Molecular epidemiology of herpes simplex virus type 1 genital infection in association with clinical manifestations

Summary.  The antigenic types of herpes simplex virus (HSV), HSV-1 and HSV-2 are considered to be the etiology of genital herpes. Symptoms of primary HSV-1 and HSV-2 genital infections are similar, however, recurrence of the infection is less frequent after the HSV-1-related genital infection. We determined genotypes of 79 HSV-1 strains isolated from genital lesions in women (43 from primary and 36 from recurrent infections), by analyzing restriction fragment length polymorphism of the HSV-1 strains. Each proportion of genotypes of F1, F12, and F41 in strains isolated from recurrent genital lesions was higher than the corresponding proportion in strains from primary genital lesions. Genotypes of HSV-1 strains isolated two or more times from recurrent genital lesions of each of three subjects were genotye F1, thereby supporting the hypothesis that the F1 genotype is closely associated with recurrence. While the possibility of a genotype preference at the site of infection was not ruled out, genotypes of more than half the number of HSV-1 strains from genital lesions were the same as those from non-genital (mainly oral-facial) lesions analyzed in our foregoing studies, thus indicating that most HSV-1 genotypes are apparently shared by genital and non-genital lesions. Accepted October 1, 1999     

Role of macrophages in hepatitis induced by Herpes simplex virus types 1 and 2 in mice.

A marked difference was found between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in the induction of hepatic necrotic lesions in mice inoculated intraperitoneally. Although HSV-2 produced many large, progressive liver lesions in 4-week-old BALB/c mice, HSV-1 only occasionally induced a few, self-limiting foci, which eventually healed. This was reflected in the isolation of HSV from the liver and spleen, two organs that are rich in macrophages. Although HSV-1 could be only temporarily isolated, HSV-2 was found in the two organs until the mice died. On the other hand, no such difference was found in the isolation of virus from the brain, which contains no macrophages, and the mice eventually died from encephalitis. This difference in hepatic involvement caused by the two virus types was found to parallel a marked difference in the restriction of HSV-1 and HSV-2 replication by macrophages as measured by an infectious center assay in vitro. HSV-2 produced 17 times as many infectious centers in infected peritoneal macrophage cultures as did HSV-1. Furthermore, the HSV-2 plaques in the cell overlay were large and increasing in size, whereas the HSV-1 plaques were small and showed regression on prolonged incubation. It was shown that this diversity was unique to the macrophage population and not caused by differences in the uptake of virus by macrophages. This model involving two closely related virus types shows the importance of tissue macrophages in the primary host defense against virus infections.     

Replication of herpes simplex virus type 1 and 2 in the medulla of the adrenal gland after vaginal infection of mice

Summary After vaginal infections of mice with neuroinvasive strains of herpes simplex virus type 1 and 2 (HSV-1, HSV-2) virus replicates in the epithelium of the vagina, in the paravaginal ganglia, in the spinal cord and finally in the brain and in the adrenal glands. However, viral antigens could be demonstrated only in the medulla of the adrenal glands but not in the cortex, as assessed by immunohistochemistry (IHC). HSV could not be isolated from liver, spleen, uterus, and ovaries. This contrasts to the intraperitoneal (i.p) route of infection with replication in different visceral organs including the adrenal gland's cortex.     

Detection of herpes simplex virus type 1, herpes simplex virus type 2 and varicella-zoster virus in skin lesions. Comparison of real-time PCR, nested PCR and virus isolation.

