years of follow up; P=0.05). Similarly, women who only had receptive oral sex, without vaginal intercourse, were also more likely than sexually inactive women to acquire HSV-1 (9.8 vs. 1.2 cases per 100 woman-years of follow up; P=0.04). CONCLUSIONS: Receiving cunnilingus and vaginal intercourse are important risk factors for the acquisition of HSV-1 among young women. Genital herpes simplex prevention strategies will need to consider both the increased susceptibility for HSV-1 acquisition that young adults now have at sexual debut and the important contributions of HSV-1 to the burgeoning genital herpes simplex epidemic.

Simultaneous identification of two populations of sympathetic preganglionic neurons using recombinant herpes simplex virus type 1 expressing different reporter genes.

We generated neurotropic herpes simplex type 1 viruses expressing human placental alkaline phosphatase and studied the utility of this enzyme as a marker of infected neurons. The neurotropism of these viruses was assessed by their ability to infect sympathetic preganglionic neurons after adrenal injection in hamsters. The transneuronal transfer of these viruses was examined by their ability to cross the peripheral synapse from the kidney to renal preganglionic neurons or to cross the central synapse from the adrenal gland to the medulla oblongata. Finally, we injected an alkaline phosphatase-expressing herpes simplex virus into the adrenal gland and a beta-galactosidase-expressing herpes simplex virus (US5gal) into the muscular wall of the small intestine to label two neural circuits in one animal and to assess the feasibility of a dual-virus labelling system. The alkaline phosphatase gene was inserted into the glycoprotein J locus or the virus-induced host shut-off locus in the herpes simplex genome to create viruses which replicate (gJHAP HSV or vhsHAP HSV) or into the thymidine kinase locus to generate a virus that does not replicate in neurons in vivo (TK- HAP HSV). Each of the three viruses was retrogradely transported from the adrenal gland of hamsters to sympathetic preganglionic neurons, suggesting that the neurotropism of these viruses was maintained. gJHAP HSV travelled transneuronally from the kidney to sympathorenal preganglionic neurons and from the adrenal gland to neurons in the rostral ventrolateral medulla. Neuronal infection with alkaline phosphatase-expressing virus could be identified using histochemistry but detailed morphology of these neurons was not revealed. However, staining by anti-herpes simplex virus immunoperoxidase demonstrated that they had normal morphology. Identification of two distinct neural circuits in one animal was achieved with our dual-virus labelling system. The nonreplicating TK- HAP HSV was used in combination with US5gal to identify intestinal and adrenal sympathetic preganglionic neurons. The beta-galactosidase-expressing intestinal neurons were labelled bilaterally in the nucleus intermediolateralis, pars principalis, and alkaline phosphatase-expressing adrenal neurons were found ipsilaterally. Some clusters of sympathetic preganglionic neurons in the nucleus intermediolateralis, pars principalis contained mostly intestinal sympathetic preganglionic neurons and a few adrenal sympathetic preganglionic neurons. In other areas, the opposite pattern occurred. About 3-7% of the labelled sympathetic preganglionic neurons were double-labelled by both markers. The distinct and crisp morphology and dendritic processes of neurons stained by beta-galactosidase histochemistry contrasted with the partial staining of neurons by alkaline phosphatase, revealing beta-galactosidase as a better marker of infected neurons. In conclusion, alkaline phosphatase-expressing herpes simplex viruses are yet neurotropic after insertion of this marker enzyme into any of three different loci of the herpes simplex genome. One replicating alkaline phosphatase-expressing virus travelled transneuronally. These alkaline phosphatase-expressing herpes simplex virus can be used together with beta-galactosidase-expressing herpes simplex viruses to determine the target specificity of sympathetic preganglionic neurons controlling visceral organs or can be used to express two different recombinant genes in two targeted neuronal populations. This study suggests that sympathetic preganglionic neurons controlling the intestine and adrenal gland are almost completely distinct.

