Stability of glycoprotein gene sequences of herpes simplex virus type 2 from primary to recurrent human infection, and diversity of the sequences among patients attending an STD clinic.

BACKGROUND: Herpes simplex virus type 2 (HSV-2) is sexually transmitted, leading to blisters and ulcers in the genito-anal region. After primary infection the virus is present in a latent state in neurons in sensory ganglia. Reactivation and production of new viral particles can cause asymptomatic viral shedding or new lesions. Establishment of latency, maintenance and reactivation involve silencing of genes, continuous suppression of gene activities and finally gene activation and synthesis of viral DNA. The purpose of the present work was to study the genetic stability of the virus during these events. METHODS: HSV-2 was collected from 5 patients with true primary and recurrent infections, and the genes encoding glycoproteins B,G,E and I were sequenced. RESULTS: No nucleotide substitution was observed in any patient, indicating genetic stability. However, since the total number of nucleotides in these genes is only a small part of the total genome, we cannot rule out variation in other regions. CONCLUSIONS: Although infections of cell cultures and animal models are useful for studies of herpes simplex virus, it is important to know how the virus behaves in the natural host. We observed that several glycoprotein gene sequences are stable from primary to recurrent infection. However, the virus isolates from the different patients were genetically different.

Identification and characterization of two zebrafish nectin-1 genes that are differentially expressed in the developing eye and brain.

Nectins are cell adhesion molecules of the immunoglobulin type that play important roles in the development of the nervous system. We have characterized two paralogous zebrafish nectin-1 genes, nectin-1a and nectin-1b, that differ in expression. Nectin-1a expression is first found in the anterior neural keel and later in the optic cup. In the retina, nectin-1a appears in the outer part and extends inwards, while nectin-1b starts in the inner part and spreads outwards. Only nectin-1a was detected in the cornea, the lens, and in the region of photoreceptor cell differentiation in the retina. Both genes were expressed in ganglion cells and inner nuclear neurons. In the brain, nectin-1a was restricted to the epiphysis and a cluster of cells in the posterior hindbrain, whereas nectin-1b was found in several brain areas. Zebrafish may, therefore, be a useful model for identifying different functions of nectin-1 in the developing eye and nervous system.

HIV-1, HSV-2 and syphilis among pregnant women in a rural area of Tanzania: prevalence and risk factors.

BACKGROUND: Evidence suggests that a substantial proportion of new HIV infections in African countries are associated with herpes simplex virus type 2 (HSV-2). Thus, the magnitude of HSV-2 infection in an area may suggest the expected course of the HIV epidemic. We determined prevalence of genital herpes, syphilis and associated factors among pregnant women from a remote rural Tanzanian community that has a low but increasing HIV prevalence. METHODS: We analysed 1296 sera and responses to a standard structured questionnaire collected from pregnant women aged between 15-49 years, attending six different antenatal clinics within rural Manyara and Singida regions in Tanzania. Linked anonymous testing (with informed consent) of the serum for specific antibodies against HSV-2 was done using a non-commercial peptide- 55 ELISA. Antibodies against syphilis were screened by using rapid plasma reagin (RPR) and reactive samples confirmed by Treponema pallidum haemagglutination assay (TPHA). RESULTS: Previous analysis of the collected sera had shown the prevalence of HIV antibodies to be 2%. In the present study the prevalence of genital herpes and syphilis was 20.7% (95% CI: 18.53-23.00) and 1.6% (95% CI: 1.03-2.51), respectively. The presence of HSV-2 antibodies was associated with polygamy (OR 2.2, 95% CI: 1.62 - 3.01) and the use of contraceptives other than condoms (OR 1.7, 95% CI: 1.21 - 2.41). Syphilis was associated with reporting more than one lifetime sexual partner (OR 5.4, 95% CI: 1.88 - 15.76) and previous spontaneous abortion (OR 4.3, 95% CI: 1.52-12.02). CONCLUSION: The low prevalence of HIV infection offers a unique opportunity for strengthening HIV prevention in a cost-effective manner. The identification and control of other prevalent curable STIs other than syphilis and specific intervention of HSV-2 in specific populations like pregnant women would be one among approaches towards preventing incident HIV infections.

