Multiplex real-time PCR for the simultaneous detection of herpes simplex virus, human herpesvirus 6, and human herpesvirus 7

A simultaneous detection system to quantify HSV, HHV-6, and HHV-7 DNA via multiplex real-time PCR using different fluorochromes was developed. The minimum quantitative level established via this multiplex assay was four copies per reaction for HSV type 1, four copies for HHV-6, and three copies for HHV-7, respectively. The dynamic range encompassed at least six orders of magnitude. The system was specific and reproducible even in the presence of large amounts of other viral DNA. We then applied this multiplex real-time PCR assay to 105 CSF specimens obtained from subjects less than 15 years old in whom a diagnosis of viral encephalitis/encephalopathy was suspected on clinical grounds. The detection rate for each viral DNA was 6.7% for HSV, 9.5% for HHV-6, and 1.9% for HHV-7. These results indicate that our system is reliable and may be useful for the rapid diagnosis of viral encephalitis/encephalopathy.     

siRNA干扰HSV1 gD糖蛋白基因的研究

以HSV1 gD糖蛋白基因为靶点,设计合成5对siRNA,并构建pEGFP-N1-gD融合表达质粒,脂质体法共转染Vero细胞,利用绿色荧光蛋白(EGFP)报告基因的特征,流式细胞仪筛选特异沉默gD表达的siRNA。然后有效siRNA转染Vero细胞并感染HSV1,通过空斑减数实验,Real-time PCR和子代病毒滴度评价其对HSV-1感染的作用。结果显示siRNA能有效抑制gD-EGFP融合蛋白和感染细胞内gD糖蛋白的表达,但对HSV-1的感染性无明显抑制作用,故选择gD作为siRNA抗病毒靶点还需进一步的论证。     

Herpesvirus DNA in Peripheral Blood Mononuclear Cells of Some Patients with Meniere's Disease

Herpes simplex virus‐1 (HSV) or varicella zoster virus (VZV) DNA was detected by nested polymerase chain reaction in peripheral blood mononuclear cells of patients with Meniere's disease (one of 28 patients for HSV‐1,2 of 28 patients for VZV) during acute illness (within 5 days after onset). On the other hand, neither HSV‐1 DNA or VZV DNA was detected in PBMCs of 50 age‐ and sex‐matched healthy individuals and 50 pregnant women. These findings may imply that reactivation of HSV‐1 or VZV may be associated with the development of some cases of Meniere's disease.     

Diagnosis of Genital Herpes by Polymerase Chain Reaction Amplification

A new polymerase chain reaction (PCR) method employing type-specific primers and probes was applied to 114 clinical specimens obtained from 58 female patients with genital lesions or who had a history of genital herpes. Ten and 15 specimens, respectively, were positive for herpes simplex virus (HSV)-1 and HSV-2 by cell culture. All of 10 culture-confirmed HSV-1 cases and 11 of 15 (73%) culture-confirmed HSV-2 cases were identified by PCR. Although there were several cases with discrepancy between cell culture and PCR for HSV-2, the results suggest that this PCR procedure could be applied to clinical specimens from the female genital tract.     

Moxibustion activates host defense against herpes simplex virus type I through augmentation of cytokine production

Moxibustion is a technique used in traditional oriental medicine, the aim of which is to cure and/or prevent illness by activating a person's ability for self‐healing. In this study, we assessed how moxibustion would affect the immune system and whether it would augment protective immunity. Mice were treated with moxibustion at Zusanli (ST36) acupoints; we analyzed mortality and cytokine activity in sera after infection with herpes simplex virus type 1 (HSV‐1), and cytokine gene expression in the skin and the spleen without a virus challenge. Our study demonstrates that pretreatment of BALB/c mice with moxibustion resulted in a marked increase in the survival rate after infection with lethal doses of HSV‐1, and elevated serum levels of IL‐1β and IFN‐γ on days 1 and 6 post‐infection with HSV‐1. Semi‐quantitative RT‐PCR assay showed that moxibustion treatment augmented the expression of IL‐1α, IL‐1β, IL‐6, universal‐IFN‐α, MIP‐1α, and TNF‐α mRNA in the skin, and IL‐1α, IL‐1β, IL‐12p40, IL‐15, u‐IFN‐α, MIP‐1α, and TNF‐α mRNA in the spleen. Moreover, moxibustion induces augmentation of natural killer cell activity. Collectively, our study demonstrates that moxibustion activates protective responses against HSV‐1 infection through the activation of cytokine production including IFN, and of NK cells.     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

