Asymptomatic seminal infection of herpes simplex virus:impact on male infertility

In more than half of infertile men,the cause of their infertility is unknown.Several studies revealed the role of viral infections in male infertility.The aim of the present study was to determine the prevalence of herpes simplex virus-1 (HSV-1) and HSV-2 in semen from asymptomatic infertile male patients,and its association with altered semen parameters.A total of 70 semen samples were collected from infertile men who attended the Research and Clinical Center for Infertility in Yazd,Iran.Semen analysis and diagnostic real-time PCR using specific primers and probes for HSV-1 and HSV-2 DNA were performed.Comparison of semen parameters between virally infected and non-infected samples were performed with independent t-test and Mann-Whitney test.Semen analysis showed that infertile men fell into two groups,the male factor group and the unexplained group.HSV-1 and HSV-2 DNA was detected in 16 (22.9%) and 10 (14.3%) of 70 semen samples,respectively.All HSV-positive samples had abnormal semen parameters (the male factor group).Although HSV infection was not associated with sperm motility and morphological defects,it was correlated with lower sperm count in the seminal fluid.The findings suggest that asymptomatic seminal infection of HSV plays an important role in male infertility by adversely affecting sperm count.     

Case report: benefits of quantitative polymerase chain reaction in the clinical management of herpes simplex virus 1 infection with prominent hepatitis and unusual secondary progression

Herpes simplex virus (HSV) infection is rarely considered in the differential diagnosis of severe acute hepatitis and disseminated infection in immunocompetent adults. A case of disseminated HSV-1 infection in an 82-year-old woman initially presenting with neurological problems, signs of meningitis and prominent hepatitis was investigated. Initial diagnosis, monitoring, and follow-up were based on the application of molecular methods to cerebrospinal fluid, serum, and liver tissue samples from this patient. Following an initial full recovery, the patient presented delayed intracerebral haemorrhage and diffuse arthralgia. This atypical case, with delayed secondary progression, highlights the wide range of clinical features of HSV infection and the benefits of monitoring viral load by quantitative real-time polymerase chain reaction (PCR) during patient management.     

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.     

Relationship between cervical disease and infection with human papillomavirus types 16 and 18, and herpes simplex virus 1 and 2

Persistent infection with high‐risk HPV, particularly Type HPV 16 and 18, is necessary in the development of cervical cancer, but apart from HPV infection, other causative factors of most cervical cancers remain unknown. The aim of this study was to determine the prevalence of HPV 16 and HPV 18 and HSV 1 and HSV 2 in cervical samples, and to assess the role of HSVs in cervical carcinogenesis. Two hundred thirty‐three healthy controls and 567 cases (333 of cervicitis, 210 of cervical intraepithelial neoplasia, and 24 of squamous cell carcinoma) in cervical exfoliative cells were tested for HPV 16, HPV 18, HSV 1, and HSV 2 DNA using the triplex real‐time polymerase chain reaction method. In contrast to healthy women, positive rate of HPV is related significantly to cervical lesions (odds ratios (ORs) = 4.1, P < 0.01 for cervical intraepithelial neoplasia; ORs = 24.9, P < 0.01 for squamous cell carcinoma), but not cervicitis (ORs = 2.3, P > 0.05). HSV 2 prevalence in cervical intraepithelial neoplasia and squamous cell carcinoma was higher than in healthy women (ORs = 4.9, P < 0.05 for cervical intraepithelial neoplasia; ORs = 4.7, P < 0.05 for squamous cell carcinoma). HSV 2 coinfection with HPV in cervical intraepithelial neoplasia and squamous cell carcinoma was strongly higher than in healthy women (ORs = 34.2, P < 0.01 for cervical intraepithelial neoplasia; ORs = 61.1, P < 0.01 for squamous cell carcinoma). The obtained results indicated that the presence of HPV is associated closely with cervical cancer, and that HSV 2 infection or co‐infection with HPV might be involved in cervical cancer development, while HSV 1 might not be involved. J. Med. Virol. 84:1920–1927, 2012. © 2012 Wiley Periodicals, Inc.     

