Application of real-time PCR for quantitative detection of Clostridium botulinum type A toxin gene in food

The TaqMan real-time PCR method for the quantitative detection of C. botulinum type A was developed based on sequence-specific hybridization probes. The validity of this assay was verified by using 10 genera of 20 strains, including reference strains of C. botulinum types A, B, C, D, E and F. The detection limit of this assay was evaluated on C. botulinum type A, using a 10-fold dilution series of DNA and spores . The DNA and spores were detected up to level of 0.1 ng/ml and 10(2)spores/ml, respectively. Spore spiked food sample preparation prior to the real-time PCR was performed by two methods, heat treatment and GuSCN. The detection limits after heat treatment showed 10(2) spores/ml for spiked sausage slurry, and 10(3) spores/ml for spiked canned corn slurry, while detection limits after GuSCN precipitation showed 10(2) spores/ml in both sausage and canned corn. Therefore the real-time PCR assay after GuSCN precipitation is useful for the quantification of C. botulinum type A because it showed identical CT values in both pure spore solutions and food slurries. We suggest that quantitative analysis of C. botulinum type A by TaqMan real-time PCR can be a rapid and accurate assessment method for botulinal risk in food samples.     

Uncoating the herpes simplex virus genome

Initiation of infection by herpes simplex virus (HSV-1) involves a step in which the parental virus capsid docks at a nuclear pore and injects its DNA into the nucleus. Once "uncoated" in this way, the virus DNA can be transcribed and replicated. In an effort to clarify the mechanism of DNA injection, we examined DNA release as it occurs in purified capsids incubated in vitro. DNA ejection was observed following two different treatments, trypsin digestion of capsids in solution, and heating of capsids after attachment to a solid surface. In both cases, electron microscopic analysis revealed that DNA was ejected as a single double helix with ejection occurring at one vertex presumed to be the portal. In the case of trypsin-treated capsids, DNA release was found to correlate with cleavage of a small proportion of the portal protein, UL6, suggesting that UL6 cleavage may be involved in making the capsid permissive for DNA ejection. In capsids bound to a solid surface, DNA ejection was observed only when capsids were warmed above 4 degrees C. The proportion of capsids releasing their DNA increased as a function of incubation temperature with nearly all capsids ejecting their DNA when incubation was at 37 degrees C. The results demonstrate heterogeneity among HSV-1 capsids with respect to their sensitivity to heat-induced DNA ejection. Such heterogeneity may indicate a similar heterogeneity in the ease with which capsids are able to deliver DNA to the infected cell nucleus.     

Interactome analysis of herpes simplex virus 1 envelope glycoprotein H

Herpes simplex virus 1 (HSV‐1) envelope glycoprotein H (gH) is important for viral entry into cells and nuclear egress of nucleocapsids. To clarify additional novel roles of gH during HSV‐1 replication, host cell proteins that interact with gH were screened for by tandem affinity purification coupled with mass spectrometry‐based proteomics in 293T cells transiently expressing gH. This screen identified 123 host cell proteins as potential gH interactors. Of these proteins, general control nonderepressive‐1 (GCN1), a trans‐acting positive effector of GCN2 kinase that regulates phosphorylation of the α subunit of translation initiation factor 2 (eIF2α), was subsequently confirmed to interact with gH in HSV‐1‐infected cells. eIF2α phosphorylation is known to downregulate protein synthesis, and various viruses have evolved mechanisms to prevent the accumulation of phosphorylated eIF2α in infected cells. Here, it was shown that GCN1 knockdown reduces phosphorylation of eIF2α in HSV‐1‐infected cells and that the gH‐null mutation increases eIF2α in HSV‐1‐infected cells, whereas gH overexpression in the absence of other HSV‐1 proteins reduces eIF2α phosphorylation. These findings suggest that GCN1 can regulate eIF2α phosphorylation in HSV‐1‐infected cells and that the GCN1‐binding viral partner gH is necessary and sufficient to prevent the accumulation of phosphorylated eIF2α. Our database of 123 host cell proteins potentially interacting with gH will be useful for future studies aimed at unveiling further novel functions of gH and the roles of cellular proteins in HSV‐1‐infected cells.     