BACKGROUND: Herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2) and varicella-zoster virus (VZV) cause a wide range of signs and symptoms, varying from trivial mucocutaneous lesions to life-threatening infections, especially in immuno-suppressed patients. Since antiviral drugs are available, rapid and sensitive laboratory diagnosis of these virus infections is important. OBJECTIVE: To set up and evaluate HSV-1, HSV-2 and VZV qualitative real-time PCR on the Lightcycler system and to compare the results with those of the 'in-house' nested PCR and virus isolation. STUDY DESIGN: 110 consecutive samples from dermal or genital lesions from patients suspected of having HSV infections and another 110 samples from patients with suspected VZV infections were tested with real-time PCR, nested PCR and virus isolation. RESULTS: 24 samples (22%) were positive for HSV-1 by virus isolation and nested PCR, whereas 26 (24%) were positive by real-time PCR. HSV-2 was detected in 28 samples (25%) by virus isolation, in 41 (37%) by nested PCR and in 40 (36%) by real-time PCR. VZV was isolated in 15 samples (14%) and VZV DNA was detected in 51 samples (46%) by nested PCR as well as by real-time PCR. Nucleic acid amplification increased the detection rate of HSV-2 and VZV DNA in particular compared to virus isolation. No significant difference in sensitivity was found between real-time PCR and nested PCR. CONCLUSION: Real-time PCR has the advantage of rapid amplification, a reduced risk for contamination and it is a suitable method for diagnosis of VZV and HSV in specimens from skin lesions.     

Herpes simplex virus type 1 and 2 in the adrenal glands: Replication and histopathology

Summary The adrenal glands were shown to be the most severely infected organs in the early phase of HSV-1 infections (up to 10 days p. i.) after i. p. infections in mice. Virus could be isolated from the adrenal glands as early as one hour after infection with pathogenic and apathogenic strains. Infection of the adrenal glands is a result of viremia. The content of HSV-1 (5 strains) was much higher in the adrenals than in spleen and liver. It peaked at 3–4 days p. i. compared to 1–2 days in spleen and liver. Only strain 17 syn⁺ produced low tissue titres in the adrenal glands.Morphologic alterations by HSV-1 infections commenced with distinct foci 2 days after infection in the zona fasciculata, detected immunohistochemically by HSV-specific peroxidase-staining. Necrotic cells could be observed. The foci became confluent until day 4 and remained in this status up to day 7 p. i. During infection immunocompetent cells (macrophages, granulocytes, many T-helper — but only few T-cytotoxic/suppressor lymphocytes) could be observed. On day 10 p. i. the viral antigen had been completely eliminated.In contrast, intraperitoneal infections with 5 strains of HSV-2 resulted in infection of the adrenal glands only to a low degree. The titer of virus was low (exception: strain HG 52). This correlates well with the type of disease produced by either HSV-1 or 2. By comparing the replication of different strains of HSV-1 and 2, three types of tropism after i. p. infection of mice can be distinguished.With 7 FiguresIn partial fulfillment of his M. D.'s degree.     

Interference between strains of type 1 and type 2 herpes simplex virus.

Herpes simplex virus type 2 (HSV-2) markedly interferes with the replication of herpes simplex virus type 1 (HSV-1) upon simultaneous infection of HEp-2 cells, even under conditions where HSV-1 infection precedes that of HSV-2 by 3 hr. Interference required infective HSV-2 virus. The HSV-1 progeny of mixed infections were found to be phenotypically mixed. In most cases, prior infection with homologous strains slightly inhibits the replication of a superinfecting HSV-1 strain. An exception to this finding is HSV-1 strain MP, whose replication is accelerated by prior infection with nondefective HSV-1 strains. The implications of these findings as related to studies of defective HSV, to studies of the genetic interactions between HSV-1 and HSV-2, and to clinical infections with HSV are discussed.     