Suppression of generation and replication of acyclovir-resistant herpes simplex virus by a sensitive virus

The role of acyclovir-sensitive herpes simplex virus (HSV) was analyzed in the process of its replacement by a resistant virus in vitro and in vivo in the aspect of acyclovir therapy. The mode of replacement of acyclovir-sensitive HSV with acyclovir-resistant HSV was examined by the passages of acyclovir-sensitive wild type HSV in Vero cells under acyclovir-treatment. The development of resistance was monitored more adequately by counting the number of acyclovir-resistant viruses in 10,000 plaque forming units than by the conventional susceptibility assay. The resistance increased with the proportion of thymidine kinase-deficient (TK(-)) viruses, when the susceptibilities of acyclovir-treated HSV population to 5'-iodo-2'deoxyuridine and phosphonoacetic acid were examined. The increased resistance was due to the increased proportion of acyclovir-resistant virus but not intermediately resistant virus. Infection with mixtures of TK(-) and acyclovir-sensitive strains rendered TK(-) sensitive to acyclovir, and virus yields were reduced to the levels of acyclovir-sensitive virus in Vero cells. Their yield reduction depended on the proportion of acyclovir-sensitive viruses and induction of TK activity. This reduction in virus yields of the mixture of TK(-) and acyclovir-sensitive strains was confirmed by acyclovir treatment in the skin of mice with cutaneous infection. Acyclovir treatment combined with superinfection of acyclovir-sensitive virus delayed the development of herpetic skin lesions due to acyclovir-resistant virus and reduced virus yields in the infected skin. Acyclovir-sensitive virus plays an important role in suppressing the generation and replication of acyclovir-resistant virus during acyclovir therapy.     

Quantitative detection of herpes simplex virus DNA in the lower respiratory tract

Quantitation of herpes simplex virus (HSV) DNA in bronchoalveolar lavage specimens could indicate an infectious role in the lower respiratory tract. The aim of this study was to compare quantitative HSV DNA results from adult bronchoalveolar lavage specimens to clinical outcome. Quantitative real-time PCR assays targeting HSV and other herpes viruses were performed on adult bronchoalveolar lavage specimens obtained from a largely immunocompromised population during a 1-year period. The results were compared to patient characteristics and outcome. HSV DNA was detected in 11 (19%) of 57 bronchoalveolar lavage specimens with a mean viral level of 5.6 log genome equivalents/ml (range, 2.9-8.1 log). A threshold of HSV DNA levels equal or higher than 5.0 log (n = 7) was associated with mortality within 28 days following hospital admission (odds ratio [OR], 6.8; 95% confidence interval [CI], 1.2-39.2). A threshold level of 5.5 log was associated with mortality within 28 days of sampling (OR 8.5; 95% CI 1.2-62.1), only after excluding patients receiving specific antiviral medication. Patients with HSV DNA levels equal or higher than 7.5 log had severe respiratory failure. Viral pneumonia was histologically proven in one patient with 8.0 log at autopsy. No patient with HSV DNA levels below 5.5 log (n = 5) or DNA levels higher than 5.0 log of cytomegalovirus (CMV) (n = 3), Epstein-Barr virus (EBV) (n = 9), varicella-zoster virus (VZV) (n = 1), or human herpesvirus 6 (HHV-6) (n = 0) died within 28 days of hospital admission. We conclude that quantitative detection of HSV DNA in bronchoalveolar lavage fluid is a potential diagnostic tool for detection of relevant viral infection of the lower respiratory tract.     

Autopsy findings in two cases of neonatal herpes simplex virus infection: detection of virus by immunohistochemistry, in situ hybridization and the polymerase chain reaction

We describe the pathological findings in two fatal cases of neonatal infection with herpes simplex virus. One had an encephalitis caused by herpes simplex virus type 2 (HSV-2); the other had a disseminated infection with herpes simplex virus type 1 (HSV-1). Confirmation of the diagnosis was obtained by use of the polymerase chain reaction to amplify viral DNA from paraffin sections of autopsy tissues. By using primers which amplify fragments of the HSV-1 thymidine kinase gene and HSV-2 glycoprotein gene respectively it was possible to discriminate between infection with HSV-1 and HSV-2. In contrast, immunohistochemistry and in situ hybridization using commercially available reagents did not distinguish between HSV-1 and HSV-2 infection. However, immunohistochemistry and in situ hybridization are probably more reliable than the polymerase chain reaction for assessment of the distribution of virus in different tissues.     

Effect of centrifugation on herpes simplex virus isolation

The effects of high-speed centrifugation on the isolation of herpes simplex virus (HSV) were studied. Aliquots of laboratory or clinical specimens were inoculated into test tubes and flat-bottomed tubes containing HEp2 monolayers. Test tubes were incubated at 35 degrees C on roller drums (standard method), and flat-bottomed tubes were centrifuged at 15,000g at 35 degrees C for 1 hr, before being incubated at 35 degrees C without rolling (centrifuged method). Centrifugation of clinical and laboratory specimens of HSV type 1 and HSV type 2 produced significantly increased isolation rates compared with the standard method. When clinical and laboratory specimens were diluted, the centrifuged method was more sensitive at all dilutions. When 20 specimens were used for end-point titrations, the centrifuged method was 10 times more sensitive for 15 specimens and 100 times more sensitive for five specimens. There was no difference in the time taken for the appearance of cytopathic effect (CPE) between the standard and centrifuged methods.     

