Is hepatitis B-virucidal validation of biocides possible with the use of surrogates?

The hepatitis B virus(HBV)is considered to be a major public health problem worldwide,and a significant number of reports on nosocomial outbreaks of HBV infections have been reported.Prevention of indirect HBV transmission by contaminated objects is only possible through the use of infection-control principles,including the use of chemical biocides,which are proven to render the virus non-infectious.The virucidal activity of biocides against HBV cannot be predicted;therefore,validation of the virucidal action of disinfectants against HBV is essential.However,feasible HBV infectivity assays have not yet been established.Thus,surrogate models have been proposed for testing the efficacy of biocides against HBV.Most of these assays do not correlate with HBV infectivity.Currently,the most promising and feasible assay is the use of the taxonomically related duck hepatitis B virus(DHBV),which belongs to the same Hepadnaviridae virus family.This paper reviews the application of DHBV,which can be propagated in vitro in primary duck embryonic hepatocytes,for the testing of biocides and describes why this model can be used as reliable method to evaluate disinfectants for efficacy against HBV.The susceptibility levels of important biocides,which are often used as ingredients for commercially available disinfectants,are also described.     

Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection

Infections by high-risk human papillomaviruses (HPV) are the causative agents for the development of cervical cancer. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV), many host cellular proteins have been implicated in the process. The proprotein convertase furin has been demonstrated to cleave the minor capsid protein, L2, post-attachment to host cells and is required for infectious entry by HPV16 PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that tissue-derived HPV16 native virus (NV) initiates infection independent of cellular furin. We show that HPV16 L2 is cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 is also not dependent on cellular furin, but two other alpha papillomaviruses, HPV18 and HPV31, are dependent on the activity of cellular furin for infection.     

Exogenous Vimentin Supplementation Transiently Affects Early Steps during HPV16 Pseudovirus Infection

Understanding and modulating the early steps in oncogenic Human Papillomavirus (HPV) infection has great cancer-preventative potential, as this virus is the etiological agent of virtually all cervical cancer cases and is associated with many other anogenital and oropharyngeal cancers. Previous work from our laboratory has identified cell-surface-expressed vimentin as a novel HPV16 pseudovirus (HPV16-PsVs)-binding molecule modulating its infectious potential. To further explore its mode of inhibiting HPV16-PsVs internalisation, we supplemented it with exogenous recombinant human vimentin and show that only the globular form of the molecule (as opposed to the filamentous form) inhibited HPV16-PsVs internalisation in vitro. Further, this inhibitory effect was only transient and not sustained over prolonged incubation times, as demonstrated in vitro and in vivo, possibly due to full-entry molecule engagement by the virions once saturation levels have been reached. The vimentin-mediated delay of HPV16-PsVs internalisation could be narrowed down to affecting multiple steps during the virus’ interaction with the host cell and was found to affect both heparan sulphate proteoglycan (HSPG) binding as well as the subsequent entry receptor complex engagement. Interestingly, decreased pseudovirus internalisation (but not infection) in the presence of vimentin was also demonstrated for oncogenic HPV types 18, 31 and 45. Together, these data demonstrate the potential of vimentin as a modulator of HPV infection which can be used as a tool to study early mechanisms in infectious internalisation. However, further refinement is needed with regard to vimentin’s stabilisation and formulation before its development as an alternative prophylactic means.     

Virucidal activity of ortho-phthalaldehyde solutions against hepatitis B and C viruses

Ortho-phthalaldehyde (OPA), a high-level disinfectant alternative to glutaraldehyde, was tested for efficacy against human hepatitis B virus (HBV) and hepatitis C virus (HCV) using surrogate animal viruses. HBV and HCV are the most prevalent human bloodborne viruses but have not yet been propagated in the laboratory. The surrogate viruses, duck hepatitis B virus (DHBV) and bovine viral diarrhea virus (BVDV), were used to assess the virucidal efficacy of OPA on HBV and HCV, respectively. After a timed exposure to the test disinfectant, the surrogate virus dried on a hard surface was neutralized and assayed to detect viable viruses using appropriate cell lines. A greater than 4-log(10) reduction in virus titer was demonstrated using dilute OPA solutions against dried DHBV and BVDV after 5 minutes of exposure at 20 degrees C. OPA was shown to be efficacious against surrogate viruses for human hepatitis B and hepatitis C virus. This is the first time that OPA efficacy has been demonstrated for HBV and HCV.     