Isolation of the Enhanced Neurovirulent HSV-1 Strains from Korean Patients

Herpes simplex virus type 1 (HSV-1) is a neurotropic DNA virus which has latency in human. In this study, we isolated various HSV-1 strains, named KHS, from the skin lesions of Korean patients and characterized the specific features of each strain. We found that KHS strains produced small, cell associated and nonsyncycial plaques in Vero cells. We classified KHS strains into two substrains, KHS 1 which had highly condensed plaques and KHS 2 which had less condensed plaques. Since gD protein of HSV-1 plays important roles in viral plaque formation, we determined the nucleotide sequences of gD genes of KHS strains. According to deduced amino acid sequences of gD protein in KHS strains compared with prototype strains KOS and F, we found that gD of KHS strains have more putative O-glycosidic sites, serine in KHS 1 and threonine in KHS 2, respectively.To find out the establishment of viral latency, we infected each virus strain into eyes of mice and carried out trigerminal ganglia explanting experiment. We found that both KHS strains established latent infections stably just as did the prototype KOS and F strains. The eye swab experiments were carried out to check the viral replication in vivo. KHS 1 exhibited a longer shedding time in eyes of mice. We also found that KHS 1 has a higher neurotropic affinity by determining the time it took for the virus to reach the trigerminal ganglia from the eyes. Currently, we are studying the possible mechanism of high neuroinvasiveness of KHS 1 strain.     

Asymptomatic vaginal herpes simplex virus infections in mice: virology and pathohistology

Summary One of the causes of genital tract infections in humans are herpes simplex virus types 1 and 2 (HSV-1, HSV-2). Although primary and recurrent infections can be clinically apparent and in part very serious, many infections are asymptomatic and result only in temporary genital shedding of virus (recurrences). During our investigations of vaginitis, strain IES of HSV-1 produced an asymptomatic infection. Replication in the murine vaginal (vag.) epithelium as well as antibody formation after vag. infection was comparable to those of survivors after infection with highly virulent strains. Titration of liver, spleen, ovaries, adrenal glands, spinal cord, or brain after vag. IES infection revealed no virus, whereas after i.p. infection virus could be demonstrated in many organs examined. Histological examination with a DNA probe (in situ hybridisation), HSV antibodies (immunohistochemistry), and haematoxylin and eosin (HE) staining showed only small focal HSV lesions of the vaginal epithelium in early stages of the infection, never exceeding to the subepithelial tissue. Severe infiltrations and ulcerations after infection with highly virulent strains (17syn⁺, ER^–) could never be demonstrated after IES vag. infection. Identical replication rates of both groups of HSV despite much greater areas of epithelial necrosis with the virulent strains may be explained by the large number of virus inactivating granulocytes induced by the virulent strains, thus inactivating the hypothetical higher virus load.     

Thymidine kinase sequence analysis of herpes simplex virus type 1 strains present in different compartments in an atypical impetiginous rash on the lesional skin of a burn patient

We report the case of a 23-year-old male burn patient with an unusual herpes simplex virus (HSV) skin manifestation. The clinical symptoms and results of HSV type 1 (HSV-1) UL23 polymorphism analysis from saliva and lesional skin underscores the need for performing molecular analysis of HSV-1 infections in burned patients presenting unusual skin lesions.     

Comparison of the association with eczema herpeticum in the two predominant genotypes of herpes simplex virus type 1

Eczema herpeticum, sometimes called Kaposi's varicelliform eruption, is usually caused by a disseminated herpes simplex virus infection in a patient whose underlying skin disease is atopic dermatitis. Herpes simplex virus type 1 (HSV-1), a widespread infectious agent in human populations, is the etiologic agent of eczema herpeticum. Analyses of restriction fragment length polymorphism (RFLP) of HSV-1 strains isolated in Japan, using restriction endonucleases, revealed the presence of two predominant genotypes of F1 and F35. The number of HSV-1 strains of F1 genotype was over twice that of the F35 genotype, and the nucleotide change between F1 and F35 was estimated to be 1.5%. The question of whether the genomic difference between two predominant genotypes could influence clinical manifestations remained to be addressed. On the basis of RFLP, we determined genotypes of HSV-1 strains isolated from the patients in Japan, including those with eczema herpeticum. Two of four HSV-1 strains of F35 genotype were from patients with eczema herpeticum, whereas none of 12 HSV-1 strains of F1 genotype was from those with eczema herpeticum. Thus, the F35 genotype seemed to be associated more frequently with eczema herpeticum than the F1 genotype. © 1996 Wiley-Liss, Inc.     

