Application of shRNA-containing herpes simplex virus type 1 (HSV-1)-based gene therapy for HSV-2-induced genital herpes

HSV-1-based vectors have been widely used to achieve targeted delivery of genes into the nervous system. In the current study, we aim to use shRNA-containing HSV-1-based gene delivery system for the therapy of HSV-2 infection. Guinea pigs were infected intravaginally with HSV-2 and scored daily for 100 days for the severity of vaginal disease. HSV-2 shRNA-containing HSV-1 was applied intravaginally daily between 8 and 14 days after HSV-2 challenge. Delivery of HSV-2 shRNA-containing HSV-1 had no effect on the onset of disease and acute virus shedding in animals, but resulted in a significant reduction in both the cumulative recurrent lesion days and the number of days with recurrent disease. Around half of the animals in the HSV-2 shRNA group did not develop recurrent disease 100 days post HSV-2 infection. In conclusion, HSV-2 shRNA-containing HSV-1 particles are effective in reducing the recurrence of genital herpes caused by HSV-2.     

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.     

巢式PCR检测龈下菌斑中HCMV、EBV、HSV-1与慢性牙周炎活动性的关系

目的　建立临床检测龈下菌斑标本中人巨细胞病毒 (HCMV)、Epstein Barr病毒 (EBV)、单纯疱疹病毒 1型 (HSV 1 )巢式PCR方法 ,研究这 3种病毒与慢性牙周炎活动性的关系。方法　收集6 2例慢性牙周炎患者 (男性 2 7例、女性 35例 ,平均年龄 5 3岁 )的牙周炎活动部位、牙周炎静止部位的龈下菌斑 ,提取DNA后使用巢式PCR检测HCMV、EBV、HSV 1 ,比较分析其在同一病人不同部位的检出率。结果　牙周炎活动部位的HCMV检出率为 38.7%,EBV的检出率为 5 8%,HSV 1的检出率为30 .6 %,两种以上病毒合并感染的检出率为 4 0 .3%;牙周炎静止部位的HCMV检出率为 1 4 .5 %,EBV为 2 2 .6 %,HSV 1为 1 1 .3%,两种以上病毒合并感染的检出率为 1 1 .3%。这 3种病毒及其合并感染在牙周炎活动部位的检出率均高于牙周炎静止部位 ,差异有统计学意义 (P <0 .0 5 )。结论　提示HC MV、EBV、HSV 1与慢性牙周炎的活动性相关。     

Differentiation of herpes simplex virus types 1 and 2 in clinical samples by a real-time taqman PCR assay

While the clinical manifestations of HSV-1 and -2 overlap, the site of CNS infection, complications, response to antivirals, frequency of antiviral resistance, and reactivation rate on mucosal surfaces varies between HSV-1 and -2. Detection of HSV DNA by PCR has been shown to be the most sensitive method for detecting HSV in clinical samples. As such, we developed a PCR-based assay to accurately distinguish HSV-1 from HSV-2. Our initial studies indicated the assay using type specific primers was slightly less efficient for detecting HSV-1 and -2 DNA than the high throughput quantitative PCR assay we utilize that employs type common primers to gB. We subsequently evaluated the type specific assay on 3,131 specimens that had HSV DNA detected in the type common PCR assay. The typing results of these specimens were compared with the monoclonal antibody staining results of culture isolates collected from the same patients at the same time, and the HSV serologic status of the patient. The typing assay accurately identified both HSV-1 and -2 with a specificity of >99.5% and was significantly more sensitive than typing by culture and subsequent monoclonal antibody assays. Complete concordance was seen between the typing assay and HSV serologic status of the patient. Dual (HSV-1 and -2) infection in clinical samples was recognized in 2.6% of clinical samples using the new typing assay. This assay, when used in combination with the type common assay, can now accurately type almost all mucosal and visceral HSV isolates by molecular techniques.     

