Inactivation of laboratory animal RNA-viruses by physicochemical treatment

Eight commonly used chemical disinfectants and physical treatments (UV irradiation and heating) were applied to both enveloped RNA viruses (Sendai virus, canine distemper virus) and unenveloped RNA viruses (Theiler's murine encephalomyelitis virus, reo virus type 3) to inactivate infectious virus particles. According to the results, alcohols (70% ethanol, 50% isopropanol), formaldehyde (2% formalin), halogen compounds (52ppm iodophor, 100ppm sodium hypochlorite), quaternary ammonium chloride (0.05% benzalkonium chloride) and 1% saponated cresol showed virucidal effects giving more than 99.95% reduction in the infectivity of virus samples of Sendai virus and canine distemper after 10 minutes exposure. There was no significant difference in the effects on the two enveloped RNA viruses. The susceptibility of unenveloped RNA viruses to chemical disinfectants and physical treatments differed greatly from the enveloped viruses. The two unenveloped viruses showed distinct resistance to 50% isopropanol, 2% formalin, 1% saponated cresol and to physical treatments (heating at 45, 56, 60 degrees C, and UV irradiation). These results indicate that using physicochemical methods to inactivate RNA viruses in laboratory animal facilities should be considered in accordance with the characteristics of the target virus. For practical purposes in disinfecting enveloped RNA viruses, 70% ethanol, 0.05% quaternary ammonium chloride and 1% saponated cresol diluted in hot water (greater than 60 degrees C) are considered as effective as UV irradiation. For unenveloped RNA viruses, halogen compounds, more than 1,000 ppm sodium hypochlorite or 260 ppm iodophor are recommended over a period of 10 minutes for disinfecting particles, although these compounds result in an oxidation problem with many metals.     

Thermal Inactivation of Enteric Viruses and Bioaccumulation of Enteric Foodborne Viruses in Live Oysters (Crassostrea virginica)

Human enteric viruses are among the main causative agents of shellfish-associated outbreaks. In this study, the kinetics of viral bioaccumulation in live oysters and the heat stabilities of the predominant enteric viruses were determined both in tissue culture and in oyster tissues. A human norovirus (HuNoV) GII.4 strain, HuNoV surrogates (murine norovirus [MNV-1], Tulane virus [TV]), hepatitis A virus (HAV), and human rotavirus (RV) bioaccumulated to high titers within oyster tissues, with different patterns of bioaccumulation for the different viruses. We tested the thermal stability of each virus at 62, 72, and 80°C in culture medium. The viruses can be ranked from the most heat resistant to the least stable as follows: HAV, RV, TV, MNV-1. In addition, we found that oyster tissues provided protection to the viruses during heat treatment. To decipher the mechanism underlying viral inactivation by heat, purified TV was treated at 80°C for increasing time intervals. It was found that the integrity of the viral capsid was disrupted, whereas viral genomic RNA remained intact. Interestingly, heat treatment leading to complete loss of TV infectivity was not sufficient to completely disrupt the receptor binding activity of TV, as determined by the porcine gastric mucin–magnetic bead binding assay. Similarly, HuNoV virus-like particles (VLPs) and a HuNoV GII.4 strain retained some receptor binding ability following heat treatment. Although foodborne viruses have variable heat stability, 80°C for >6 min was sufficient to completely inactivate enteric viruses in oysters, with the exception of HAV.     

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.     

Interaction of infectious viral particles with a quaternary ammonium chlorid (QAC) surface

The antiviral activity of a surface-bonded quaternary ammonium chloride (QAC) was examined in this study. The mechanism of inactivation was elucidated by a combination of infectivity assay, radioactive labeling assay, and sedimentation analysis. Although the virions are still infectious when attached onto the chemically modified surface, we found these viruses are inactivated if they are eluted from the surface. The inactivation is caused by the disruption of the viral envelope with subsequent release of the nucleocapsid. No evidence indicates the released nucleocapsid is further disrupted. An enveloped virus shows a much higher affinity for the QAC-treated surface than a nonenveloped one due to hydrophobic interaction. The QAC-treated beads can effectively remove the enveloped viruses at low protein concentrations. The titer of herpes simplex virus was reduced by a factor of nearly 5 logarithm units in a 0.5 wt % bovine serum albumin solution with less that 10% protein loss. However, the presence of proteins in the solution reduced both the rate and capacity of this nonspecific adsorption-inactivation process. As a consequence, the removal efficiency is relatively poor in solutions with high protein content.     