High seroprevalence of herpes simplex virus type 2 infection in French human immunodeficiency virus type 1-infected outpatients

Using commercially available herpes simplex virus (HSV) type-specific serological diagnostic tests, HSV type 2 (HSV-2) antibody prevalence was assessed in two parallel prospective studies including 534 human immunodeficiency virus type 1 (HIV-1)-infected outpatients living in two areas of northern France. In the first cohort of 434 subjects, 223 (51%) individuals demonstrated a positive HSV-2 serological status while 66 (66%) of 100 subjects in the second cohort were seropositive for HSV-2 (51 versus 66%; P = 0.08). Among the 223 HSV-2-seropositive subjects identified in the first study cohort, only 22 (10%) had suffered from recurrent anogenital lesions during the past 12 months while 154 (69%) had no clinical history of herpesvirus infection. Our findings demonstrate high proportions of subclinical and undiagnosed HSV-2 infection in HIV-1-infected individuals and suggest that HSV type-specific serological testing in the French HIV-1-infected subpopulation could be an efficient strategy to diagnose clinically asymptomatic HSV-2 infections.     

Vaginal infection of mice with HSV type 2 variant ER-: a new animal model for human primary genital HSV type 2 infections.

Studying the pathogenesis of vaginal infections in mice with two variants of Herpes simplex virus type 2 (HSV-2) strain ER we observed that both variants ER+ and ER- caused severe vaginitis but only ER+ invaded the CNS leading to lethal neurological disease. In contrast, mice infected with ER- cleared the virus from the vagina and recovered from infection. ER+ and ER- expressed equal levels of thymidine kinase (TK) indicating a TK-independent difference in neurovirulence. Using the non-neurovirulent variant ER-, we were able to investigate humoral immune responses later after infection. Vaginal infection with ER- suppressed serum antibody formation after a secondary systemic HSV-1 infection. Fresh isolates of HSV-1 and HSV-2 caused uniformly a lethal neurological disease after vaginal inoculation of mice. However, some animals survived an intraperitoneal infection with these isolates. Infection with HSV-1 isolates stimulated a strong antibody production, whereas infection with HSV-2 isolates suppressed antibody formation, thus supporting earlier results from our group obtained with laboratory strains. Since suppression of antibody formation could be demonstrated with clinical HSV-2 isolates and likewise after vaginal infection with HSV-2 variant ER- we consider this phenomenon to be of relevance in human genital HSV-2 infections. Vaginal infection of mice with variant ER- represents a new model for primary genital HSV-2 infections; this model could be useful for histopathological, virological, immunological and drug testing studies.     

Experimental encephalitis in the newborn rat due to herpes simplex virus type 2.

Neonatal Wistar rats were inoculated intracranially with one of two strains of Herpes simplex virus type 2 (HSV-2). Death due to herpetic encephalitis occurred in many animals inoculated at 0 to 15 days of age. In rats inoculated at 0 to 10 days, lesions were destructive in nature, whereas in older animals with demonstrable CNS lesions, there was usually a prominent inflammatory cell response. Typical herpesvirus particles were observed in neurons, astrocytes, and oligodendrocytes by electron microscopy and HSV-2 was frequently isolated from the CNS of inoculated animals. An age-related resistance to HSV-2 was observed in rats inoculated at 20 or more days of age. A significant difference in susceptibility to high and low titers of virus administered was observed in older suckling animals, but not in rats inoculated at 0 or 5 days of age. In view of the age-related resistance to HSV-2 and the histopathological findings, the newborn rat appears to be a suitable animal model to study perinatal HSV-2 encephalitis in infants.     