人乳头状瘤病毒16型伪病毒载体对肝癌细胞系HepG2细胞的杀伤研究

目的 构建基于人乳头状瘤病毒（HPV）16型的伪病毒载体,并检测其对肝癌细胞系HepG2细胞的杀伤活性。方法利用昆虫什状病毒表达系统表达病毒蛋白,在体外将病毒蛋白包装白喉毒（DT）A链表达型质粒,形成伪病毒。透射电镜观察病毒样颗粒（VLP）的结构,并转染肝癌细胞系HepG2。乳酸脱氢酶释放法检测对肝癌细胞系HepG2的杀伤能力。结果透射电镜观察显示病毒蛋白可自我组装成VLP,在转染肝癌细胞系HepG2后,乳酸脱氢酶释放法成功检测到伪病毒埘肝癌细胞系HepG2的杀伤作用。结论基于HPV16的新型伪病毒载体能成功转染肝癌细胞系HepG2,并产生杀伤活性,为肝癌的基因治疗提供了一种可选择的方法。     

Virucidal activity of a quaternary ammonium compound disinfectant against feline calicivirus: a surrogate for norovirus

BACKGROUND: Norovirus, formerly known as Norwalk virus, is an important cause of gastroenteritis outbreaks in hospitals, food services, schools, and cruise ships. Infection control practices by using disinfectants to eliminate noroviruses from surfaces and environmental samples reduce the morbidity and spread of virus outbreaks. There are not many commercial disinfectants effective against norovirus. Noroviruses cannot be cultivated in vitro. However, feline calicivirus can be used as a surrogate to determine disinfectant efficacy against noroviruses. Feline calicivirus was used in a virucidal effectiveness test protocol as a surrogate for norovirus to determine the virucidal efficacy of R-82, a quaternary ammonium compound disinfectant cleaner. METHODS: Feline calicivirus suspensions containing at least 5% fetal bovine serum were dried on carriers and treated with 1:256 dilutions of R-82 disinfectant in water, with a hardness of 400 ppm as calcium carbonate, for 10 minutes. Hypochlorite concentrations of 100 +/- 10 and 1,000 +/- 10 ppm, respectively, were also analyzed as internal control standards. After contact period, the test agents were neutralized with 2 mL of appropriate neutralizer, and mixtures were scraped from carrier surfaces with a cell scraper. Selected dilutions of the neutralized inoculum/test agent mixtures were added to cultured cell monolayers of appropriate host cells. Postincubation, the infectious feline calicivirus was scored microscopically by observing virus-specific cytopathic effects produced by replicating infectious virus. The performance criterion was a minimum of 4-log(10) reduction in cytopathic effects of feline calicivirus. RESULTS: After a 10-minute contact time, formulation R-82 diluted 1:256 showed a 6.6- and 6.4-log(10) reductions in cytopathic effects of feline calicivirus during initial and confirmatory testing, respectively, demonstrating complete inactivation of the virus. A hypochlorite solution of 1,000 ppm exhibited similar log(10) reductions to the quaternary ammonium disinfectant, demonstrating the reproducibility of the protocol. CONCLUSION: Formulation R-82, a quaternary ammonium compound, is a 1-step disinfectant cleaner, which exhibited virucidal activity against feline calicivirus suspensions dried on hard surface carriers. Surfaces are vectors for virus transmission during outbreaks by transferring the virus to people or other environmental surfaces. Therefore, treatment of contaminated surfaces with formulation R-82 will optimize disinfection of noncritical and critical surfaces in health care institutions, reducing the possibility of virus transmission during outbreaks.     

The SV40 Pseudovirus: Its Potential for General Transduction in Animal Cells

The rates of reassociation of DNA from a monkey cell line (Vero), from SV40 pseudovirus (consisting of Vero DNA and virus protein coat), and from mature SV40 were measured. The results show that the host DNA in the pseudovirus contains repeated and unique sequences in the same proportions as normal host DNA; hence, no one portion of the host genome is preferentially incorporated in the pseudovirus. It was also shown that the Vero DNA in the pseudoviruses enters the nuclei of mouse embryo cells without loss of its physical integrity.     