Proteomics in immunity and herpes simplex encephalitis

The genetic theory of infectious diseases has proposed that susceptibility to life-threatening infectious diseases in childhood, occurring in the course of primary infection, results mostly from individually rare but collectively diverse single-gene variants. Recent evidence of an ever-expanding spectrum of genes involved in susceptibility to infectious disease indicates that the paradigm has important implications for diagnosis and treatment. One such pathology is childhood herpes simplex encephalitis, which shows a pattern of rare but diverse disease-disposing genetic variants. The present report shows how proteomics can help to understand susceptibility to childhood herpes simplex encephalitis and other viral infections, suggests that proteomics may have a particularly important role to play, emphasizes that variation over the population is a critical issue for proteomics and notes some new challenges for proteomics and related bioinformatics tools in the context of rare but diverse genetic defects.     

Transcutaneous immunisation with herpes simplex virus stimulates immunity in mice

Herpes simplex virus (HSV) is common throughout the world and is a target for vaccine development. Transcutaneous immunisation is a novel technique that uses the application of vaccine antigens in solution on the skin in the presence of cholera toxin (CT) as an adjuvant. This study investigated the potential of transcutaneous immunisation in C3H mice, using CT co-administered with whole inactivated HSV-1 (CT+HSVi) or HSV-1 antigens extracted from infected Vero cells (CT+HSVag) or a control protein (CT+BSA). The application of any of the three vaccines on to bare mouse skin resulted in the migration of Langerhans cells from the epidermis and in the production of serum antibodies to CT. Both HSV preparations generated serum and mucosal (faecal) antibodies to HSV, with the CT+HSVi vaccine being a more potent stimulator of humoral immunity. The CT+HSVag vaccine, however, was the more potent stimulator of cell-mediated immunity, giving rise to a strong delayed type hypersensitivity response and lymphocyte proliferation in vitro. When the mice were challenged by epidermal inoculation of HSV, the CT+HSVag vaccine induced a higher level of protection than the CT+HSVi vaccine, a result which may indicate that the efficacy of HSV vaccines depends on stimulation of cell-mediated rather than humoral responses. The success of topical vaccination suggests that the transcutaneous route may offer a promising potential for novel vaccine delivery which merits further investigation.     

Role of herpes simplex virus 1 Us3 in viral neuroinvasiveness

Us3 is a serine–threonine protein kinase that is encoded by herpes simplex virus 1 (HSV‐1). In experimental animal models of HSV infection, peripheral and intracranial inoculations can be used to study viral pathogenicity in peripheral sites (e.g., eyes and vagina) and central nervous systems (CNSs), respectively. In addition, peripheral inoculation can be used to investigate this virus' ability to invade the CNS (neuroinvasiveness) from peripheral sites. HSV‐1 Us3 has previously been shown to be critical for viral pathogenicity in both peripheral sites and CNSs of mice. However, the role of HSV‐1 Us3 in viral neuroinvasiveness has not yet been elucidated. In the present study, the yields of a Us3 null mutant virus and its repaired virus in the eyes, trigeminal ganglia, and brains of mice following ocular inoculation were examined. It was found that, although the repaired virus appeared in the brains of mice 3 days after infection, peak replication occurring 7 days after infection, no viral replication of the Us3 null mutant virus was detectable. These findings indicate that HSV‐1 Us3 plays a crucial role in the ability of the virus to invade the brain from the eyes. Thus, HSV‐1 Us3 is a significant neuroinvasiveness factor in vivo.     

Autophagy interaction with herpes simplex virus type-1 infection

More than 50% of the U.S. population is infected with herpes simplex virus type-I (HSV-1) and global infectious estimates are nearly 90%. HSV-1 is normally seen as a harmless virus but debilitating diseases can arise, including encephalitis and ocular diseases. HSV-1 is unique in that it can undermine host defenses and establish lifelong infection in neurons. Viral reactivation from latency may allow HSV-1 to lay siege to the brain (Herpes encephalitis). Recent advances maintain that HSV-1 proteins act to suppress and/or control the lysosome-dependent degradation pathway of macroautophagy (hereafter autophagy) and consequently, in neurons, may be coupled with the advancement of HSV-1-associated pathogenesis. Furthermore, increasing evidence suggests that HSV-1 infection may constitute a gradual risk factor for neurodegenerative disorders. The relationship between HSV-1 infection and autophagy manipulation combined with neuropathogenesis may be intimately intertwined demanding further investigation.     