Herpes simplex viral infection in human neonates: an immunohistochemical and electron microscopic study

Specimens obtained at autopsy from six neonates with herpes simplex virus (HSV) infections were examined microscopically, electron microscopically, and immunohistochemically. Coagulative necrosis with inclusions was found in the livers and adrenal glands in all cases, as well as in various other organs, including the spleen, bone marrow, lungs, esophagus, tongue, and thymus, in some cases. Distinct hemorrhagic diathesis was found in three cases. No characteristic clinical findings, such as skin rashes or elevated titers of the antibody to HSV, were found, and clinical diagnosis was therefore difficult. In three cases isolation and typing of the causative virus were performed virologically, and type 1 HSV (HSV-1) was identified as the causative virus. Immunohistochemically, the type and distribution of the virus were evaluated in all cases with type-specific antisera to types 1 and 2 (HSV-2) antigens by the peroxidase-antiperoxidase method. In five cases the infections were found to be due to HSV-1 and in only one case to HSV-2. In the placenta in one case of HSV-2 infection, HSV antigen was demonstrated in the chorionic villi. Electron microscopic study confirmed the existence of viral particles in the placenta in that case and, thus, the possibility of a transplacental route of infection.     

Herpes simplex virus infections and current methods for laboratory detection

Herpes simplex virus (HSV) is endemic in all societies throughout the world and produces year-round infections in all age groups. While orofacial and genital skin infections predominate, a significant minority of individuals develop more serious herpetic disease in the eye, meninges, and brain tissue. Perinatal infection of the newborn has a high likelihood of dissemination to multiple visceral organs, and immunocompromised patients can develop aggressive necrotizing skin lesions, as well as disseminated disease. HSV is not fastidious and is easily propagated in vitro; virus culture methods are straightforward and currently are offered in many community hospital microbiology laboratories. However, amplified nucleic acid probe assays have now been developed that are significantly more sensitive than culture, have fewer specimen collection and transport constraints, and can generate reliable results the same day of testing. Recent innovations in HSV antibody assays that use serotype 1- and serotype 2- specific reagents can now provide accurate serological separation of HSV-1 and HSV-2 infections by ELISA and Western blot (immunoblot) methods. Community hospitals should anticipate more frequent requests for these more sensitive and specific diagnostic tests. When reliable commercial assays become available, new serological and molecular tests may be within the scope of the community hospital laboratory.     

Improved sensitivity of restriction endonuclease analysis of herpes simplex virus type 2 DNA with polyacrylamide gradient gel electrophoresis

Polyacrylamide gradient gel electrophoresis was used to resolve fragments of herpes simplex virus type 2 (HSV-2) DNA, produced by the restriction endonucleases Alu I, Bam HI, Pst I, and Sma I, which cleave the HSV-2 DNA into more than 30 fragments each. HSV-2 strains isolated from different individual patients could be easily distinguished from each other by the endonucleases Bam HI and Sma I. Successive virus isolates from a single person, analyzed using Alu I and Sma I, showed variability of fragment patterns. The effect of passaging the virus in cell cultures for several cycles was evaluated with the restriction endonuclease Alu I. No differences were found after 29 successive passages in VERO cells. Polyacrylamide gradient gel analysis of restriction endonuclease digests of HSV-2 DNA enables the use of enzymes that cleave the DNA into a great number of fragments, thus improving the sensitivity of analysis.     

Characterization of herpes simplex virus strains isolated from patients with various diseases

Summary Herpes simplex virus strains (HSV) were isolated from various herpetic diseases. These HSV-strains isolated in Bulgaria (687) and in other countries (13) were studied by the neutralization test using standard type specific rabbit antisera and human gamma globulin. The serotype distribution of all strains showed: HSV-1 = 323 strains, HSV-2 = 372 strains, 5 strains behaved antigenically intermediate. A close correlation between the serotype of the strains and the localization of the lesions was established. All but four strains (1.24 per cent) isolated from the head belong to HSV-1, and only six HSV-strains (1.9 per cent) from lesions with other localizations were not HSV-2. Within six months to 8 years 2 to 4 herpes strains were isolated repeatedly from 30 patients. In 23 of these cases they were identical and in the other they showed different biological (3) or antigenical (4) properties. The results suggest, that under natural conditions some HSV-intermediate strains may exist.With 1 Figure     

Pathogenicity and latency competence for rabbits of the herpes simplex virus type 1 ANGpath gC and gE defective mutants.

