Lars Haarr

Identification and characterization of the virion protein products of herpes simplex virus type 1 gene UL47

We have identified two encoded proteins with an antiserum raised against a synthetic oligopeptide corresponding to amino acids 671 to 684 of the predicted protein product of gene UL47 of herpes simplex virus type 1 (HSV-1). They have apparent Mrs of 82,000 and 81,000 and are both major virion components located in the tegument. The 82/81K proteins were first detected in infected cells in minor amounts 6 h after infection at 37 degrees C but were later (from 10 h until 24 h after infection) present in large amounts. UL47 regulation was investigated using phosphonoacetic acid (PAA), an inhibitor of DNA synthesis: the amounts of the 82/81K protein synthesized were compared with those of 65KDBP, an early gene product, and 21K/22K, a true late gene product. The data showed that UL47 is regulated as a true late gene.

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

ABSTRACT 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.

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

BackgroundEvidence 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.MethodsWe 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).ResultsPrevious 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).ConclusionThe 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.

The herpes simplex virus type 1 particle: structure and molecular functions

This review is a summary of our present knowledge with respect to the structure of the virion of herpes simplex virus type 1. The virion consists of a capsid into which the DNA is packaged, a tegument and an external envelope. The protein compositions of the structures outside the genome are described as well as the functions of individual proteins. Seven capsid proteins are identified, and two of them are mainly present in precursors of mature DNA‐containing capsids. The protein components of the 150 hexamers and 12 pentamers in the icosahedral capsid are known. These capsomers all have a central channel and are connected by Y‐shaped triplexes. In contrast to the capsid, the tegument has a less defined structure in which 11 proteins have been identified so far. Most of them are phosphorylated. Eleven virus‐encoded glycoproteins are present in the envelope, and there may be a few more membrane proteins not yet identified. Functions of these glycoproteins include attachment to and penetration of the cellular membrane. The structural proteins, their functions, coding genes and localizations are listed in table form.

Prevalence of Antibodies against Herpes Simplex Virus Types 1 and 2 in Children and Young People in an Urban Region in Tanzania

ABSTRACT 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.

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, and risk factors for, HSV-2 antibodies in sexually transmitted disease patients, healthy pregnant females, blood donors and medical students in Tanzania and Norway

The prevalence of specific HSV-2 antibodies was studied in Tanzanian and Norwegian sexually transmitted disease (STD) patients (1095) and non-STD patients (488). Correlates to demographic and behavioural factors were evaluated. Seropositivity was determined by the non-commercial peptide-55 enzyme-linked immunoassay. The prevalence of HSV-2 antibodies was 70% in Tanzanian and 17% in Norwegian STD patients, 35% in Tanzanian blood donors and pregnant women, and 4, 7 and 14% in Norwegian medical students, blood donors and pregnant women respectively. A higher HSV-2 prevalence was associated with female sex, increasing age, previous STDs, history of genital HSV infection, coitarchal age (age at first intercourse) <15 years and HIV seropositivity. Compared to previous data, the prevalence of HSV-2 antibodies in Tanzanian STD patients has increased remarkably. In Norwegian STD patients our results are consistent with, or lower than, the prevalence previously reported in Western Europe. Demographic rather than behavioural factors were associated with higher prevalence of HSV-2 antibodies in STD patients.

LABELING OF SURFACE PROTEINS OF HERPES SIMPLEX VIRUS TYPE 1 USING A MODIFIED BIOTIN-STREPTAVIDIN SYSTEM

Methods of labeling surface proteins on herpes simplex virus (HSV) which have minimal effect on the biological activity of the virus are useful for the study of both the localization and function(s) of surface proteins. The present work describes a procedure using a water-soluble biotin compound, sulfo-NHS-biotin, which is unable to penetrate biological membranes and reacts with primary amines in proteins. Labeled proteins were detected by binding of [125I]streptavidin. Specific reaction with surface proteins was shown in Western blots using antibodies against selected proteins in the envelope or in the tegument. Proteins susceptible to iodination were also biotinylated, but the efficiency of labeling varied from one protein to another. As a result of freezing and thawing of the virus, as well as the manipulations involved in Ficoll gradient purification, internal proteins were labeled. The infectivity of the virus was reduced by approximately 40% after biotinylation. Labeled viruses were visualized by fluorescein isothiocyanate-conjugated streptavidin, and seen as distinct spots on the surface of the cells.

