Herpes Simplex Virus Isolates Research Articles

Novel Resistance-Associated Mutations of Thymidine Kinase and Dna Polymerase Genes of Herpes Simplex Virus Type 1 and Type 2

Studies to verify correlations between phenotypes and genotypes of herpes simplex virus (HSV) are an important tool to establish a database of resistance-associated mutations. In this study, 32 acyclovir (ACV)-resistant clinical HSV-1 and 4 ACV-resistant clinical HSV-2 isolates were examined in parallel by both phenotypic and genotypic resistance testing. Additionally, five non-viable HSV-1 strains and two non-viable HSV-2 strains with clinical resistance were included in genotypic resistance analysis. All ACV-resistant HSV isolates showed cross-resistance to brivudin and penciclovir, and were sensitive to foscarnet and cidofovir. Acyclovir resistance was assigned to frameshift and single non-synonymous mutations of the thymidine kinase (TK) gene in 32 out of 37 HSV-1 strains and in 4 out of 6 HSV-2 strains. In three HSV-1 isolates, there were resistance-associated amino acid substitutions of the DNA polymerase (pol). Six substitutions in the TK and two in the DNA pol gene could not be attributed without doubt to either ACV resistance or natural gene polymorphism. Altogether, 10 resistance-related mutations in the TK and 1 in the DNA pol gene have not been reported previously. The novel non-synonymous mutations found in this study enrich the knowledge about the genetic alterations of TK and DNA pol genes in ACV-resistant clinical HSV strains. Together with data from the literature, the findings justify the generation of a HSV database that contains resistance mutations associated with ACV resistance phenotype.

Herpes simplex virus: A marker of severity in bacterial ventilator-associated pneumonia

PurposeVentilator-associated pneumonia (VAP) is the most frequent nosocomial infection in intensive care units and has a high morbidity and mortality rate. It is mainly a bacterial disease, although the potential role of viruses as pathogens or copathogens in VAP is under discussion. Our study aims were to determine the incidence of herpes simplex virus (HSV) in the lower respiratory tract (LRT) secretions in patients with bacterial VAP and to assess its potential clinical relevance. Material and MethodsThis is a prospective observational study carried out over a 14-month period. All LRT samples of adult patients with VAP were sent for bacterial culture and virus isolation. We compared patients with bacterial VAP with isolation of HSV and those without. ResultsOne-hundred seventy-seven patients had confirmed bacterial VAP. Herpes simplex virus was present in 13.4% of them. Patients with HSV had more severe underlying conditions and worse outcome. They consumed more antibiotics for the VAP episode, had more Clostridium difficile infection, spent a longer time on mechanical ventilation, had a longer intensive care unit and hospital stay, and had greater mortality than those without. ConclusionsHerpes simplex virus excretion in LRT secretions is not infrequent in VAP, and it is associated with greater severity and worse prognosis.

Genotypic Characterization of UL23 Thymidine Kinase and UL30 DNA Polymerase of Clinical Isolates of Herpes Simplex Virus: Natural Polymorphism and Mutations Associated with Resistance to Antivirals

The molecular mechanisms of herpes simplex virus (HSV) resistance to antiviral drugs interfering with viral DNA synthesis reported so far rely on the presence of mutations within UL23 (thymidine kinase [TK]) and UL30 (DNA polymerase) genes. The interpretation of genotypic antiviral resistance assay results requires the clear distinction between resistance mutations and natural interstrain sequence variations. The objectives of this work were to describe extensively the natural polymorphism of UL23 TK and UL30 DNA polymerase among HSV-1 and HSV-2 strains and the amino acid changes potentially associated with HSV resistance to antivirals. The sequence analysis of the full-length UL23 and UL30 genes was performed. Ninety-four drug-sensitive clinical isolates (43 HSV-1 and 51 HSV-2) and 3 laboratory strains (KOS, gHSV-2, and MS2) were studied for natural polymorphism, and 25 clinical isolates exhibiting phenotypic traits of resistance to antivirals were analyzed for drug resistance mutations. Our results showed that TK and DNA polymerase are highly conserved among HSV strains, with a weaker variability for HSV-2 strains. This study provided a precise map of the natural polymorphism of both viral enzymes among HSV-1 and HSV-2 isolates, with the identification of 15 and 51 polymorphisms never previously described for TK and DNA polymerase, respectively, which will facilitate the interpretation of genotypic antiviral-resistant testing. Moreover, the genotypic characterization of 25 drug-resistant HSV isolates revealed 8 new amino acid changes located in TK and potentially accounting for acyclovir (ACV) resistance.