Virucidal activities of novel hand hygiene and surface disinfectant formulations containing EGCG-palmitates (EC16)

BackgroundNon-toxic hand hygiene and surface disinfectant products with virucidal activity against alcohol-resistant nonenveloped norovirus are in urgent need.MethodAlcohol-based formulations were made with epigallocatechin-3-gallate-palmitate (EC16), an FDA accepted food additive. Based on in-house testing of formulations, 3 prototypes, PTV80 hand gel, PST70 surface disinfectant spray and PST70 surface disinfectant wipe, were selected from in-house tests for independent testing at GLP (good laboratory practice) laboratories according to EN 14476:2019 (hand gel), ASTM test method E1053-20 (spray), and ASTM E2362-15, E1053, and ASTM E2896-12 (wipe).ResultsThe PTV80 hand gel prototype demonstrated a >99.999% reduction of murine norovirus S99 infectivity in 60 seconds. Carrier testing of the PST70 surface spray and surface wipe demonstrated reduction of feline calicivirus infectivity by >99.99% in 60 seconds. In addition, testing with human coronavirus and human herpes simplex virus demonstrated >99.99% efficacy in 60 seconds, consistent with broad spectrum virucidal activity.ConclusionsThe novel non-toxic prototypes containing EC16 were found to be suitable for use in future hand sanitizer gel, surface disinfectant spray and wipe products against norovirus. Products based on these formulations could be used safely to help prevent and control norovirus and other emerging virus outbreaks, pending future studies.     

Evaluating the virucidal activity of four disinfectants against SARS-CoV-2

BackgroundThe recent COVID-19 pandemic highlights the need for efficacious virucidal products to limit the spread of SARS-CoV-2. Several studies have suggested that alcohol-based sanitizers and some disinfectants are effective. While virucidal activity data of low-level disinfectants are lacking and some conclusions are not clear yet.MethodsWe evaluated the virucidal activity of 2 quaternary ammonium compounds (QAC) disinfectants (MICRO-CHEM PLUS and FWD), W30 (an amphoteric surfactant), and Medical EtOH against SARS-CoV-2. Suspension tests covering different concentration and contact time were performed using the integrated cell culture-qPCR method.ResultsEach of disinfectants was effective at inactivating SARS-CoV-2. MCP and FWD are highly effective within 15 seconds. W30 is also efficient within 2 minutes at concentration of 1%. Consistent with previous report, our results also demonstrated that 38% ethanol was sufficient to completely inactivate virus, which proved the method used in this study is feasible.Conclusions and DiscussionQAC disinfectants, MCP and FWD, are highly effective for the inactivation of SARS-CoV-2, which making them practical for use in health care setting and laboratories where prompt disinfection is important. The low-level disinfectant based on amphoteric surfactant, W30, which may present in commonly available household hygiene agents is also able to inactivate SARS-CoV-2.     

Papillomavirus Infectious Pathways: A Comparison of Systems

The HPV viral lifecycle is tightly linked to the host cell differentiation, causing difficulty in growing virions in culture. A system that bypasses the need for differentiating epithelium has allowed for generation of recombinant particles, such as virus-like particles (VLPs), pseudovirions (PsV), and quasivirions (QV). Much of the research looking at the HPV life cycle, infectivity, and structure has been generated utilizing recombinant particles. While recombinant particles have proven to be invaluable, allowing for a rapid progression of the HPV field, there are some significant differences between recombinant particles and native virions and very few comparative studies using native virions to confirm results are done. This review serves to address the conflicting data in the HPV field regarding native virions and recombinant particles.     

Simian virus (SV) 40 like sequences in cell lines and tumour biopsies from Australian malignant mesotheliomas

Background : Simian virus (SV) 40 sequences have been found in some, but not all studies of mesotheliomas. This virus is known to cause tumours in rodents but its role in human oncogenesis remains controversial.
Aims : The aim of this study therefore was to determine whether SV40 is associated with the development of mesotheliomas in Australia. The absence of the virus or its gene products in tissue derived from mesotheliomas would detract from this possibility.
Methods : We used polymerase chain reaction from three pairs of primers to amplify different regions of the large T antigen from DNA from cell lines and cDNA from both cell lines and an independent set of tumour biopsies from patients with mesothelioma.
Results : We examined five human mesothelioma cell lines that were established in our laboratories. In addition, we examined several tumour biopsies from seven different patients. SV40 like sequences were present in all the cell lines and in at least one sample from each of the patients examined.
Conclusions : The large T antigen of SV40 or an SV40 like virus is expressed in Australian mesotheliomas and therefore could be aetiologically-associated with tumourigenesis. Alternatively, these sequences could be expressed subsequent to the development of the disease.     