Complement component C3b binds directly to purified glycoprotein C of herpes simplex virus types 1 and 2

Cells infected with herpes simplex virus type 1 (HSV-1), but not HSV-2, express on their surfaces a receptor for the complement component C3b. Receptor activity is markedly enhanced by treatment of the infected cells with neuraminidase. Employing a direct binding assay, consisting of purified HSV glycoproteins immobilized on nitrocellulose and iodinated C3b as a probe, we found that C3b binds directly to gC-1, as well as to gC-2, but not to gB or gD from either serotype. C3b binding was enhanced by treatment of gC-1 or gC-2 with neuraminidase. Endo F or endo H treatment of gC-1 had no effect on C3b binding. However, treatment of gC-2 with these endoglycosidases had a marked negative effect on C3b binding. These results suggest that N-linked oligosaccharides are involved in binding of C3b to gC-2, but not gC-1. Alternatively, removal of N-linked oligosaccharides from gC-2 might adversely affect polypeptide conformation. Glycoprotein C-2 also differs from gC-1 in its effects on the complement cascade. Whereas gC-1 accelerated the decay of the alternative pathway C3 convertase and impaired the efficiency of lysis by the components C5 through C9, gC-2 stabilized the active C3 convertase and had little effect on the late-acting components. The dissimilarity of gC-1 and gC-2 with regard to their effects on the complement cascade may have implications regarding the role of these glycoproteins in confronting the host immune response.     

Analysis of varicella-zoster virus and herpes simplex virus in various clinical samples by the use of different PCR assays

Rapid and reliable detection of varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and -2 (HSV-2) is of clinical significance in immunocompromised patients and patients with infections of the central nervous system. This paper describes the detection of VZV and HSV using the commercially available Affigene^® VZV and Affigene^® HSV 1/2 tracer kits in comparison to “in-house” polymerase chain reaction (PCR) assays. For sample preparation, Qiagen (Hilden, Germany) and Affigene^® (Cepheid AB, Bromma, Sweden) DNA extraction kits were used. 175 samples were analyzed for VZV and 352 samples for HSV-1 and -2. Generally more positive results were obtained using the Affigene^® assays compared to the “in-house” methods independent of the DNA preparation method used. There were significant differences in sensitivity between the Affigene^® HSV 1/2 tracer and the “in-house” PCR assays for the detection of both HSV-1 and -2 in cerebrospinal fluid and vesicle/skin swabs. The Affigene^® HSV 1/2 and VZV tracers are very sensitive assays for detection of VZV and HSV. A wide variety of clinical samples can be examined in combination with either the Qiagen or the Affigene^® DNA extraction kits for preparation.     

Suitability and clinical application of a multiplex nested PCR assay for the diagnosis of herpes simplex virus infections

A novel multiplex nested polymerase chain reaction (PCR) assay was designed and evaluated for routine diagnosis of herpes simplex virus (HSV) infections in patients with either putative HSV infection of the central nervous system or suspected HSV keratitis. Single-tube amplification of HSV type 1 (HSV-1) or type 2 (HSV-2) DNA extracted from cerebrospinal fluid (CSF) or from keratectomy specimens was followed by differentiation of the virus type-specific PCR products either by agarose gel analysis or by DNA enzyme immunoassay. Among 417 CSF specimens obtained from 395 consecutive patients with clinically suspected HSV infection, 11 (2.6%) were positive for HSV-1 DNA and four (1.0%) probes were positive for HSV-2 DNA. None of the specimens was positive for both HSV-1 and HSV-2 DNA. The genome of HSV-2 was detected in a CSF sample obtained from a woman with meningoencephalitis and genital herpes. The presence of PCR inhibitors was detected in six of 111 (5.4%) reconstructed CSF samples. Inhibition could be removed following extraction with a commercial kit. HSV-1 DNA, but no HSV-2 DNA, was detected in corneal buttons obtained from patients with suspected herpetic keratitis. No contamination has been recorded during the 2-year routine use of this test, which has met the specific requirements of a diagnostic laboratory. © 1996 Wiley-Liss, Inc.     