Detection of viral genomes of the Herpesviridae family in multiple sclerosis patients by means of the polymerase chain reaction (PCR)

BACKGROUND: The multiple sclerosis seems to be the junction between genetics alteration and an unknown environmental factor, that they would originate an autoimmune alteration, that they would be the reason of the inflammation and demyelinization responsible of the disease. Our objective has been the determination of this possible environmental factor and to reach it, we have studied the appearance of Herpesviridae family viruses. MATERIALS AND METHODS: 204 blood samples were studied: 102 from relapsing-remitting multiple sclerosis patients (43 were undergoing beta-interferon treatment), and 102 from blood donors with the same age and sex than multiple sclerosis patients. From this samples, we extracted the DNA of peripheral blood mononuclear cells (PBMCs), and we analyzed by polymerase chain reaction (PCR) to detect the appearance of herpes simplex virus, varicella zoster virus, Epstein-Barr virus, cytomegalovirus, human herpes simplex virus 6 (HHV-6), human herpes simplex virus 7 and human herpes simplex virus 8. RESULTS: a) we only found significative difference (p = 0.0001) in HHV-6: 21.5% donors positive samples (22/102), opposite to 49.02% of positivity in mulytiple sclerosis patients (50/102); b) we didn't found significative differences in none of other viruses studied, between patients treated with beta-interferon and non-treated ones. CONCLUSIONS: Our results suggest us that HHV-6 can play an important role in the multiple sclerosis development. The beta-interferon treatment doesn't affect to DNA prevalence of none of studied viruses.

N-terminal sequence analysis of equine herpes simplex virus 1 glycoproteins D and B and evidence for internal cleavage of the gene 71 product.

Signal cleavage sites of equine herpes simplex virus 1 (EHV-1) glycoproteins D and B (gD and gB) and an endoproteolytic cleavage site of EHV-1 gB were determined by N-terminal amino acid sequencing and compared with known cleavage sites of homologues in other herpes simplex virus. Signal cleavage of EHV-1 gD occurred between Arg35 and Ala36 in a region of basic amino acids resembling the endoproteolytic cleavage sites of viral glycoproteins, nine amino acids downstream of the predicted site, while EHV-1 gB was cleaved as predicted between Ala85 and Val86. Endoproteolytic cleavage of EHV-1 gB occurred between Arg548 and Ala549, 28 amino acids downstream of the cleavage site predicted from conserved sequences of other herpes simplex virus gB homologous. One interpretation of these data is that EHV-1 gB is cleaved internally at both sites, a possibility which was supported by the apparent molecular masses of the unglycosylated gB subunits produced in the presence of tunicamycin. This double cleavage would release a stretch of amino acids which is not present in sequenced gB molecules of other herpes simplex viruses. Experiments with glycosylation inhibitors indicated that cleavage of EHV-1 gB can occur in the absence of glycosylation. N-terminal sequencing also determined that a 42 kDa EHV-1 glycoprotein was a product of internal cleavage of the protein encoded by gene 71. Staggered endoproteolytic cleavage after adjacent arginine residues 506 and 507 separates the 42 kDa C-terminal subunit containing all the cysteine residues from the serine and threonine rich N-terminal region.

The genome of a very virulent Marek's disease virus.

Here we present the first complete genomic sequence, with analysis, of a very virulent strain of Marek's disease virus serotype 1 (MDV1), Md5. The genome is 177,874 bp and is predicted to encode 103 proteins. MDV1 is colinear with the prototypic alphaherpes virus herpes simplex virus type 1 (HSV-1) within the unique long (UL) region, and it is most similar at the amino acid level to MDV2, herpes simplex virus of turkeys (HVT), and nonavian herpes simplex viruses equine herpes simplex viruses 1 and 4. MDV1 encodes 55 HSV-1 UL homologues together with 6 additional UL proteins that are absent in nonavian herpes simplex viruses. The unique short (US) region is colinear with and has greater than 99% nucleotide identity to that of MDV1 strain GA; however, an extra nucleotide sequence at the Md5 US/short terminal repeat boundary results in a shorter US region and the presence of a second gene (encoding MDV097) similar to the SORF2 gene. MD5, like HVT, encodes an ICP4 homologue that contains a 900-amino-acid amino-terminal extension not found in other herpes simplex viruses. Putative virulence and host range gene products include the oncoprotein MEQ, oncogenicity-associated phosphoproteins pp38 and pp24, a lipase homologue, a CxC chemokine, and unique proteins of unknown function MDV087 and MDV097 (SORF2 homologues) and MDV093 (SORF4). Consistent with its virulent phenotype, Md5 contains only two copies of the 132-bp repeat which has previously been associated with viral attenuation and loss of oncogenicity.

Expression and roles of herpes simplex virus entry mediators A and C in cells of oral origin.