Disinfectants against African Swine Fever: An Updated Review

African Swine Fever (ASF), a hemorrhagic disease with a high mortality rate in suids, is transmitted via direct and indirect contact with infectious animals and contaminated fomites, respectively. ASF reached Europe in 2014, affecting 14 of the 27 EU countries including, recently, the Italian peninsula. The fast and unprecedented spread of ASF in the EU has highlighted gaps in knowledge regarding transmission mechanisms. Fomites, such as contaminated clothing and footwear, farming tools, equipment and vehicles have been widely reported in the spread of ASF. The absence of available vaccines renders biosecurity measures, cleaning and disinfection procedures an essential control tool, to a greater degree than the others, for the prevention of primary and secondary introductions of ASF in pig farms. In this review, available data on the virucidal activity of chemical compounds as disinfectants against the ASF virus (ASFV) are summarized together with laboratory methods adopted to assess the virucidal activity.     

高危型人乳头瘤病毒感染率及亚型分布与宫颈病变程度的相关性分析

目的 探讨妇女高危型人乳头瘤病毒(HR-HPV)感染率及亚型分布与宫颈病变程度的相关性.方法 回顾性分析2018年1月至2019年12月绍兴市3 307例妇女细胞学检查和HPV筛查结果.细胞学检查采用液基薄层细胞检测(TCT),HPV筛查采用聚合酶链式反应(PCR)-反向点杂交法,采用SPSS 18.0软件进行统计学分...     

Comparative efficacy of ethanol and isopropanol against feline calicivirus, a norovirus surrogate

BACKGROUND: Improper disinfection of environmental surfaces contaminated by the feces or vomitus of infected patients is believed to be a major cause of the spread of noroviruses (NoV) in close institutional settings. Although several disinfectants are available, the search for safe and effective disinfectant continues. Because alcohol and alcohol-based products have been used as antiseptics and their efficacy against several enveloped viruses has been documented, we wanted to determine their efficacy against nonenveloped calicivirus. METHODS: Feline calicivirus (FCV) was used as a surrogate for NoVs, using the carrier test. We evaluated the virucidal efficacy of various concentrations of ethanol and isopropanol against FCV, dried on an inanimate, nonporous contact surface for contact times of 1, 3, and 10 minutes. The virus was eluted after alcohol treatment and titrated in feline kidney cells. Percentage virus inactivation was calculated by comparing these titers with those obtained with virus eluted from controls. RESULTS: Ethanol at 70% and 90% and isopropanol at 40% to 60% concentrations were found to be the most effective, killing 99% of FCV within a short contact time of 1 minute. CONCLUSION: Isopropanol was more efficacious than ethanol at 40% to 60% concentrations, suggesting that the use of an appropriate concentration of isopropanol or ethanol would help in controlling the transmission of NoVs from environmental contact surfaces.     

人乳头瘤病毒16型L1的表达、病毒样颗粒组装及其免疫原性研究

目的 在原核表达系统中表达HPV16 L1蛋白,纯化后在体外自组装成VLPs并鉴定。方法 优化GenBank中HPV16 L1基因序列并截短C末端25个氨基酸,构建至原核表达载体pET-28a上,获得重组表达载体pET28a-16L1△C25。采用镍亲和层析法纯化超声上清,于体外解组装-重组装HPV16 VLPs,采用动态光散射和透射电镜进行形貌分析,纯化后于第0、2和4周免疫小鼠,假病毒中和试验检测HPV16 VLPs免疫后血清中和抗体。结果 双酶切和测序结果表明成功构建pET28a-16L1△C25重组质粒,诱导表达后,经SDS-PAGE和Western blotting分析显示表达的L1蛋白大部分以可溶性形式存在,纯化后的蛋白样品于体外重新组装,动态光散射和透射电镜能够观察到形态与天然病毒颗粒相似的VLPs,第6周小鼠血清中和抗体滴度Log10平均值达到4.43。结论 利用原核表达系统成功表达了截短型HPV16 L1蛋白,并于体外组装成结构完整的VLPs,且具有较好的免疫原性,为低成本HPV预防性疫苗的研发奠定基础。     

Malaria infection and anemia prevalence in Zambia's Luangwa District: an area of near-universal insecticide-treated mosquito net coverage

Vaccinia virus (VACV) is the cause of bovine vaccinia (BV), an emerging zoonotic disease that affects dairy cows and milkers. Some chemical disinfectants have been used on farms affected by BV to disinfect cow teats and milkers' hands. To date, there is no information about the efficacy of disinfectants against VACV. Therefore, this study aimed to assess the virucidal activity of some active disinfectants commonly used in the field. Sodium hypochlorite, quaternary ammonium combined with chlorhexidine, and quaternary ammonium combined with glutaraldehyde were effective in inactivating the virus at all concentrations tested. Iodine and quaternary ammonium as the only active component were partially effective. The presence of bovine feces as organic matter and light decreased the effectiveness of sodium hypochlorite. These results show that an appropriated disinfection and asepsis of teats and hands may be helpful in the control and prevention of BV and other infections with VACV.     