Index of Suspicion in the Nursery

A woman presents with decreased fetal movement, and ultrasonography reveals hydrops fetalis. Specific findings include massive ascites and a small pericardial effusion that was not present on ultrasonography 1 week ago. A 37 weeks' gestation boy is delivered via emergent cesarean section. Apgar scores are 7 and 9 at 1 and 5 minutes, respectively. The baby is intubated endotracheally 10 minutes after delivery because of increased work of breathing from his markedly distended abdomen and transported to the neonatal intensive care unit (NICU). Physical examination findings are normal, except for increased work of breathing and a tight, distended abdomen with visibly dilated veins. Initial laboratory results include a white blood cell count of 18.3×103/mcL (18.3×109/L), hemoglobin of 9.7 g/dL (97 g/L), and platelet count of 331×103/mcL (331×109/L). Chemistry results are within normal limits. Blood is sent for cultures, and empiric therapy with ampicillin and gentamicin is initiated. The baby also receives a 15-mL/kg packed red blood cell transfusion. A Replogle tube is placed to low intermittent suction. The infant is made NPO and started on intravenous fluids. Paracentesis is performed to relieve the pressure on his diaphragm and improve breathing, and 755 mL of green/brown fluid with particulate matter that has the appearance of meconium is aspirated. Umbilical lines are placed and pediatric surgery is consulted. Abdominal radiography shows intra-abdominal calcifications. An 11-day-old term girl whose mother had a positive cervical culture for human papillomavirus (HPV) is referred for admission because of a rectal temperature of 38.6°C and a history of “gasping for air.” Four days after birth, the baby became fussy, especially with feedings, and developed an intermittent cough and hoarse cry. Over several days, she became sleepier, only waking every 4 to 5 hours to breastfeed. On the pediatric ward, the baby …

Sequence Variability in Clinical and Laboratory Isolates of Herpes Simplex Virus 1 Reveals New Mutations

Herpes simplex virus 1 (HSV-1) is a well-adapted human pathogen that can invade the peripheral nervous system and persist there as a lifelong latent infection. Despite their ubiquity, only one natural isolate of HSV-1 (strain 17) has been sequenced. Using Illumina high-throughput sequencing of viral DNA, we obtained the genome sequences of both a laboratory strain (F) and a low-passage clinical isolate (H129). These data demonstrated the extent of interstrain variation across the entire genome of HSV-1 in both coding and noncoding regions. We found many amino acid differences distributed across the proteome of the new strain F sequence and the previously known strain 17, demonstrating the spectrum of variability among wild-type HSV-1 proteins. The clinical isolate, strain H129, displays a unique anterograde spread phenotype for which the causal mutations were completely unknown. We have defined the sequence differences in H129 and propose a number of potentially causal genes, including the neurovirulence protein ICP34.5 (RL1). Further studies will be required to demonstrate which change(s) is sufficient to recapitulate the spread defect of strain H129. Unexpectedly, these data also revealed a frameshift mutation in the UL13 kinase in our strain F isolate, demonstrating how deep genome sequencing can reveal the full complement of background mutations in any given strain, particularly those passaged or plaque purified in a laboratory setting. These data increase our knowledge of sequence variation in large DNA viruses and demonstrate the potential of deep sequencing to yield insight into DNA genome evolution and the variation among different pathogen isolates.

Phenotypic and genotypic characterization of acyclovir-resistant clinical isolates of herpes simplex virus

Sixteen herpes simplex virus type 1 (HSV-1) and four type 2 (HSV-2) isolates resistant to acyclovir (ACV) were characterized retrospectively for drug resistance. Phenotypic testing was performed by means of tetrazolium reduction assay and genotypic analysis was carried out by sequencing of thymidine kinase (TK) and DNA-polymerase (pol) genes. All strains were characterized as cross-resistant to penciclovir, brivudin and susceptible to cidofovir. In addition, three strains were resistant to foscarnet. Genotypic analysis revealed two to seven non-synonymous mutations in the TK gene of HSV-1 and one to seven non-synonymous mutations in the DNA pol gene of HSV-1 and 2 associated with the gene polymorphism. Seventeen strains contained at least one non-synonymous resistant-related mutation in the TK gene and three strains, which were additionally foscarnet-resistant, revealed one resistance-associated mutation in the DNA pol gene. In most strains, resistant-related mutations in TK gene represented frameshift mutations and single non-synonymous nucleotide substitutions of conserved gene regions. However, numerous amino acid changes could not be interpreted clearly as accounting for resistance. In conclusion, further studies, e.g. site-directed mutagenesis experiments are required to characterize mutations of the TK and DNA pol genes in ACV-resistant viral strains as part of viral gene polymorphism or as cause of drug resistance.