Divergence and recombination of clinical herpes simplex virus type 2 isolates.

Herpes simplex virus type 2 (HSV-2) infects the genital mucosa and is one of the most common sexually transmitted viruses. Here we sequenced a segment comprising 3.5% of the HSV-2 genome, including genes coding for glycoproteins G, I, and E, from 27 clinical isolates from Tanzania, 10 isolates from Norway, and 10 isolates from Sweden. The sequence variation was low compared to that described for clinical HSV-1 isolates, with an overall similarity of 99.6% between the two most distant HSV-2 isolates. Phylogenetic analysis revealed a divergence into at least two genogroups arbitrarily designated A and B, supported by high bootstrap values and evolutionarily separated at the root. Genogroup A contained isolates collected in Tanzania, and genogroup B contained isolates collected in Tanzania and Scandinavia, implying that the genetic variability of HSV-2 is higher in Tanzania than in Scandinavia. Recombination network analysis and bootscan analysis revealed a complex pattern of phylogenetically conflicting informative sites in the sequence alignments. These signals were present in synonymous and nonsynonymous sites in all three genes and were not accumulated in specific regions, observations arguing against positive selection. Since the PHI test applied solely to synonymous sites revealed a high statistical probability of recombination, we suggest as a novel finding that homologous recombination is, as reported earlier for HSV-1 and varicella-zoster virus, a prominent feature in the evolution of HSV-2.

Herpes simplex virus infection and genital ulcer disease among patients with sexually transmitted infections in Dar es Salaam, Tanzania.

The relative importance of Haemophilus ducreyi and Treponema pallidum in genital ulcer disease in Africa has decreased recently, whereas that of herpes simplex virus (HSV) type 2 has increased. We analysed 301 lesional specimens from Tanzanian patients with genital ulcer disease for the presence of H. ducreyi, T. pallidum and HSV-1/HSV-2 by performing a separate PCR for each pathogen. Infectious agents were detected in 211 (70%) of the cases. A single pathogen was found in 191 samples and two or more pathogens in the remaining 20. HSV-2 represented 83% of all identified pathogens, HSV-1 8%, T. pallidum 4% and H. ducreyi 5%. HSV-1 was identified as a single pathogen in four samples, in combination with others in an additional 14 samples. Thus, HSV-1 can also be the cause of genital ulcer disease in Africa. Regular surveillance of genital ulcer disease aetiology is important in programs for management of genital ulcer disease and HIV in Africa.

Prevalence of antibodies against herpes simplex virus types 1 and 2 in children and young people in an urban region in Tanzania.

Herpes simplex virus type 1 (HSV-1) is transmitted by close contact, both sexual and nonsexual, and infections are acquired during childhood and adolescence. Herpes simplex virus type 2 (HSV-2), however, is thought to be transmitted mainly by sexual contact. Most HSV-2 infections are consequently expected to occur after the onset of sexual activity. Recent reports indicate an increasing prevalence of HSV-2 on the African continent, but most studies have been performed on adult cohorts. In the present study, we collected sera from Tanzanian children and young persons from 1 to 20 years old, with at least 100 individuals in each age group. Antibodies against HSV-1 and HSV-2 were detected by an in-house Western blot method which was shown to perform well in comparison with a commercial Western blot assay. Type-specific antibodies were also analyzed by two noncommercial enzyme-linked immunosorbent assay methods based upon the antigenicities of branched synthetic oligopeptides corresponding to epitopes in glycoprotein G of HSV-1 or HSV-2. The prevalence of HSV-1 antibodies increased gradually from 73% for the age group of 1 to 4 years to 92% for the age group of 17 to 20 years. The prevalence of HSV-2 antibodies was unexpectedly high, as 15% of the children were infected by the age of 8 years, with the incidence increasing gradually to 40% in the age group of 17 to 20 years. The reason for this unexpectedly high frequency is not clear but could suggest that nonsexual transmission of HSV-2 is more common than previously thought. There was no statistically significant association between seropositivities for HSV-2 and human immunodeficiency virus.