Concurrent reactivation of varicella zoster virus and herpes simplex virus in an immunocompetent child

Latency within the nervous system is a characteristic feature of herpesviridae infection. It is reactivated by triggering factors such as UV exposure, stress, and trauma. Simultaneous reactivation of herpes simplex and herpes zoster is uncommon, however, an observation provably explained by differences in the triggering mechanism. Concurrent reactivation of herpes simplex virus (HSV) and varicella zoster virus (VZV) is occasionally encountered in immunosuppressed patients; on the other hand, it is rarely reported in immunocompetent individuals. We present the case of an immunocompetent 8-yr-old female patient with concurrent reactivation of HSV on the face and VZV on the right L2 dermatome.     

Age-specific prevalence of antibodies to pyrimidine kinase enzymes of herpes simplex virus types 1 and 2, and of varicella zoster virus

The age-specific prevalence of antibodies to pyrimidine kinase enzymes of herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) and of varicella zoster virus (VZV) was measured in serum specimens from 360 persons. Specific inhibition of viral enzyme activity by the serum was used as an indication of the presence of antibodies to the enzyme. For HSV-1 and HSV-2 together the overall prevalence of positive sera increased with age and reached about 50% in the older age groups, the major part of the positive sera being HSV-1 positive. Sera inhibitory to the VZV pyrimidine kinase enzyme were detected only sporadically. Six percent of the tested sera repeatedly inhibited the cytosolar corresponding enzyme of uninfected host cells by an unidentified mechanism. The results of the pyrimidine kinase antibody-assay test were compared to those obtained by complement fixation (CF) and enzyme immunoassay (EIA) in a separate set of 50 sera. The correlation between the CF and the EIA tests was good in this selected serum set with CF titres from less than 8 to 64. Twenty-four percent of the CF positive sera were negative by the pyrimidine kinase antibody assay, while all pyrimidine-kinase-antibody-positive sera also had antibodies detectable by the two other methods. These results confirm and extend earlier observations on the occurrence of antibodies in human sera to HSV and VZV pyrimidine kinase enzymes.     

Transgenic reporter mouse for bioluminescence imaging of herpes simplex virus 1 infection in living mice

Bioluminescence imaging allows spatial and temporal progression of viral infection to be detected and quantified in living mice, thereby providing a new approach for studies of viral-host pathogenesis. It has been necessary to construct and validate recombinant reporter viruses that express firefly luciferase to investigate viral replication and spread with this imaging technology. This strategy greatly limits the ability to analyze multiple strains of virus and/or existing viral mutants, and reporter viruses also may be attenuated relative to the respective parental viruses. To facilitate bioluminescence imaging of herpes simplex virus type 1 (HSV-1), we developed a transgenic reporter mouse that uses the promoter from HSV-1 thymidine kinase to control expression of firefly luciferase. Infection with HSV-1 activated expression of firefly luciferase in corneal and flank models of infection, and amounts of bioluminescence increased in proportion to increasing input titers of virus. Imaging could detect infection with three different strains of HSV-1 with the following relative rank order of bioluminescence produced at the site of infection: McKrae > 17 > KOS. Corneal infection with as few as 1 x 10(3) pfu strain McKrae was detectable above background levels. By comparison, infection with vaccinia virus did not affect bioluminescence in the reporter mouse. Collectively, these data establish a new transgenic reporter mouse for infection with HSV-1, thereby enabling in vivo bioluminescence imaging studies of HSV-1 pathogenesis without constructing new reporter viruses.     

Simultaneous detection of herpes simplex virus types 1 and 2 by real-time PCR and Pyrosequencing.