Isolation and Preliminary Characterization of Herpes Simplex Virus 1 Primary Enveloped Virions from the Perinuclear Space

Herpes simplex virus 1 (HSV-1) nucleocapsids exit the nucleus by budding into the inner nuclear membrane, where they exist briefly as primary enveloped virions. These virus particles subsequently fuse their envelopes with the outer nuclear membrane, permitting nucleocapsids to then enter the cytoplasm and complete assembly. We have developed a method to isolate primary enveloped virions from HSV-1-infected cells and subjected the primary enveloped virion preparation to MALDI-MS/MS (matrix-assisted laser desorption ionization-tandem mass spectrometry) analyses. We identified most capsid proteins, a tegument protein (VP22), a glycoprotein (gD), and a cellular protein (annexin A2) in the primary enveloped virion preparation. We determined that annexin A2 does not play an essential role in infection under our experimental conditions. Elucidating the structure and biochemical properties of this unique virus assembly intermediate will provide new insights into HSV-1 biology.

A MOLECULAR METHOD FOR TYPING HERPES SIMPLEX VIRUS ISOLATES AS AN ALTERNATIVE TO IMMUNOFLUORESCENCE METHODS

Background: Typing of Herpes simplex virus (HSV) isolates is required to identify the virus isolated in culture. The methods available for this include antigen detection by immunofluorescence (IF) assays and polymerase chain reaction (PCR). This study was undertaken to standardize a molecular method for typing of HSV and compare it with a commercial IF reagent for typing. Objectives: To compare a molecular method for typing HSV isolates with a monoclonal antibody (MAb) based IF test. Study design: This cross-sectional study utilized four reference strains and 42 HSV isolates obtained from patients between September 1998 and September 2004. These were subjected to testing using an MAb-based IF test and a PCR that detects the polymerase (pol) gene of HSV isolates. Results: The observed agreement of the MAb IF assay with the pol PCR was 95.7%. Fifty four point eight percent (23/42) of isolates tested by IF typing were found to be HSV-1, 40.5% (17/42) were HSV-2, and two (4.8%) were untypable using the MAb IF assay. The two untypable isolates were found to be HSV-2 using the pol PCR. In addition, the cost per PCR test for typing is estimated to be around Rs 1,300 (USD 30), whereas the cost per MAb IF test is about Rs 1,500 (USD 35) including all overheads (reagents, instruments, personnel time, and consumables). Conclusion: The pol PCR is a cheaper and more easily reproducible method for typing HSV isolates as compared to the IF test. It could replace the IF-based method for routine typing of HSV isolates as availability of PCR machines (thermal cyclers) is now more widespread than fluorescence microscopes in a country like India.

Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses

The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6-99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.

Efficacy of anise oil, dwarf-pine oil and chamomile oil against thymidine-kinase-positive and thymidine-kinase-negative herpesviruses

Abstract The effect of anise oil, dwarf-pine oil and chamomile oil against different thymidine-kinase-positive (aciclovir-sensitive) and thymidine-kinase-negative (aciclovir-resistant) herpes simplex virus type 1 (HSV-1) strains was examined. Clinical HSV-1 isolates containing frameshift mutations in the thymidine kinase (TK) gene, an insertion or a deletion, yield a non-functional thymidine kinase enzyme resulting in phenotypical resistance against aciclovir. The inhibitory activity of three different essential oils against herpes simplex virus isolates was tested in-vitro using a plaque reduction assay. All essential oils exhibited high levels of antiviral activity against aciclovir-sensitive HSV strain KOS and aciclovir-resistant clinical HSV isolates as well as aciclovir-resistant strain Angelotti. At maximum noncytotoxic concentrations of the plant oils, plaque formation was significantly reduced by 96.6–99.9%, when herpesviruses were preincubated with drugs before attachment to host cells. No significant effect on viral infectivity could be achieved by adding these compounds during the replication phase. These results indicate that anise oil, dwarf-pine oil and chamomile oil affected the virus by interrupting adsorption of herpesviruses and in a different manner than aciclovir, which is effective after attachment inside the infected cells. Thus the investigated essential oils are capable of exerting a direct effect on HSV and might be useful in the treatment of drug-resistant viruses. Chamomile oil did not reveal any irritating potential on hen's egg chorioallantoic membrane, demonstrated the highest selectivity index among the oils tested and was highly active against clinically relevant aciclovir-resistant HSV-1 strains.