Tampon culture for quantitation of cervicovaginal herpes simplex virus.

From 10(1) to 10(6) TCID50 (mean tissue culture infective doses) per ml HSV I and II can be quantitatively recorded from vaginal tampons. Tampons and sterile cotton swabs are equally sensitive in recovering HSV I and II. Direct cotton swab cultures of the cervix and cervicovaginal tampon cultures from the same patients recovered similar quantities of HSV, ranging from 1.5 to 6.5 log10 TCID50/ml in 5 patients.     

The stability of herpes simplex virus on vaginal tampons.

Herpes simplex virus can be quantitatively recovered from vaginal tampons suspended in tissue culture medium and stored over a wide range of temperatures. Because herpes simplex virus is stable for several days with refrigeration or after freezing, this method of culture of cervicovaginal virus lends itself to epidemiologic studies.     

Isolation of high-molecular-weight infectious DNA from type 1 herpes simplex virus

Isolation of DNA from type 1 herpes simplex virus (strain L2) is described; the DNA possessed the characteristics of an intact molecule: sedimentation rate, physical length, and infectivity. Data on infectivity of preparations of this DNA were obtained in cultures of chick embryonic fibroblasts.D. I. Ivanovskii Institute of Virology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. D. Solov'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 1, pp. 26–28, January, 1977.     

Growth of herpes simplex virus in epidermal keratinocytes determines cutaneous pathogenicity in mice

Herpes simplex viruses (HSV)-1 and -2 isolated from genital lesions were examined for cutaneous pathogenicity and its correlation with cellular tropism. HSV-1 caused vesiculation, erosion/ulcer, and zosteriform lesions successively, but skin lesions of HSV-2 developed without vesiculation in some mice, and with statistically significantly less frequent vesiculation than HSV-1. Thus, the virological type of HSV was correlated with its cutaneous pathogenicity. The growth characteristics of HSV-1 and -2 were compared in cultured human embryonic lung (HEL) fibroblasts, human lung cancer A549 cells, human neonatal epidermal keratinocytes, human neonatal dermal fibroblasts, HeLa cells, and Vero cells. HSV-2 produced plaques that were 72% times the size of HSV-1 plaques in epidermal keratinocytes but 230%-500% the size in the other cells. The difference between HSV-1 and -2 in the ratio of plaque size to virus yield in epidermal keratinocytes was much larger (502 times) than the ratio of the other cells (5.57-28.8 times). Keratinocytes are the major constituent of the epidermal layer of the skin and the cells in which vesiculation and erosion/ulceration occur histologically. Therefore, the smaller spread of HSV-2 in keratinocytes of the epidermal layer and the greater spread in other cells of the dermal layer might reflect its lesser invasiveness in the epidermal layer despite larger invasiveness in the dermal layer, which is reflected in the low incidence of erosion/ulcer of the skin compared to HSV-1. Thus, the growth of HSV in epidermal keratinocytes appeared to correlate with the cutaneous pathogenicity causing vesiculation in the skin.     

In vitro reactivation of latent herpes simplex virus by the demethylating compound hexamethylene-bis-acetamide

We have characterized previously a model of herpes simplex virus (HSV) infection of rat dorsal root ganglia (DRG) following cutaneous infection. During acute infection HSV can be isolated from co-cultivated rat ganglia in (mean ± S.E.M) 4.8 ± 0.33 days (d) and from latently infected ganglia in 7.8 ± 0.53 (d) (P 0.0001). We treated co-cultivated rat ganglia from acute and latent infected rats with the demethylating compound hexamethylene-bis-acetamide (HEX) to see what effect, if any, it would have on HSV infection. HEX-treated ganglia from rats with acute infection did not differ significantly from controls in the proportion of rats, skin or ganglia positive for HSV. The mean time to detect virus was not different between treated (3.6 ± 0.38 d) and control (3.1 ± 0.46 d) (P> 0.05) groups. In latent infected rats there was no difference between treated and controlled groups in the proportions of rats, skin and ganglia positive for HSV. There was a significant difference in the mean time to CPE between the HEX and control groups respectively (4.5 ± 0.72 d vs 8.92 ± 1.42 d, P < 0.01). We conclude that HEX converted latent HSV infection to a productive one.     