HPV in anal squamous cell carcinoma and anal intraepithelial neoplasia (AIN)

Background and aims Majority of cases of anal squamous cell carcinoma are human papilloma virus (HPV)-induced and result from anal intraepithelial neoplasia (AIN). This study was conducted to examine methods which may enable the routine diagnosis of HPV-induced changes in the anal rim and the consequences of such detection especially in view of a more sensitive diagnosis of AIN. Results were clinically correlated.Methods The study included biopsy samples from 87 patients who had been diagnosed with the following disease patterns: 47 invasive anal carcinoma, 33 AIN of varying severity and seven condylomatous lesions. In 52 of these cases, a tumour was clinically suspected. All biopsies were retrospectively examined for microscopic indications of HPV infection. After microdissection, additional HPV analysis via PCR was carried out.Results In 38 of 47 cases of anal carcinoma, HPV DNA could be detected via PCR (80.9%), the majority of which were HPV 16 (33/38=86.8%). In 29 of the 33 cases of AIN, HPV DNA was detected (87.9%), most of these in AIN III (15/16=93.8%). Histological markers of HPV infection were detected in all 87 cases.Discussion In our series, the clinical diagnosis of the invasive anal carcinoma had a high sensitivity of 93.6%, with a specificity of 80%. The positive predictive value was 84.6%, and the negative predictive value 91.4%. In contrast, AIN had been detected clinically in none of the cases. In this situation, especially with high-risk patients, our findings recommend anal HPV screening in combination with anal cytology and anoscopy.Conclusion Based on our results, we urgently recommend for any histological report on excision of anal lesions to include a statement whether histological markers of HPV infection were detected. In individual cases, validation via HPV PCR must be considered.     

Pathogenesis of malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) results from neoplastic transformation of mesothelial cells. Past asbestos exposure represents the major risk factor for MPM, as the link between asbestos fibres and MPM has been largely proved by epidemiological and experimental studies. Asbestos fibres induce DNA and chromosome damage linked to oxidative stress following phagocytosis. Recently, simian virus 40 (SV40) has been implicated in the aetiology of MPM. The origin of human infection has been associated with SV40-contaminated polio vaccines, although to date, no epidemiological data supports this hypothesis. SV40 may act as a coactivator of asbestos in mesothelial oncogenesis. The transforming potency of SV40 results from the activity of two viral proteins, large T and small t antigens. SV40 infection stimulates production of growth factors elsewhere implicated in autocrine growth of mesothelioma cells and inactivates RASSF1, a gene silenced in MPM. Roles for ionising radiation, chemicals or genetic factors have also been suggested from the observation of sporadic MPM cases or animal studies. Genetic alterations in the tumour suppressor genes, P16/CDKN2A and neurofibromatosis 2 (NF2), are found both in human MPM and in asbestos-exposed Nf2-deficient mice. MPM is still of great international concern. Despite a ban on asbestos use in Western countries, the incidence of MPM is increasing, due to the long delay between asbestos exposure and diagnosis. Moreover, asbestos is still used in developing countries. The implication of other risk factors, especially SV40, supports a need for further research into MPM.     

Human papillomaviruses in colorectal cancers: A case-control study in western patients

BackgroundColorectal cancer (CRC) is one of the most common cancers. As in other cancer locations, the involvement of human papillomaviruses (HPV) has been suggested but remains highly debated with wide differences among reported prevalence of HPV infection in CRCs.AimTo determine the actual prevalence of high risk HPV16 and 18 in a large case-control study.MethodsCRC specimens were used for analysis of both tumor and distant healthy tissue. As a non-malignant control group, samples from sigmoid diverticulosis resections were studied. Detection of HPV16 and HPV18 DNA was performed using a real time polymerase chain reaction (qPCR). Ten percent of tumor samples were also randomly subjected to a complete HPV genotyping using the INNO-LiPA technique.Results467 samples were analyzed: 217 tumor samples from 210 CRCs, 210 distant healthy tissue samples, and 40 sigmoid samples. HPV18 DNA was never amplified and HPV16 was amplified only three times in tumor tissues with viral loads under or at the limit of quantification. New extraction from the same tumor blocks for these samples revealed no HPV with qPCR and INNO-Lipa assays.ConclusionWith adequate procedures and reliable techniques, no HPV was detected in the largest case-control study so far, bringing more evidence on the absence of involvement of HPV in CRCs.