Diagnosis and treatment of herpes simplex virus (HSV) infection in the newborn

Neonatal herpes simplex virus (HSV) disease is a rare but sometimes highly lethal infection. The reported incidence in Australia is approximately four cases per 100,000 live births. HSV type 2 (HSV-2) is the predominant serotype that causes infection in the newborn in the United States, whereas in Australia 1 neonatal infection is usually caused by HSV type 1 (HSV-1), most likely due to greater prevalence of oral and genital HSV-1 disease in this country 3. Diagnosis of neonatal infection requires a high index of clinical suspicion as signs are non-specific, and is usually confirmed by isolation of HSV from skin vesicle or detection of HSV DNA in the cerebrospinal fluid, blood or surface swab. Treatment requires intravenous aciclovir for 14-21 days depending on the form of disease.

In vitro Antiviral Activity of Distamycin A against Clinical Isolates of Herpes Simplex Virus 1 and 2 from Transplanted Patients

Objective(s): Herpes simplex virus (HSV) infections in immunocompromised individuals may require prolonged antiviral therapy resulting in the emergence of viral strains resistant to the currently employed antiviral drugs. Distamycin A (DA), a basic antibiotic belonging to the lexitropsin DNA minor groove binding drugs, exhibits antiviral properties. In this study we evaluated the in vitro cytotoxicity and antiviral activity of DA against HSV type 1 and HSV type 2 clinical isolates from transplanted patients and compared them with those of acyclovir (ACV) in search of alternative antiviral drugs. Methods: Viral detection and typing was performed by multiplex PCR and immunofluorescence assay; the in vitro cytotoxicity of DA and the antiviral activity of ACV and DA was evaluated respectively by neutral red uptake assay and plaque reduction assay for HSV2 isolates and fluorescence reduction assay for HSV1 isolates. Results: Tissue culture 50% cytotoxic concentration of DA was 58 µM. Tissue culture 50% inhibitory concentration values ranged from 0.16 to 7.4 µM for the ACV-sensitive and from 5.4 to 32 µM for the ACV-resistant viral strains. Conclusions: In spite of the lower activity against ACV-resistant strains, DA may be used as an antiherpetic drug.

Genetic susceptibility to herpes simplex virus 1 encephalitis in mice and humans

Herpes simplex encephalitis is a rare complication of herpes simplex virus 1 infection that strikes otherwise healthy individuals. Its pathogenesis has long remained elusive. We highlight the investigations dealing with the genetic basis of herpes simplex encephalitis in mice and humans. Mouse models have revealed the impact of various host genes on protective immunity to herpes simplex encephalitis through strain-dependent variability (forward genetics) and via targeted knockouts (reverse genetics). These studies established in particular the crucial role of IFNalpha/beta in immunity to herpes simplex virus 1, paving the way towards the elucidation of the genetic cause of human herpes simplex encephalitis. Two children with rare, specific STAT1 or NEMO mutations displayed a broad impairment of IFNalpha/beta and IFNlambda-mediated immunity and predisposition to several infectious diseases including herpes simplex encephalitis. In contrast, children with UNC93B1 and TLR3 mutations displayed a selective impairment of dsRNA-induced IFNalpha/beta and IFNlambda production and predisposition to isolated herpes simplex encephalitis. Herpes simplex encephalitis results from a series of monogenic primary immunodeficiencies that impair the TLR3 and UNC-93B-dependent production of IFNalpha/beta and IFNlambda in the central nervous system, at least in a fraction of children. This is not only crucial for the understanding of immunity to herpes simplex virus 1, but also for the diagnosis and treatment of herpes simplex encephalitis.