A total of 139 rabbits was infected to the right scarified cornea with HSV type 1 strains Kupka, ANG, ANGpath and their gE defective (ANGpathI2-4), gC defective (ANGpathgC18), gC/gE negative (ANGpathCI-8) and gC/ICP4 deletion (ANGpathY1) mutants. Strains ANG, ANGpath, ANGpathgC18 and ANGpathY1 were, in contrast to the two gE negative mutants, highly lethal, but 79% of rabbits infected with the non-encephalitogenic Kupka strain survived. Strain Kupka and strain ANGpath gE-negative mutants I2-4 and gCI-8 were tested for their latency competence. While Kupka established latency in the homolateral trigeminal ganglia from 80% of infected rabbits, I2-4 did so in one of 10 animals only, and the gC/gE mutant gCI-8 was not harboured in any of infected animals in an inducible form. Significant correlation was found between shedding into the culture fluid of reactivated virus from the explanted ganglion and brain stem fragments at one hand and the presence of the viral DNA in these organs on the other hand as judged by spot blot hybridization with the HSV-1 strain 17 Kpn I fragments h and i to DNA extracts prepared from these organs. Hybridizations were predominantly negative with the DNA from the corresponding non-cultured organs, except in a few cases of non-cultured ganglion and brain stem from rabbits previously infected with the gE deletion mutants which displayed positive hybridization, although no virus reactivation could be observed in corresponding explants.     

Discriminant analysis of DNA polymorphisms in herpes simplex virus type 1 strains involved in primary compared to recurrent infections

The restriction fragment length polymorphism (RFLP) was analyzed using a set of herpes simplex virus type 1 (HSV-1) strains isolated from oro-facial lesions (oro-facial site collection), which was composed of two subsets: one subset consisted of 57 strains from primary oro-facial lesions, and the other of 47 strains from recurrent oro-facial lesions of patients complicated by factors possibly triggering the recurrence (e.g. malignancy, operation, and treatment of dental caries). RFLP analysis was carried out previously on two other sets of HSV-1 strains: one set was from genital lesions (genital site collection), and the other was from non-genital lesions (non-genital site collection). Discriminant analysis was carried out on the three sets of HSV-1 strains: the criterion variable had two values of primary infection or recurrence, and the predictor variables were 20 RFLPs. The degrees of separation between primary infection and recurrence increased in the order oro-facial site collection, genital site collection, and non-genital site collection. The results of discriminant analysis in this study confirmed that reactivation of HSV-1 infection is influenced by triggering factors and the site of infection, thereby suggesting the capabilities of multivariate analysis (including discriminant analysis) with DNA polymorphisms for molecular epidemiological studies.     

Evaluation of mixed infection cases with both herpes simplex virus types 1 and 2

Herpes simplex virus type 1 (HSV-1) is isolated principally from the upper half of the body innervated by the trigeminal ganglia whereas herpes simplex virus type 2 (HSV-2) is generally isolated from the lower half of the body innervated by the sacral ganglia. However, recent reports suggest that HSV-1 and HSV-2 can each infect both the upper and lower half of the body causing a variety of symptoms and there is a possibility that HSV-1 and HSV-2 infections can occur simultaneously with both causing symptoms. HSV type in clinical isolates from 87 patients with genital herpes and 57 with ocular herpes was determined by the polymerase chain reaction (PCR), and six cases of mixed infection with both HSV-1 and HSV-2 were identified. Of the six cases, three were patients with genital herpes and three were ocular herpes patients. Analysis of the copy number of the HSV-1 and HSV-2 genome by a quantitative real time PCR demonstrated that HSV-1 was dominant at a ratio of approximately 100:1 in the ocular infections. In contrast, the HSV-2 genome was present at a 4-40 times higher frequency in isolates from genital herpes patients. There was no obvious difference between the clinical course of mixed infection and those of single HSV-1 or HSV-2 infections. This study indicated that the frequency of mixed infection with both HSV-1 and HSV-2 is comparatively higher than those of previous reports. The genome ratio of HSV-1 and HSV-2 reflects the preference of each HSV type for the target organ.