一次PCR扩增单纯疱疹和巨细胞病毒基因的检测方法

宫内感染是影响优生优育质量的一个重要因素,由病毒引起的宫内感染主要有以下四类病原：ＨＳＶ、ＣＭＶ、ＴＯＸＯ和风疹病毒（ＲＶ）,其中ＨＳＶ和ＣＭＶ的感染率较高。孕期这两种病原的感染会影响胎儿的正常发育,严重的可导致流产甚至胎儿畸形。目前已有针对这两种病...     

Quantitation of herpes simplex virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis by the polymerase chain reaction

Background: Previous studies have shown the diagnostic utility of qualitative detection of herpes simplex virus (HSV) DNA by the polymerase chain reaction (PCR) in cerebrospinal fluid samples (CSF) from patients with herpes simplex encephalitis (HSE).Objectives: To determine whether quantitation of HSV DNA in CSF could be useful for monitoring efficacy of antiviral therapy and provide prognostic indications.Study design: A quantitative PCR assay using an internal control for evaluation of PCR efficiency and detection of PCR inhibitors was developed and used for retrospective testing of 98 CSF samples from 26 patients with serologically diagnosed HSE during the period 1980–1995.Results: HSV DNA was detected in 36 CSF samples from 23 patients. PCR positivity was 100% for CSF samples collected within 10 days after onset, and 30.4 and 18.7% for samples collected 11–20 and 21–40 days later, respectively. The 3 PCR-negative patients had their first CSF collected 14, 16, and 28 days after onset, respectively. Three of 98 (3.1%) CSF samples were completely or partially inhibitory to PCR. Initial DNA levels were not significantly different in patients with HSE due to either primary or recurrent HSV infection. In addition, they were not related to severity of clinical symptoms nor were predictive of the outcome. A progressive decrease in viral DNA levels was observed both in patients who received acyclovir therapy and in a small number of untreated patients.Conclusions: This study: (i) confirms the high sensitivity of PCR for the diagnosis of HSE; (ii) emphasizes the need for an internal control of amplification to achieve maximal sensitivity and perform reliable quantitation of viral DNA; and (iii) suggests that CSF might not be the best specimen to investigate in studies of the natural history of HSE.     

白斑综合症病毒实时荧光LAMP检测方法的建立及应用

研究利用ESE-Quant tube scanner检测平台, 建立了一套基于环介导等温扩增技术(Loop-Mediated Isothermal Amplification, LAMP)的实时荧光检测方法, 用于白斑综合征病毒(White Spot Syndrome Virus, WSSV)的检测; 并在此基础上, 与巢式PCR、Real-time PCR和其他已发表的4种LAMP方法在检测灵敏度、实际应用方面进行比较. 结果显示, 研究建立的实时荧光LAMP检测方法在63℃恒温反应30min可检测到最低为105倍稀释的基因组DNA模板, 与Real-time PCR检测方法的灵敏度相当, 高于巢式PCR和其他已发表的4种LAMP方法的检测灵敏度; 而且特异性较好, 与传染性皮下及造血组织坏死病毒等5种常见对虾病原DNA均无交叉反应. 通过构建质粒进一步进行灵敏度测试显示, 本研究建立的实时荧光LAMP检测方法最低检测限度为24个拷贝质粒DNA, 检出时间亦为30min. 通过对66份待检样品的检测结果显示, 实时荧光LAMP检测方法的检出阳性率为7.57%, 准确率为100%, 高于其他WSSV的检测方法. 因此, 研究建立的WSSV实时荧光LAMP检测方法, 操作简单, 反应速度快, 特异性好, 灵敏度高, 成本低廉, 可以直观、实时地观察反应的进行情况, 适合对虾养殖现场及诊断实验室的WSSV快速检测.       