BACKGROUND: Up to 80% of the US adult population has been exposed to herpes simplex virus (HSV) type 1, primarily during childhood. Also, approximately 20% of the US population has contracted genital herpes from HSV-2 infections. Clinical symptoms can present as fever, headache, malaise, myalgia, and cold sores/lesions that cause pain, itching, dysuria, and vaginal or urethral discharge. A recurrence of infection is common. HSV culturing is characterized by low sensitivity with variable success rates due to shipping conditions. OBJECTIVE: To design and validate a real-time PCR assay capable of simultaneously detecting each HSV subtype. STUDY DESIGN: ATCC-purchased HSV-1 and HSV-2 positive samples and HSV-1 and HSV-2 infected clinical specimens were assayed simultaneously with shared amplification primers and subtype-specific probes against the HSV glycoprotein B gene on a Rotor-Gene 3000 platform. Separately, two PCR reactions were performed in which one primer contained a 5' biotin modification. Single-stranded DNA from the amplicon was purified and Pyrosequenced. RESULTS: The quantitative range of the assay extended from 10(8) through 10(0) copies of each virus (r(2) > 0.991) and specificity was determined by non-amplification of 37 different human pathogens, including other herpesviruses such as VZV, CMV, and EBV. Sensitivity and specificity values of 100% were calculated by concordance analysis between the real-time PCR and the DNA Pyrosequencing results (HSV-1: n = 119, HSV-2: n = 120). Application of this assay to 4581 cervical swab specimens collected from women visiting physicians primarily in six states provided detection rates of 3.1% for HSV-1 and 7.6% for HSV-2. The average age of women infected with HSV-1 was 29.5 versus 35.6 for HSV-2. CONCLUSIONS: This procedure was demonstrated as both highly sensitive and specific for the detection of HSV-1 and HSV-2 in a single reaction. Also, the integration of Pyrosequencing analysis permitted an innovative and rapid verification for each subtype.     

Comparison of the Simplexa HSV1 & 2 Direct kit and laboratory-developed real-time PCR assays for herpes simplex virus detection

BackgroundRapid detection and differentiation of herpes simplex viruses (HSV) is important for patient management and treatment, especially in HSV meningoencephalitis.ObjectivesResults of Simplexa HSV1 & 2 Direct kit (Focus Diagnostics), an FDA-cleared sample-to-result method providing results in ∼75 min, were compared to those of laboratory-developed real-time PCR assays (LDT) for detection of HSV1 and HSV2.Study designSamples tested included 168 cerebral spinal fluid (CSF) collected prospectively and 150 tested retrospectively: 81 from clinical testing and 69 from subjects in a neonatal herpes study; and 53 plasma and sera. Each sample was tested by both methods on the same day.ResultsThree of 318 CSF had invalid Simplexa Direct results and negative LDT results. Three neonatal samples with low HSV viral loads by LDT could not be typed; two were HSV2 positive and one was negative by Simplexa Direct. Of 312 CSF with valid, type-specific results, HSV1 was detected in 16 by LDT and in 17 by Simplexa Direct; HSV2 was detected in 48 by LDT and in 49 by Simplexa Direct. Concordance rates were 98.4% (κ 0.84) and 97.1% (κ 0.89) for HSV1 and HSV2, respectively. Positive percent agreements were 87.5% for HSV1 and 91.7% for HSV2. Two and four CSF were positive only by LDT and three and five were positive only by Simplexa Direct for HSV1 and HSV2, respectively.ConclusionsSimplexa HSV1 & 2 assay performed well compared to an established LDT. The faster turn-around-time compared to LDT will allow for more rapid antiviral treatment and better patient management.     

Challenges in designing a Taqman-based multiplex assay for the simultaneous detection of herpes simplex virus types 1 and 2 and Varicella-zoster virus

To optimise molecular detection of herpesviruses an internally controlled multiplex Taqman-PCR for the detection of Herpes simplex virus 1 (HSV1), Herpes simplex virus 2 (HSV2) and Varicella-zoster virus (VZV) was developed. The selection of the dye combination working on the ABI 7700 cycler for this multiplex PCR revealed crosstalk phenomena between several combinations of reference dyes and reporter dyes. A final dye combination with CY5 as reference dye and FAM/JOE/TXR as reporter dyes was selected. The influence of the concentration of the internal positive control (IPC) concentration on the quantitative results of HSV1, HSV2 and VZV positive patient samples was analysed. The results indicate that high IPC concentrations are detrimental for the sensitivity of the multiplex assay and that the presence of the IPC molecule narrows the dynamic range of the duplex PCRs between any of the virus PCRs and the IPC-PCR. The optimised multiplex assay detecting HSV1, HSV2 and VZV using 10(3) IPC molecules showed a performance and sensitivity comparable to that of the individual assays.     