DNA- and RNA-binding proteins of chromatin from Escherichia coli

The different proteins present in chromatin of Escherichia coli have been analyzed by a variety of techniques. The chromatin was isolated using a previously published procedure (Sjåstad, K., Fadnes, P., Krüger, P.G. Lossius, I. and Kleppe, K. (1982) J. Gen. Microbiol. 128, 3037) and solubilized by the action of micrococcal nuclease or DNAase I. The DNA-protein and RNA-protein complexes thus obtained were purified by sucrose gradient centrifugation and isopycnic gradient centrifugation in metrizamide in low ionic strength. The protein: DNA ratio of the DNA-protein complexes was estimated from the latter method and found to be approx. 1.75. The protein components were analyzed further by one- and two-dimensional gel electrophoresis. Approx. 15 major polypeptides were detected in the DNA-protein complex, whereas 10 were present in the RNA-protein complex. The majority of the polypeptides in both complexes had acidic isoelectric pH. The polypeptides in the two complexes differed markedly and only two polypeptides, having molecular weights of 57,000 and 37,000, respectively, were found to be common in both complexes. In agreement with earlier studies, the basic protein HU was not present in the DNA-protein complex. Affinity studies of the proteins from chromatin using DNA- and RNA-Sepharose columns in general confirmed the above conclusions. The two-dimensional gel electrophoretic patterns of the proteins in the different complexes were compared with those of proteins in the inner and outer membranes. Only one of the major polypeptides present in the inner membrane, having a molecular weight of 57,000, was enriched in the DNA-protein complex.

Processing of herpes simplex virus type 1 glycoproteins: two-dimensional gel analysis using monoclonal antibodies.

The number of discrete species of glycoprotein induced by herpes simplex virus type 1 (HSV-1, strain 17 syn+) and their processing has been examined by a combination of immunoprecipitation with monoclonal antibodies and analysis of immune precipitates by two-dimensional (2D) gel electrophoresis. Seventeen different monoclonal antibodies directed against glycoproteins A/B, C, D and E were used. Polypeptides intermediate in the synthesis of gA/B, C and D were visualized and two early intermediates of glycoprotein A/B (pgA/B1 and pgA/B2), both mannose-containing, were identified. Comparison on 2D gels of polypeptides synthesized in vitro from HSV-1-infected cell mRNA with those synthesized in pulse-chase experiments in vivo has allowed identification of early precursors of pgA/B and pgD. Their apparent mol. wt. were 105000 and 47000 respectively. The 2D gel analysis of glycoproteins induced in HFL cells infected with 17 syn+ revealed a number of previously unreported glycoprotein species. One had mobility on both gradient and single concentration SDS-polyacrylamide gels similar to that of gC but was resolved from gC on non-equilibrium pH gradient gels. This glycoprotein was produced in relatively large amounts and was not precipitated by any of the monoclonal antibodies used in this study. The data suggest that this glycoprotein either has a polypeptide chain unrelated to those of glycoproteins A/B, C, D or E or alternatively is derived from one of them and modified in such a way as to mask or remove the antigenic sites with which the monoclonal antibodies interact. Sixteen other previously unreported glycoprotein species not obviously related, as judged by their electrophoretic mobility, to gA/B, C, D or E were also identified: two reacted with gA/B-specific monoclonal antibodies and five with glycoprotein C-specific monoclonal antibodies. The origin of the remaining nine new glycoproteins has still to be ascertained.