Demographic and behavioural factors in Tanzanian and Norwegian patients with sexually transmitted infections.

To evaluate whether differences in demographic or behavioural factors might explain differences in reported or diagnosed sexually transmitted infections (STI), we have compared data from 1097 Tanzanian and Norwegian STI patients. Most demographic data were similar, whereas some behavioural data differed. Norwegian patients reported significantly higher numbers of sexual partners than Tanzanian. Thirty-three percent of Tanzanian patients tested positive for HIV antibodies, females more often (43%) than males (26%). Approximately one-third and two-thirds of the female HIV-positive Tanzanian STI patients had already seroconverted at the age of 25 and 30 years, respectively. The national differences encountered probably reflect cultural differences, different panoramas of STI and a lower accessibility to optimal health services in Tanzania. Lack of expected statistical associations between some of the data in the Tanzanian STI group might question the validity of the retrospectively collected data in this group, or indicate that questions not included in the questionnaire might be of importance.

A branched, synthetic oligopeptide corresponding to a region of glycoprotein G of HSV-1 reacts sensitively and specifically with HSV-1 antibodies in an ELISA.

Herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), which are common worldwide, are so similar that antibodies directed against one serotype may crossreact with antigens from the other one. Methods for specific detection of antibodies against HSV-1 or HSV-2 are based upon the antigenicities of glycoproteins G. However, due to the cost, the available commercial methods may not readily be used in developing countries. A different enzyme-linked immunosorbent assay (ELISA) method, based upon a synthetic oligopeptide corresponding to an immunogenic region in glycoprotein G of HSV-2, has been used recently and successfully for detection of HSV-2 antibodies. In the present study, the sequences of a newly identified immunogenic and type-specific region in glycoprotein G of HSV-1 was used to synthesize three different, branched oligopeptides. The performances of these peptides in an ELISA were investigated by testing Scandinavian and African sera which were characterized by commercial ELISA and Western blotting methods and divided into four groups either lacking HSV antibodies, containing antibodies against one or the other virus, or against both types. The peptide which corresponded in sequence to the immunodominant region was as specific and sensitive by an ELISA as were the commercial methods. The method is inexpensive and reliable.

The core 2 beta-1,6-N-acetylglucosaminyltransferase-M encoded by bovine herpesvirus 4 is not essential for virus replication despite contributing to post-translational modifications of structural proteins.

The Bo17 gene of bovine herpesvirus 4 (BoHV-4) is the only virus gene known to date that encodes a homologue of the cellular core 2 beta-1,6-N-acetylglucosaminyltransferase-mucine type (C2GnT-M). Recently, our phylogenetic study revealed that the Bo17 gene has been acquired from an ancestor of the African buffalo around 1.5 million years ago. Despite this recent origin, the Bo17 sequence has spread to fixation in the virus population possibly by natural selection. Supporting the latter hypothesis, it has been shown by our group for the V. test strain that Bo17 is expressed during BoHV-4 replication in vitro, and that Bo17 expression product (pBo17) has all three enzymic activities exhibited by cellular C2GnT-M, i.e. core 2, core 4 and I branching activities. In the present study, firstly it was investigated whether encoding a functional C2GnT-M is a general property of BoHV-4 strains. Analysis of nine representative strains of the BoHV-4 species revealed that all of them express the Bo17 gene and the associated core 2 branching activity during virus replication in vitro. Secondly, in order to investigate the roles of Bo17, its kinetic class of expression was analysed and a deleted recombinant strain was produced. These experiments revealed that Bo17 is expressed as an early gene which is not essential for virus replication in vitro. However, comparison of the structural proteins, produced by the wild-type, the revertant and the deleted viruses, by 2D gels demonstrated that pBo17 contributes to the post-translational modifications of structural proteins. Possible roles of Bo17 in vivo are discussed.