Incidence and distribution of herpes simplex virus types 1 and 2 from genital lesions in college women.

During a 9-month period, 9,772 women were treated at the student health center's gynecology clinic. Herpes simplex virus was isolated from 30 of 57 patients clinically diagnosed as suffering from a herpetic or herpetic-like genial infection for a virological incidence rate of 0.31%. Using virus plaque diameter in chick embryo cells and heat stability of viral thymidine kinase, 37% of the isolates were classified as herpes simplex virus type 1 and 63% were classified as herpes simplex virus type 2.     

Effects of S-acetylglutathione in cell and animal model of herpes simplex virus type 1 infection

Intracellular glutathione (GSH) plays an important regulatory role in the host response to viral infections. Replenishment of intracellular GSH is a desirable yet challenging goal, since systemic GSH supplementation is rather inefficient due to a short half-life of GSH in blood plasma. Further, GSH is not taken up by cells directly, but needs to be broken down into amino acids and resynthesized to GSH intracellularly, this process often being impaired during viral infections. These obstacles may be overcome by a novel glutathione derivative S-acetylglutathione (S-GSH), which is more stable in plasma and taken up directly by cells with subsequent conversion to GSH. In the present study, in vitro effects of supplementation with S-GSH or GSH on intracellular GSH levels, cell survival and replication of human herpes simplex virus type 1 (HSV-1) were studied in human foreskin fibroblasts. In addition, in vivo effects of supplementation with S-GSH or GSH on HSV-1-induced mortality were studied in hr/hr mice. In cell culture, viral infection resulted in a significant decrease of intracellular GSH levels. S-GSH efficiently and dose-dependently (5 and 10 mM tested) restored intracellular GSH, and this replenishment was more efficient than with GSH supplementation. In mice, S-GSH, but not GSH, significantly decreased HSV-1-induced mortality (P<0.05). The data suggest that S-GSH is a suitable antiviral agent against HSV-1 both in vitro and in vivo, indicating that this drug may be of benefit in the adjunctive therapy of HSV-1 infections.     

栀子提取物ZG对单纯疱疹病毒1型吸附量的影响

目的 观察栀子提取物ZG对单纯疱疹病毒1型（HSV-1）吸附过程中病毒吸附量的影响，以探讨其抗病毒作用机理。方法 采用异硫氰酸荧光素（fluorescein isothiocyanate，FTrc）作为荧光探针标记病毒，以肝素钠为对照，借助流式细胞仪，通过收集多种不同的加药方式后细胞表面的荧光强度，来考察病毒吸附过程中病毒吸附量的变化及栀子提取物ZG对此变化的影响。结果 单纯疱疹病毒1型在Hep-2细胞表面吸附存在饱和性、特异性；肝素钠3种加药方式均能明显降低病毒吸附量，先加药后吸附及吸附和加药同时进行两种方式，优于先吸附后加药的方式，其吸附抑制率分别为84．76％和82．02％；栀子提取物ZG3种加药方式均能明显降低病毒吸附量，其中以吸附和加药同时进行组最为显著，吸附抑制率为68．46％。结论 栀子提取物ZG可能是通过作用于病毒吸附的多个环节来达到抑制HSV-1的吸附作用。     

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.     

Molecular basis for resistance of herpes simplex virus type 1 mutants to the sulfated oligosaccharide inhibitor PI-88

Herpes simplex virus type 1 variants selected by virus propagation in cultured cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to the presence of PI-88 during their initial infection of cells and/or their cell-to-cell spread. Nucleotide sequence analysis revealed that the deletion of amino acids 33-116 of gC but not lack of gC expression provided the virus with selective advantage to infect cells in the presence of PI-88. Purified gC (Delta33-116) was more resistant to PI-88 than unaltered protein in its binding to cells. Alterations that partly contributed to the virus resistance to PI-88 in its cell-to-cell spread activity were amino acid substitutions Q27R in gD and R770W in gB. These results suggest that PI-88 targets several distinct viral glycoproteins during the course of initial virus infection and cell-to-cell spread.     

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.