A nested real-time PCR assay has an increased sensitivity suitable for detection of viruses in aerosol studies

Aims:  Influenza is commonly spread by infectious aerosols; however, detection of viruses in aerosols is not sensitive enough to confirm the characteristics of virus aerosols. The aim of this study was to develop an assay for respiratory viruses sufficiently sensitive to be used in epidemiological studies.
Method:  A two-step, nested real-time PCR assay was developed for MS2 bacteriophage, and for influenza A and B, parainfluenza 1 and human respiratory syncytial virus. Outer primer pairs were designed to nest each existing real-time PCR assay. The sensitivities of the nested real-time PCR assays were compared to those of existing real-time PCR assays. Both assays were applied in an aerosol study to compare their detection limits in air samples.
Conclusions:  The nested real-time PCR assays were found to be several logs more sensitive than the real-time PCR assays, with lower levels of virus detected at lower Ct values. The nested real-time PCR assay successfully detected MS2 in air samples, whereas the real-time assay did not.
Significance and Impact of the Study:  The sensitive assays for respiratory viruses will permit further research using air samples from naturally generated virus aerosols. This will inform current knowledge regarding the risks associated with the spread of viruses through aerosol transmission.     

Axin expression delays herpes simplex virus‐induced autophagy and enhances viral replication in L929 cells

Axin, a negative regulator of the Wnt signaling pathway, plays a critical role in various cellular events including cell proliferation and cell death. Axin‐regulated cell death affects multiple processes, including viral replication. For example, axin expression suppresses herpes simplex virus (HSV)‐induced necrotic cell death and enhances viral replication. Based on these observations, this study investigated the involvement of autophagy in regulation of HSV replication and found axin expression inhibits autophagy‐mediated suppression of viral replication in L929 cells. HSV infection induced autophagy in a time‐ and viral dose‐dependent manner in control L929 cells (L‐EV), whereas virus‐induced autophagy was delayed in axin‐expressing L929 cells (L‐axin). Subsequent analysis showed that induction of autophagy by rapamycin reduced HSV replication, and that inhibiting autophagy by 3‐methyladenine (3MA) and beclin‐1 knockdown facilitated viral replication in L‐EV cells. In addition, preventing autophagy with 3MA suppressed virus‐induced cytotoxicity in L‐EV cells. In contrast, HSV replication in L‐axin cells was resistant to changes in autophagy. These results suggest that axin expression may render L929 cells resistant to HSV‐infection induced autophagy, leading to enhanced viral replication.     

Evaluation of Herpes Simplex Detection in Corneal Scrapings by Three Molecular Methods

Herpes simplex virus (HSV)-1 keratitis (HSK) is a sight-threatening ocular infection with worldwide occurrence. A prompt laboratory diagnosis is often very useful. The purpose of this study was to evaluate molecular methods as rapid diagnostic tools compared with cell culture of HSK. Corneal scrapings from patients with clinically suspected HSK were tested by direct immunofluorescence assay (IFA) for HSV-1 antigen and by polymerase chain reaction (PCR) for HSV-1 DNA, and an attempt for viral isolation was performed on Vero cell line culture. Positive samples by cell culture were 20.8%, whereas PCR was positive in 29.2%, and IFA was positive in 33.3%. IFA had better sensitivity (80%) and negative predictive value (81.8%) than PCR (70% and 76.9%, respectively); however, PCR had better specificity (71.4%) and positive predictive value (63.6%). This indicates that a combination of cell culture, IFA and PCR constitutes the best set of tools for diagnosis of clinically suspected cases of HSK. Documented infection can be further assessed by cell-culture technique or PCR depending laboratory availability.     

Polymerase chain reaction for identification of herpes simplex virus (HSV-1), cytomegalovirus (CMV) and human herpes virus-type 6 (HHV-6) in oral swabs

Considering that sensitive and specific methods to detect HSV-1, CMV and HHV-6 on oral mucosa have a great impact on oral diagnosis practice and research, together with the evidence that PCR is a rapid and reliable method, the purpose of the present study was to develop primer sets to detect HSV-1, CMV and HHV-6 in oral swabs by nested polymerase chain reaction (nested PCR). We developed a practical method for sample collection without tissue trauma, and the swabs were stored until used for DNA extraction. After the nested PCR a DNA fragment of 241 bp corresponding to HSV-1 was amplified. DNA fragments of 224 and 369 bp were amplified corresponding to CMV and HHV-6, respectively. DNA sequencing analysis confirmed the expected sequences of each virus. In conclusion, it was demonstrated that these new primer sets are able to identify HSV-1, CMV and HHV-6 in oral swab using nested PCR.     