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.     

单纯疱疹病毒II型糖蛋白G主要抗原表位的表达和抗原性检测*

利用PCR拼接技术,合成含单纯疱疹病毒II型糖蛋白G(gG2)抗原表位（氨基酸序列第561～578位）的片段,并进一步利用基因工程技术获得该表位的双拷贝片段,克隆入pET-KDO表达载体进行原核表达。经IPTG诱导后,高效表达出分子量大小约为39,000D的融合蛋白,经Western-blot检测具有良好的抗原性。表达的融合蛋白经凝血酶切割和亲和层析纯化,得到双拷贝gG2（561～578aa）目的蛋白,经ELISA检测具有良好的灵敏度和特异性。该重组抗原的构建和表达可用于HSV-2特异性血清学诊断的研究。     

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.     

Nested real-time PCR for hepatitis A detection

Aims:  The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods.
Methods and Results:  By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe.
Conclusions:  We have developed a novel NRT-PCR method capable of detecting as little as 0·2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system.
Significance and Impact of the Study:  NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.     

Nuclear accumulation of a heat-shock 70-like protein during herpes simplex virus replication

A monoclonal antibody defines an antigen, p68, related to hsp70, which is located in nuclei of uninfected exponential cells. Nuclear p68 is released by DNase but not RNase treatment suggesting an association with DNA. Lytic productive infection of confluent quiescent BHK 21 cells with herpes simplex virus type-2 causes p68 to accumulate in nuclei. The effect is specific for HSV-2, and does not occur in HSV-1 infected cells. Maximum nuclear accumulation of p68 requires virus DNA synthesis although a significant accumulation occurs in the absence of such synthesis. It is suggested that the nuclear accumulation of p68 is an aspect of a cellular stress response to lytic infection with HSV-2.Imperial Cancer Research Fund, Tumour Immunology Unit.     

Evidence for a protein(s) bound to herpes simples virus DNA.

The $\text{Xba}$ I cleavage pattern of highly purified, but not specifically deproteinized, herpes simplex virus DNA does not match published patterns. If the purified herpes simplex virus DNA is first extracted with phenol and then digested with $\text{Xba}$ I, the cleavage pattern matches the published patterns. This comparison is taken as supportive of the hypothesis that there is a protein(s) bound to herpes simplex virus DNA.     

Imaging and therapy of malignant pleural mesothelioma using replication-competent herpes simplex viruses

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive cancer that is refractory to current treatment modalities. Oncolytic herpes simplex viruses (HSV) used for gene therapy are genetically engineered, replication-competent viruses that selectively target tumor cells while sparing normal host tissue. The localized nature, the potential accessibility and the relative lack of distant metastasis make MPM a particularly suitable disease for oncolytic viral therapy. METHODS: The infectivity, selective replication, vector spread and cytotoxic ability of three oncolytic HSV: G207, NV1020 and NV1066, were tested against eleven pathological types of MPM cell lines including those that are resistant to radiation therapy, gemcitabine or cisplatin. The therapeutic efficacy and the effect on survival of NV1066 were confirmed in a murine MPM model. RESULTS: All three oncolytic HSV were highly effective against all the MPM cell lines tested. Even at very low concentrations of MOI 0.01 (MOI: multiplicity of viral infection, ratio of viral particles per cancer cell), HSV were highly effective against MPM cells that are resistant to radiation, gemcitabine and cisplatin. NV1066, an oncolytic HSV that expresses green fluorescent protein (GFP), was able to delineate the extent of the disease in a murine model of MPM due to selective infection and expression of GFP in tumor cells. Furthermore, NV1066 was able to reduce the tumor burden and prolong survival even when treatment was at an advanced stage of the disease. CONCLUSION: These findings support the continued investigation of oncolytic HSV as potential therapy for patients with therapy-resistant MPM.