Destruction of Cucumber green mottle mosaic virus by heat treatment and rapid detection of virus inactivation by RT-PCR

Heat treatment is commonly used to control viral contamination of seeds. To study virus inactivation, virus was purified from seeds contaminated with Cucumber green mottle mosaic virus (CGMMV) after various heat treatments. CGMMV particles were observed to be physically disrupted by high temperature. Analysis of viral RNA revealed that the 5' and 3' termini of the genome were protected whereas regions between 2-2.5, 3.2-3.7 and 4-4.8 kb from the 5' terminus were not. Heat inactivation of virus on seeds was confirmed by RT-PCR using primers for a heat-sensitive region. The RT-PCR approach developed here may prove preferable to time- and labor-intensive bioassays for assessing virus heat inactivation.     

Association of a cellular 21-kDa transmembrane protein (VAP21) with enveloped viruses.

We reported previously that the rabies virions contained a 21-kDa cellular transmembrane protein (referred to as VAP21) as a minor component (Sagara, J. et al, Microbiol. Immunol. 41(12): 947-955, 1997). In this study, we further examined the possible interactions of VAP21 with other enveloped viruses, including the vesicular stomatitis virus (VSV; negative-stranded RNA virus), Sindbis virus (positive-stranded RNA virus) and herpes simplex virus type 1 (HSV-1; double-stranded DNA virus). An immunoblot analysis demonstrated that all of these enveloped viruses contained VAP21 in the virion as a minor component. Immunoprecipitation studies suggested that VAP21 was associated with certain viral proteins in the cell, such as the matrix (M) protein of VSV, a capsid protein of Sindbis virus, and at least a capsid protein (VP5) of HSV-1. The association was disrupted by treatment with 0.5% sodium dodecyl sulfate, but resistant to the treatment with 1% NP-40 plus 1% deoxycholate. These results suggest that: 1) VAP21 is not primarily associated with the viral transmembrane glycoprotein but rather with the internal viral protein, and, 2) this association would cause the efficient incorporation of VAP21 into the virion.     

Complete inactivation of Venezuelan equine encephalitis virus by 1,5-iodonaphthylazide

Hydrophobic alkylating compounds like 1,5-iodonaphthylazide (INA) partitions into biological membranes and accumulates selectively into the hydrophobic domain of the lipid bilayer. Upon irradiation with far UV light, INA binds selectively to transmembrane proteins in the viral envelope and renders them inactive. Such inactivation does not alter the ectodomains of the membrane proteins thus preserving the structural and conformational integrity of immunogens on the surface of the virus. In this study, we have used INA to inactivate Venezuelan equine encephalitis virus (VEEV). Treatment of VEEV with INA followed by irradiation with UV light resulted in complete inactivation of the virus. Immuno-fluorescence for VEEV and virus titration showed no virus replication in-vitro. Complete loss of infectivity was also achieved in mice infected with INA treated plus irradiated preparations of VEEV. No change in the structural integrity of VEEV particles were observed after treatment with INA plus irradiation as assessed by electron microscopy. This data suggest that such inactivation strategies can be used for developing vaccine candidates for VEEV and other enveloped viruses.     

Native hepatitis B virions and capsids visualized by electron cryomicroscopy

Hepatitis B virus (HBV) infects more than 350 million people, of which one million will die every year. The infectious virion is an enveloped capsid containing the viral polymerase and double-stranded DNA genome. The structure of the capsid assembled in vitro from expressed core protein has been studied intensively. However, little is known about the structure and assembly of native capsids present in infected cells, and even less is known about the structure of mature virions. We used electron cryomicroscopy (cryo-EM) and image analysis to examine HBV virions (Dane particles) isolated from patient serum and capsids positive and negative for HBV DNA isolated from the livers of transgenic mice. Both types of capsids assembled as icosahedral particles indistinguishable from previous image reconstructions of capsids. Likewise, the virions contained capsids with either T = 3 or T = 4 icosahedral symmetry. Projections extending from the lipid envelope were attributed to surface glycoproteins. Their packing was unexpectedly nonicosahedral but conformed to an ordered lattice. These structural features distinguish HBV from other enveloped viruses.     