ACICLOVIR RESISTANCE AMONG INDIAN STRAINS OF HERPES SIMPLEX VIRUS AS DETERMINED USING A DYE UPTAKE ASSAY

Resistance to aciclovir (ACV) among Herpes simplex virus (HSV) isolates is increasingly being reported in the literature particularly in immunocompromised patients. However, there is only limited data available from India despite widespread use of ACV in our hospital. A cross-sectional study was hence conducted to determine the aciclovir (ACV) susceptibility of HSV 1 and 2 isolates using a dye uptake (DU) assay. This study showed a 3.0% prevalence of ACV resistance among HSV-1 strains (2/66, median IC500.098 µg/mL) while in HSV-2 strains, it was 7.8% (5/64, median IC500.195 µg/mL). The IC50 for the HSV-1 and HSV-2 strains resistant to ACV was greater than or equal to 6.25 µg/mL.

Infections respiratoires à virus herpes simplex en réanimation

Detection of herpes simplex virus (HSV) in the upper and lower respiratory tract is well described. In the throat, the viral reactivation is probably due to the immunoparalysis observed in such patients, and/or to micro trauma. It is not known whether the isolation of HSV from lower respiratory tract samples of nonimmunocompromised ventilated patients corresponds to bronchial contamination from mouth and/or throat, local tracheobronchial excretion of HSV, or true HSV lung involvement (bronchopneumonitis) with its own morbidity/mortality. This disease seems common, but its diagnosis remains difficult because clinical, biological and radiological signs are not specific. HSV bronchopneumonitis is defined as a clinical deterioration, associated with HSV in lower respiratory tract and HSV-specific nuclear inclusions in cells of the bronchiolo-alveolar compartment. Risk factors associated with HSV bronchopneumonitis are oral–labial lesions, HSV in throat and macroscopic bronchial lesions seen during bronchoscopy. HSV bronchopneumonitis seems associated with longer duration of mechanical ventilation and ICU stays, but the efficacy of a specific antiviral agent in this population remains to be determinate.

Herpes Simplex Virus Lung Infection in Patients Undergoing Prolonged Mechanical Ventilation

It is not known whether the isolation of herpes simplex virus (HSV) from lower respiratory tract samples of nonimmunocompromised ventilated patients corresponds to bronchial contamination from the mouth and/or throat, local tracheobronchial excretion of HSV, or true HSV lung involvement (bronchopneumonitis) with its own morbidity/mortality. This prospective, single-center, observational study was conducted to define the frequency, risk factors, and relevance of HSV bronchopneumonitis. All consecutive nonimmunocompromised patients receiving mechanical ventilation for 5 days or more were evaluated. Bronchoalveolar lavage, oropharyngeal swabs, and bronchial biopsies (presence of macroscopic bronchial lesions) were obtained for all who deteriorated clinically with suspected lung infection. HSV bronchopneumonitis was defined as this deterioration, associated with HSV in bronchoalveolar lavage and HSV-specific nuclear inclusions in cells recovered during lavage or bronchial biopsies. HSV bronchopneumonitis was diagnosed in 42 (21%) of the 201 patients who deteriorated clinically, with a mean mechanical ventilation duration before diagnosis of 14 +/- 6 days. Risk factors associated with HSV bronchopneumonitis were oral-labial lesions, HSV in the throat, and macroscopic bronchial lesions seen during bronchoscopy. Patients with HSV bronchopneumonitis were comparable to those without at admission, but their courses were more complicated, with longer duration of mechanical ventilation and intensive care unit stays. HSV bronchopneumonitis is common in nonimmunocompromised patients with prolonged mechanical ventilation, is associated with HSV reactivation or infection of the mouth and/or throat, and seems to be associated with poorer outcome.

Aciclovir resistance among indian strains of <i>Herpes simplex</i> virus as determined using a dye uptake assay

Resistance to aciclovir (ACV) among Herpes simplex virus (HSV) isolates is increasingly being reported in the literature particularly in immunocompromised patients. However, there is only limited data available from India despite widespread use of ACV in our hospital. A cross-sectional study was hence conducted to determine the aciclovir (ACV) susceptibility of HSV 1 and 2 isolates using a dye uptake (DU) assay. This study showed a 3.0% prevalence of ACV resistance among HSV-1 strains (2/66, median IC 50 0.098 microg/mL) while in HSV-2 strains, it was 7.8% (5/64, median IC 50 0.195 microg/mL). The IC 50 for the HSV-1 and HSV-2 strains resistant to ACV was greater than or equal to 6.25 microg/mL.