Apoptosis participates to liver damage in HSV-induced fulminant hepatitis

Background: HSV fulminant hepatitis is a rare pathology. Rapid hepatic failure, as a consequence of extended liver damage, has generally been attributed to necrosis. As apoptosis can constitute another way for hepatocytes to die, we decided to investigate whether programmed cell death took place during HSV fulminant hepatitis. Methods: Liver sections were obtained from two cases of fulminant herpetic hepatitis as well as from hepatitis B virus and Rickettsia-infected livers. Herpes simplex virus infection was confirmed using in situ hybridization. Apoptosis was assessed by histopathological examination, p53, activated-caspase 3 and Fas immunohistochemistry and TUNEL labeling. Results: We report that the number of cells expressing activated-caspase 3 was largely increased in fulminant herpes simplex virus hepatitis, when compared to livers chronically infected by hepatitis B virus or from a Rickettsial acute hepatitis. Apoptosis of hepatocytes was confirmed by a positive double-staining for activated-caspase 3 and hepatocytes. Finally, the apoptotic process has progressed beyond the step of nuclear DNA cleavage as demonstrated by TUNEL labeling. Conclusion: These data as a whole show that apoptosis is responsible, at least partially, for liver damage during HSV fulminant hepatitis.     

A nested PCR assay to detect DNA in sera for the diagnosis of deep-seated trichosporonosis

Deep-seated trichosporonosis caused by Trichosporon asahii has a high mortality rate and a very poor prognosis. New species-specific oligonucleotide primers for T. asahii were developed from a sequence analysis of rRNA genes that included the internal transcribed spacer regions. A nested PCR assay with specific primers was used to examine 11 serum samples from 7 patients, who were diagnosed with deep-seated trichosporonosis histologically at autopsy. In addition, Trichosporon cell wall polysaccharide (PS) was detected by a latex agglutination (LA) test. Of 11 samples, seven had a positive LA test, and T. asahii DNA was also detected with the nested PCR assay. Of the four samples in which PS antigen was not detected, the nested PCR of two samples was positive. Our new nested PCR assay may be used as an adjunct to conventional methods for diagnosing T. asahii infection.     

Herpes Simplex Virus-Induced Expression of 70 kDa Heat Shock Protein (HSP70) Requires Early Protein Synthesis But Not Viral DNA Replication

The major 70 kDa heat shock protein (HSP70), which is scarcely expressed in unstressed rodent cells, was apparently induced by infection with herpes simplex virus (HSV). Infection with HSV types 1 and 2 elevated HSP70 mRNA levels within 4 hr post-infection. HSP70 synthesis and accumulation increased in HSV-infected cells. Irradiation of HSV with UV-light abolished the ability to induce HSP70 mRNA. Inhibitors of viral DNA synthesis did not affect the induction of HSP70 in infected cells. Protein synthesis within 2 hr after infection was necessary for HSP70 induction.     

浙江省献血员HGV和TTV感染情况的调查

应用逆转录套式聚合酶链反应(RT-Nested PCR)和半套式聚合酶链反应(Semi-nested PCR)分别检测来自浙江省3个地区165例献血员血清标本中的庚型肝炎病毒(HGV)RNA和输血传播性病毒(TTV)DNA.14.6%(24/165)和12.7%(21/165)的血清标本分别检出HGV RNA和TTV DNA,其中3.6%的血清标本(6/165)可同时检出HGV RNA和TTV DNA.实验结果表明,浙江省献血员中HGV和TTV的感染率较高.     

Prevalence of hepatitis B virus in chronic hepatitis B patients

The aim of the current study was to detect HBV by Real time - PCR in chronic hepatitis B patients. Fifty-eight sera of chronic hepatitis B patients were subjected during the period March 2009 to April 2010 in Ilam cities in West of Iran. Sera assayed by real-time PCR and ELISA methods. Twenty serum samples from healthy volunteers and non-hepatitis B patients and negative for hepatitis B seromarkers served as negative controls for the study. Among fifty-eight sera, ELISA showed fifty-five (94.8%) of the samples were positive for HBsAg and three (5.2%) negative results obtained while real-time PCR specified fifty-eight (100%) positive results in chronic hepatitis B patients. HBsAg status did not necessarily reflect HBV DNA level in the serum, as 5.2% of chronic Hepatitis B patients were positive for HBV DNA but negative for HBsAg. HBV DNA was not found to be positive amongst any of the negative controls. Real time - PCR is a sensitive and reproducible assay for HBV DNA quantization.