Lack of correlation between virus barosensitivity and the presence of a viral envelope during inactivation of human rotavirus, vesicular stomatitis virus, and avian metapneumovirus by high-pressure processing

High-pressure processing (HPP) is a nonthermal technology that has been shown to effectively inactivate a wide range of microorganisms. However, the effectiveness of HPP on inactivation of viruses is relatively less well understood. We systematically investigated the effects of intrinsic (pH) and processing (pressure, time, and temperature) parameters on the pressure inactivation of a nonenveloped virus (human rotavirus [HRV]) and two enveloped viruses (vesicular stomatitis virus [VSV] and avian metapneumovirus [aMPV]). We demonstrated that HPP can efficiently inactivate all tested viruses under optimal conditions, although the pressure susceptibilities and the roles of temperature and pH substantially varied among these viruses regardless of the presence of a viral envelope. We found that VSV was much more stable than most food-borne viruses, whereas aMPV was highly susceptible to HPP. When viruses were held for 2 min under 350 MPa at 4°C, 1.1-log, 3.9-log, and 5.0-log virus reductions were achieved for VSV, HRV, and aMPV, respectively. Both VSV and aMPV were more susceptible to HPP at higher temperature and lower pH. In contrast, HRV was more easily inactivated at higher pH, although temperature did not have a significant impact on inactivation. Furthermore, we demonstrated that the damage of virion structure by disruption of the viral envelope and/or capsid is the primary mechanism underlying HPP-induced viral inactivation. In addition, VSV glycoprotein remained antigenic although VSV was completely inactivated. Taken together, our findings suggest that HPP is a promising technology to eliminate viral contaminants in high-risk foods, water, and other fomites.     

Production of hepatitis C virus lacking the envelope-encoding genes for single-cycle infection by providing homologous envelope proteins or vesicular stomatitis virus glycoproteins in trans

Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the major components of viral particles. Here we developed a trans-complementation system that allows the production of infectious HCV particles in whose genome the regions encoding envelope proteins are deleted (HCVΔE). The lack of envelope proteins could be efficiently complemented by the expression of homologous envelope proteins in trans. HCVΔE production could be enhanced significantly by previously described adaptive mutations in NS3 and NS5A. Moreover, HCVΔE could be propagated and passaged in packaging cells stably expressing HCV envelope proteins, resulting in only single-round infection in wild-type cells. Interestingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently rescue the production of HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production. VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the delivery of HCV replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the production of single-cycle infectious HCV particles, which should be useful for studying individual steps of the HCV life cycle and may also provide a new strategy for HCV vaccine development.     

Nonoccluded baculovirus- and filamentous virus-like particles in the spotted cucumber beetle,diabrotica undecimpunctata (coleoptera: chrysomelid)

Nonoccluded baculovirus-and filamentous virus-like particles were found in nuclei of hemocytes or midgut cells of field-collected spotted cucumber beetles. Each type of particle was associated with a different type of virogenic stroma containing various viral components similar to those referred to as capsid, nucleocapsid, viroplasm, and viral envelope in other known baculovirus infections. Nucleocapsids of the virus which occured only in hemocytes were rod-shaped particles approximately 230 nm long and 52 nm wide and were enveloped singly by a trilaminar unit membrane. Enveloped and partly enveloped particles appeared to be released from the nucleus to the cytoplasm by budding through the nuclear envelope acquiring additional membranes. The nucleocapsids of the virus which occurred only in nuclei of midgut cells were filamentous particles with an average diameter of 25 nm and variable length up to 2 μm. Some extremely long particles were bent almost 360° near the middle, resulting in a hairpin-like configuration. The particles were always enveloped singly. No particles budding through the nuclear envelope were observed.     

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.     