Isolation of Herpes Simplex Virus From the Genital Tract During Symptomatic Recurrence on the Buttocks

To estimate the frequency of isolation of herpes simplex virus (HSV) from the genital tract when recurrent herpes lesions were present on the buttocks. Data were extracted from a prospectively observed cohort attending a research clinic for genital herpes infections between 1975 and 2001. All patients with a documented herpes lesion on the buttocks, upper thigh or gluteal cleft ("buttock recurrence") and concomitant viral cultures from genital sites including the perianal region were eligible. We reviewed records of 237 subjects, 151 women and 86 men, with a total of 572 buttock recurrences. Of the 1,592 days with genital culture information during a buttock recurrence, participants had concurrent genital lesions on 311 (20%, 95% confidence interval [CI] 14-27%) of these days. Overall, HSV was isolated from the genital region on 12% (95% CI 8-17%) of days during a buttock recurrence. In the absence of genital lesions, HSV was isolated from the genital area on 7% (95% CI 4%-11%) of days during a buttock recurrence and, among women, from the vulvar or cervical sites on 1% of days. Viral shedding of herpes simplex virus from the genital area is a relatively common occurrence during a buttock recurrence of genital herpes, even without concurrent genital lesions, reflecting perhaps reactivation from concomitant regions of the sacral neural ganglia. Patients with buttock herpes recurrences should be instructed about the risk of genital shedding during such recurrences. II-2.

Sequence-Based Methods for Identifying Epidemiologically Linked Herpes Simplex Virus Type 2 Strains

Traditional methods for confirming the identity of herpes simplex virus (HSV) isolates use restriction fragment length polymorphism (RFLP). However, RFLP is less amenable to high-throughput analyses of many samples, and the extent to which small differences in RFLP patterns distinguish between different viral strains remains unclear. Viral HSV type 2 (HSV-2) DNA isolates from 14 persons experiencing a primary HSV-2 infection and from their sexual partners were analyzed by RFLP and heteroduplex mobility assays. We also compared the HSV-2 sequences from seven regions, including noncoding regions between UL19 and UL20, UL24 and UL25, UL37 and UL38, and UL41 and UL42 and coding segments of the gC, gB, and gG genes. Although the resulting RFLP patterns of the couples were almost identical, minor banding differences existed between the source and susceptible partners in five couples. Heteroduplex mobility assays were unable to distinguish between unrelated strains. Overall, 22 sites of sequence variation were found in 1,482 bp of analyzed sequence. The DNA sequences differentiated between all unrelated infections, and epidemiologically related isolates had identical sequences in all but two pairs. Our results suggest that a multilocus assay based on several DNA sequences has the potential to be an informative tool for identifying epidemiologically related HSV-2 strains.

Low-Level Expression and Reversion both Contribute to Reactivation of Herpes Simplex Virus Drug-Resistant Mutants with Mutations on Homopolymeric Sequences in Thymidine Kinase

Many acyclovir-resistant herpes simplex virus isolates from patients contain insertions or deletions in homopolymeric sequences in the thymidine kinase (TK) gene (tk). Viruses that have one (G8) or two (G9) base insertions in a run of seven G's (G string) synthesize low levels of active TK (TK-low phenotype), evidently via ribosomal frameshifting. These levels of TK can suffice to permit reactivation from latently infected mouse ganglia, but in a majority of ganglia, especially with the G9 virus, reactivation of virus that has reverted to the TK-positive phenotype predominates. To help address the relative contributions of translational mechanisms and reversion in reactivation, we generated viruses with a base either inserted or deleted just downstream of the G string. Both of these viruses had a TK-low phenotype similar to that of the G8 and G9 viruses but with less reversion. Both of these viruses reactivated from latently infected trigeminal ganglia, albeit inefficiently, and most viruses that reactivated had a uniformly TK-low phenotype. We also generated viruses that have one insertion in a run of six C's or one deletion in a run of five C's. These viruses lack measurable TK activity. However, they reactivated from latently infected ganglia, albeit inefficiently, with the reactivating viruses having reverted to the wild-type TK phenotype. Therefore, for G-string mutants, levels of active TK as low as 0.25% generated by translational mechanisms can suffice for reactivation, but reversion can also contribute. For viruses that lack TK activity due to mutations on other homopolymeric sequences, reactivation can occur via reversion.