Immune-specific interferon production by peripheral blood mononuclear leukocytes from patients with primary and recurrent oro-labial herpes simplex virus infections

Immune-specific IFN (IFN) is produced by the peripheral blood mononuclear leukocytes (PBML) of greater than 95% of HSV-seropositive humans with infrequent recurrences of herpes labialis [Green, Yeh, and Overall, 1981]. However, herpes virus-induced immune-specific IFN was produced by PBML from only 33 of 48 (68.8%) persons with frequent recurrences (2-12 episodes a year). Two of eight subjects with primary herpes gingivostomatitis also failed to produce immune-specific IFN during either the acute or convalescent phases of their initial HSV infection. These data suggest that some persons have a defective immune-specific IFN response that exists from the time of their primary oro-labial HSV type 1 infection. This defect may predispose to a higher frequency of disease in some individuals.     

Monitoring of herpes simplex virus DNA types 1 and 2 viral load in cerebrospinal fluid by real‐time PCR in patients with herpes simplex encephalitis

A quantitative polymerase chain reaction (PCR) assay was evaluated retrospectively on 92 cerebrospinal fluid (CSF) samples from 29 patients with herpes simplex virus (HSV) encephalitis with the aim to study if the concentration of HSV genomes can be used as a prognostic marker and for monitoring of antiviral therapy. The results were compared to those obtained previously by nested PCR, and the numbers of HSV genomes/ml were evaluated in correlation to patient outcome and treatment. The aims were to compare the sensitivity of a conventional nested PCR to a quantitative PCR, to investigate the range of HSV genome concentration in initial samples and to evaluate possible relationships between the HSV DNA concentrations in CSF, neopterin levels, and outcome of disease. The 29 initial samples contained between 2 × 10² and 42 × 10⁶ HSV genomes/ml. There was no apparent correlation between the amount of HSV DNA in the initial samples and income status, initial neopterin levels, or prognosis. The number of HSV genomes/ml declined after treatment in all patients, but HSV DNA was still detectable after day 20 in 3 out of 16 patients. A long duration of genome detectability was found to correlate with poor outcome. There was no difference in sensitivity between the nested PCR and the quantitative PCR. While the quantitative PCR is more rational than a nested PCR, the quantitation of HSV genomes does not seem very useful as a prognostic marker in HSV encephalitis. J. Med. Virol. 81:1432–1437, 2009. © 2009 Wiley‐Liss, Inc.     

A nonlytic transforming mutant of herpes simplex virus type 2.

A small-plaque mutant (NO.69) of herpes simplex virus type 2 (HSV-2) strain 333 has been previously isolated and characterized in this laboratory. This mutant was shown to produce a high ratio of noninfectious to infectious particles when grown at the nonpermissive temperature in hamster embryo fibroblasts [Westmoreland D. and Rapp F. (1976). Journal of Virology [8:92--102]. In this study, we have demonstrated that it is possible to obtain noninfectious stocks of this virus which retain transforming ability in a biochemical transformation assay specific for detection of the HSV gene for thymidine kinase. This mutant contains a DNA genome that has a density identical to the DNA of wild-type virus. Virus and cell DNA synthesis after infection with the mutant at both the permissive and nonpermissive temperature are similar to that observed in cultures infected with the parental virus. Clones of mouse cells biochemically transformed by this virus contain HSV antigens and are presently being examined for oncogenicity.     

Steroid hormone alteration of herpes simplex virus type 1 replication.

The effect of steroid hormones on herpes simplex virus type 1 replication was examined. Virus replication studies revealed that various concentrations of prednisolone, hydrocortisone, dexamethasone, or progesterone could decrease virus yields up to a maximum of 99%. Using isopycnic centrifugation in CsCl to separate viral from cell DNA, it was found that virus-specific DNA synthesis was decreased by 30 to 100% depending on the hormone and concentration used. Cell-specific DNA synthesis was also adversely affected, but this did not alter cell viability or plating efficiency.     

Study of herpes simplex virus type 1 populations obtained from recurrences and primary infections

The analysis of 23 clinical isolates of herpes simplex virus type 1 (HSV-1) showed that 15 of 15 isolates that had undergone a few passages in tissue culture (fresh isolates) and two of eight isolates that had never been passaged (new isolates) were composed of a mixed population with respect to plaque morphology in Vero cells. Cloning and characterization of 10 large plaque viruses (L variants) and nine small plaque viruses (S variants), obtained from seven different isolates, showed the following. BamHI DNA restriction patterns of the L and the S variants from a single isolate differed only with respect to the electrophoretic mobility of the fragments that contain reiteration of specific sequences; they did not differ regarding the presence or the absence of restriction endonuclease cleavage sites. The L and S variants differed with respect to the electrophoretic profiles of infected cell glycoproteins, thermosensitivity of growth and plaquing efficiency at 39 degrees C, and, at least in the case of the two couples of variants that we tested, pathogenicity for the mouse. The hypothesis that the L variants might arise from the S variant during in vivo replication is discussed.