Modulation of signaling pathways by RNA virus capsid proteins

Capsid proteins are structural components of virus particles. They are nucleic acid-binding proteins whose main recognized function is to package viral genomes into protective structures called nucleocapsids. Research over the last 10 years indicates that in addition to their role as genome guardians, viral capsid proteins modulate host cell signaling networks. Disruption or alteration of intracellular signaling pathways by viral capsids may benefit replication of the virus by affecting innate immunity and in some cases, may underlie disease progression. In this review, we describe how the capsid proteins from medically relevant RNA viruses interact with host cell signaling pathways.     

芽胞杆菌及其代谢物抑制包膜病毒感染的研究进展

包膜病毒指具有一层脂质双层膜的病毒,如流感病毒、冠状病毒等,这些包膜病毒每年在世界范围内导致许多严重的疾病,严重威胁人类健康。使用抗病毒药物是预防与治疗病毒感染的主要策略,芽胞杆菌(Bacillus)及其代谢物能够抑制多种包膜病毒的感染。本文综述了芽胞杆菌代谢的粗提物、肽、酶、胞外聚合物、小双链RNA和热灭活的枯草芽胞杆菌孢子在抗包膜病毒感染中发挥的重要作用,其机制是通过直接破坏病毒包膜、阻止膜融合、与病毒基因组RNA直接配对、催化裂解病毒RNA、激活天然免疫反应等对抗病毒,期望为包膜病毒的持续预防和治疗提供参考。     

Mechanisms of inactivation of poliovirus by chlorine dioxide and iodine.

Chlorine dioxide and iodine inactivated poliovirus more efficiently at pH 10.0 than at pH 6.0. Sedimentation analyses of viruses inactivated by chlorine dioxide and iodine at pH 10.9 showed that viral RNA separated from the capsids, resulting in the conversion of virions from 156S structures to 80S particles. The RNAs release from both chlorine dioxide- and iodine-inactivated viruses cosedimented with intact 35S viral RNA. Both chlorine dioxide and iodine reacted with the capsid proteins of poliovirus and changed the pI from pH 7.0 to pH 5.8. However, the mechanisms of inactivation of poliovirus by chlorine dioxide and iodine were found to differ. Iodine inactivated viruses by impairing their ability to adsorb to HeLa cells, whereas chlorine dioxide-inactivated viruses showed a reduced incorporation of [14C]uridine into new viral RNA. We concluded, then, that chlorine dioxide inactivated poliovirus by reacting with the viral RNA and impairing the ability of the viral genome to act as a template for RNA synthesis.     

Virus inactivation in bone tissue transplants (femoral heads) by moist heat with the 'Marburg bone bank system'

Several virus inactivation procedures like heat treatment, gamma irradiation and chemical sterilization are used to increase the safety of bone tissue transplants. In this study we present data on the virus-inactivating effect of heat disinfection on human femoral heads, using the Marburg bone bank system 'Lobator sd-2'. Three enveloped viruses (human immunodeficiency virus type 2 [HIV-2], bovine viral diarrhoea virus as a model for Hepatitis C virus [HCV], and the herpesvirus pseudorabies virus), and three non-enveloped viruses (hepatitis A virus, poliomyelitis virus, and bovine parvovirus) were investigated.In a model system the central part of human femoral heads was contaminated with the respective cell-free virus suspension, establishing a direct contact between virus and native bone tissue. The core temperature in the femoral heads during the sterilization process was determined in additional model experiments. A temperature of 82.5 degrees C, given by the manufacturer as the effective temperature for virus inactivation, was maintained for at least 15 min in decartilaged femoral heads with a diameter of < or = 56 mm. Heat treatment using the Lobator sd-2 inactivated all viruses in human femoral heads below the detection limit (at least by a factor of > or =4 log(10)).By combining a well-focussed anamnesis of the donors and serological testing for relevant infection markers (anti-HIV-1/2, HBsAg, anti-HBcore, anti-HCV, TPHA) with heat treatment of femoral heads in the Lobator sd-2 system, a high safety level is achieved. To further increase virus safety of cadaveric bone transplants, it is recommended that multi-organ donors are tested by nucleic acid testing (i.e. polymerase chain reaction) for HIV, HBV and HCV genome.