Detection of viral sequences in archival spinal cords from fatal cases of poliomyelitis in 1951-1952.

Poliovirus (PV) subjected to genetic characterization is often isolated from faecal carriage. Such virus is not necessarily identical to the virus causing paralytic disease since genetic modifications may occur during replication outside the nervous system. We have searched for poliovirus genomes in the 14 fatal cases occurring during the last epidemics in Norway in 1951-1952. A method was developed for isolation and analysis of poliovirus RNA from formalin-fixed and paraffin-embedded archival tissue. RNA was purified by incubation with Chelex-100 and heating followed by treatment with the proteinase K and chloroform extraction. Viral sequences were amplified by a reverse transcriptase-polymerase chain reaction (RT-PCR), the products subjected to TA cloning and sequenced. RNA from the beta-actin gene, as a control, was identified in 13 cases, while sequences specific for poliovirus were achieved in 11 cases. The sequences from the 2C region of poliovirus were rather conserved while those in the 5'-untranslated region were variable. The developed method should be suitable also for other genetic studies of old archival material.

Performance characteristics of a glycoprotein G based oligopeptide (peptide 55) and two different methods using the complete glycoprotein as assays for detection of anti-HSV-2 antibodies in human sera.

Assays for serological diagnosis of HSV-2 infection in clinical settings have been generally available only recently. We wanted to investigate and compare the diagnostic utility of three different ELISAs for detection of anti-HSV-2 IgG antibodies, using intact glycoprotein G or an oligopeptide from a portion of the protein as antigens. HSV-1 negative/HSV-2 negative sera (n = 32), HSV-1 positive/HSV-2 negative sera (n = 30) and sera from HSV-2 culture positive individuals (n = 36), collected at least 6 months after culture verified HSV-2 genital infection were examined. Cut-off values were determined according to the manufacturer's instructions, and also by establishing new cut-off values at the level of highest diagnostic efficiency. Sensitivities and specificities were compared for each assay. In addition, test accuracies were compared using receiver-operating characteristics (ROC) methodology. Establishment of new cut-off values increased the performance characteristics for all three tests. At similarly set cut-off values, the peptide 55 assay showed the highest diagnostic sensitivity (100%) and specificity (98%). All three assays displayed high efficiency and also high agreement between the tests (kappa > 0.85 for all comparisons). The performance of all three assays were satisfactory although the highest efficiency and accuracy was obtained with the peptide 55 assay.

Detection of HSV-2 in genital ulcers from STD patients in Dar es Salaam, Tanzania.

BACKGROUND: Genital ulcer disease (GUD) is common in many developing countries. Several reports indicate that there is an association with HIV infection. Analysis by polymerase chain reaction (PCR) has demonstrated that the ulcers are frequently caused by herpes simplex type 2 (HSV-2), although HSV-1 is becoming increasingly important in many parts of the world. Comparable studies have not been performed in Tanzania. OBJECTIVES: To determine the prevalence of HSV-2 and HSV-1 in genital ulcers in Dar es Salaam, Tanzania and determine their possible association with HIV infection. STUDY DESIGN: Samples were collected from 70 consecutive patients with GUD attending a clinic for sexually transmitted diseases. Specimens from ulcers were analysed by PCR for the presence of HSV-2 and HSV-1, and sera were examined for antibodies against HSV-2 and HIV. RESULTS AND DISCUSSION: HSV-2 DNA was detected in 64% of the specimens from ulcers while HSV-1 DNA was not found in any of them. Antibodies to HSV-2 and HIV were detected in 79.7 and 42% of the patients' sera, respectively. Although there was a significant positive association between HIV and HSV-2 seropositivity, HSV-2 DNA in genital ulcers was not more prevalent among HIV seropositive than among HIV seronegative individuals. CONCLUSION: The prevalence of HSV-2 antibodies among Tanzanian patients with genital ulcers is very high, and HSV-2 is detected in most of the ulcers. There is an association between infections with HIV and HSV-2, but the relationship is not clear.