The roles of viral glycoprotein D (gD) and cellular herpes simplex virus entry mediators A (HveA) and C (HveC) in herpes simplex virus entry into oral cells were determined. Studies with purified truncated forms of gD-1, HveA and HveC indicated that these molecules may be involved in herpes simplex virus entry into oral cells. Moreover, HveA was expressed similarly in primary cultures of gingival keratinocytes and fibroblasts, whereas HveC was expressed at higher levels in gingival keratinocytes, as determined by RT-PCR and immunocytochemical staining. Further analysis using immunohistochemistry demonstrated that both HveA and HveC were expressed in epithelial cells, fibroblasts and vascular endothelial cells in gingival tissues. However, only HveC was detected in nerve fibers. Also, HveA was detected throughout the epidermis, whereas HveC was pronounced in the strata basale and spinosum. In conclusion, this study characterized HveA and HveC, molecules that may participate in entry of herpes simplex virus into oral cells.

Patient recognition of recrudescent Herpes Labialis: a clinical and virological assessment.

OBJECTIVES: The purpose of this study was to ascertain how accurate the general public was at diagnosing the condition of recrudescent Herpes Labialis. METHODS: An advertisement was placed in a local newspaper inviting patients to attend the Oral Medicine Clinic as soon as they thought they developed the clinically evident stage of Herpes Labialis. At the clinic, patients were examined to confirm the clinical presence of Herpes Labialis and also had a swab of the lesion(s) taken for virus culture. Virus culture was by the HEP-2 culture technique capable of detecting both herpes simplex Type 1 and herpes simplex Type 2. Patients also completed a detailed questionnaire concerning their knowledge of Herpes Labialis. RESULTS: In total, 41 patients attended for screening. The findings were that all patients had clinical Herpes Labialis, and herpes simplex virus was isolated in 96% of cases. In contrast, in only about 50% of cases were patients aware that their Herpes Labialis was caused by a virus. CONCLUSIONS: The general public are very good at recognizing Herpes Labialis lesions but need to be given more information about their infectivity.

Clinical application of polymerase chain reaction amplification to diagnosis of herpes simplex virus infection.

Amplification of viral DNA offers a potentially sensitive and specific method for identifying herpes simplex viruses in pathologic specimens. The purpose of this study is to assess the value of polymerase chain reaction amplification of DNA as a diagnostic test for herpes simplex virus in pathologic specimens. The purpose of this study is to assess for herpes simplex virus infections. We examined 79 paraffin-embedded tissue samples from 43 patients and 55 viral culture samples from 45 patients. Herpes virus DNA in the specimens was amplified by polymerase chain reaction. Using paraffin-embedded tissue on which a diagnosis of herpes simplex virus was made by morphologic criteria, 11 of 19 patients had herpes simplex virus DNA identified by PCR; herpes simplex virus DNA was not identified in any of 35 negative control specimens. Herpes virus DNA was also identified by polymerase chain reaction in all of the positive herpes simplex virus culture specimens. Of 29 culture negative specimens, herpes simplex virus DNA was identified in six. We conclude that polymerase chain reaction is useful to establish or confirm the presence of a herpes simplex virus infection in paraffin-embedded tissue samples and that it is more sensitive than viral culture.

Herpes Simplex virus type 2 induced retinal necrosis in BALB/c mice.

We injected herpes simplex virus type 2 of MS- or G-strain into the anterior chamber of BALB/c mice. In the contralateral eye inflammatory cell infiltration began in the ciliary body; focal retinitis, detected by day 8, led to total destruction of the retina by day 10. Contralateral disease was observed in 75% of mice inoculated with 8 x 10(3) pfu herpes simplex virus type 2, but in only 20% of mice receiving 80 pfu herpes simplex virus type 2. Still this low concentration, however, produced a suppressed delayed-type hypersensitivity response. Anti-herpes simplex virus type 2 antibody, first detected on day 8, reached high titers on day 10; by then, most of the mice had died of encephalitis. The G-strain of herpes simplex virus type 2 was more neurotoxic than the MS-strain, but produced the same incidence of contralateral retinitis. Herpes Simplex virus type 2 products contralateral necrotizing retinitis comparable to that produced by herpes simplex virus type 1. These findings, like those of other authors, suggest a role for herpes simplex virus type 2 in some cases of acute retinal necrosis in humans.