Efficacy of chemical disinfectants against snakehead rhabdovirus

The susceptibility of snakehead rhabdovirus to treatment at 20°C with 5 commercially available disinfectants was examined. No reduction in virus infectivity occurred following exposure to 5 ppm malachite green for 6 hours. Treatment of infective cell culture fluids with 2% formalin resulted in > 99.9% reduction in virus titre within 5 minutes and complete inactivation within 30 minutes, but a negligible loss in infectivity after exposure to 0.025% formalin for 1 hour. Suspensions of virus in distilled water were completely inactivated within 5 minutes by 12.5 ppm chlorine, 50 ppm iodine, or a 1:2000 dilution of a peroxygen disinfectant. In the presence of serum in infective cell culture fluids, however, > 50 ppm chlorine was required to inactivate the agent and no measurable reduction in infectivity was observed following treatment with 500 ppm iodine for 30 minutes.     

Inactivation of human and simian rotaviruses by chlorine dioxide

The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4 degrees C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10(5)-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate at neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants.     

Inactivation of human and simian rotaviruses by chlorine dioxide.

The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4 degrees C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10(5)-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate at neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants.     

Stability of snakehead (Ophiocephalus striatus) rhabdovirus under different environmental conditions

Snakehead rhabdovirus stored in cell culture medium was found to be relatively temperature resistant with ≥ 99.9% reduction in infectivity only occurring after 6 months at 15°C, 6 weeks at 25°C and 10 days at 35°C. At 25°C, virus stability was greatest in the range pH 5–7. Infectivity was less well retained at pH9 and rapidly lost at pH3 and pH11. Stability was poor in serum-free media with a 99.9% reduction in infectivity occurring in less than 48 hours at 25°C in de-ionized water, tap water, balanced salt solutions and artificial seawater.     

Absence of XMRV in Peripheral Blood Mononuclear Cells of ARV-Treatment Na?ve HIV-1 Infected and HIV-1/HCV Coinfected Individuals and Blood Donors

Background

Xenotropic murine leukemia virus-related virus (XMRV) has been found in the prostatic tissue of prostate cancer patients and in the blood of chronic fatigue syndrome patients. However, numerous studies have found little to no trace of XMRV in different human cohorts. Based on evidence suggesting common transmission routes between XMRV and HIV-1, HIV-1 infected individuals may represent a high-risk group for XMRV infection and spread.

Methodology/Principal Findings

DNA was isolated from the peripheral blood mononuclear cells (PBMCs) of 179 HIV-1 infected treatment naïve patients, 86 of which were coinfected with HCV, and 54 healthy blood donors. DNA was screened for XMRV provirus with two sensitive, published PCR assays targeting XMRV gag and env and one sensitive, published nested PCR assay targeting env. Detection of XMRV was confirmed by DNA sequencing. One of the 179 HIV-1 infected patients tested positive for gag by non-nested PCR whereas the two other assays did not detect XMRV in any specimen. All healthy blood donors were negative for XMRV proviral sequences. Sera from 23 HIV-1 infected patients (15 HCV⁺) and 12 healthy donors were screened for the presence of XMRV-reactive antibodies by Western blot. Thirteen sera (57%) from HIV-1⁺ patients and 6 sera (50%) from healthy donors showed reactivity to XMRV-infected cell lysate.

Conclusions/Significance

The virtual absence of XMRV in PBMCs suggests that XMRV is not associated with HIV-1 infected or HIV-1/HCV coinfected patients, or blood donors. Although we noted isolated incidents of serum reactivity to XMRV, we are unable to verify the antibodies as XMRV specific.     

Absence of detectable XMRV and other MLV-related viruses in healthy blood donors in the United States

Background

Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4–6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern.

Methodology/Principal Findings

To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus.

Conclusions/Significance

Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied.     

Survival of human immunodeficiency virus (HIV), HIV-infected lymphocytes, and poliovirus in water.

The potential for human immunodeficiency virus (HIV) to enter domestic sewers via contaminated body fluids such as blood has spurred interest in the survival of this virus in water and wastewater. This study focused on establishing the inactivation of HIV and productively infected lymphocytes in dechlorinated tap water. In addition, HIV survival was compared with that of poliovirus. Results indicated that either free HIV or cell-associated HIV was rapidly inactivated, with a 90% loss of infectivity within 1 to 2 h at 25 degrees C and a 99.9% loss by 8 h. In comparison, poliovirus showed no loss of infectivity over 24 h. The presence of human serum in tap water slowed the rate of HIV inactivation through 8 h but did not stabilize the virus through 24 h. In addition, blood from stage IV AIDS patients was introduced into tap water, and the recovery of HIV was monitored by using both an infectivity assay and polymerase chain reaction amplification of viral sequences. Virally infected cells were no longer detectable after 5 min in dechlorinated tap water, while little diminution in amplifiable sequences was observed over 2 h. Thus, detection of viral sequences by polymerase chain reaction technology should not be equated with risk of exposure to infectious HIV.     

Lack of Detection of XMRV in Seminal Plasma from HIV-1 Infected Men in The Netherlands

Background

Xenotropic murine leukaemia virus-related virus (XMRV) is a recently discovered human gammaretrovirus with yet unknown prevalence and transmission route(s). Its presence in prostate stromal fibroblasts and prostatic secretions suggests that XMRV might be sexually transmitted. We chose to study a compartment closely connected to the prostate, a location where XMRV was detected in independent studies. Seminal plasma samples from HIV-1 infected men were examined as they have an increased probability of acquiring sexually transmitted pathogens.

Methodology/Principal Findings

We studied the prevalence of XMRV in 93 seminal plasma samples of 54 HIV-1 infected men living in The Netherlands with a nested PCR amplification specifically targeting the XMRV gag gene. As a control for the presence and integrity of retrovirus particles, HIV-1 was amplified from the same samples with a PCR amplification targeting the env gene of the virus, or HIV-1 was quantified with a real-time PCR amplifying part of the pol gene.

Conclusions/Significance

Although HIV-1 was amplified from 25% of the seminal plasma samples, no XMRV was detected, suggesting that either the prevalence of XMRV is very low in The Netherlands, or that XMRV is not naturally present in the seminal plasma.     

Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of Cryptosporidium parvum Oocysts

Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4′,6′-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.     

Stability of Minute Virus of Mice to Chemical and Physical Agents

Minute virus of mice (MVM), a single-stranded deoxyribonucleic acid Parvovirus, was subjected to various inactivation procedures, including chemical disinfectants, heat, and ultraviolet radiation. MVM was found to be less stable than has been reported for other Parvoviruses. This virus was readily inactivated by a variety of chemical disinfectants, including alcohols, formaldehyde, glutaraldehyde, and chloroform. MVM was more sensitive to ultraviolet radiation than was Kilham's rat virus. MVM was more sensitive to heating at temperatures of 35 to 100 C than has been reported for other Parvoviruses. More than 95% of MVM infectivity was inactivated by heating (45, 60, or 100 C) for 60 min, acid (pH 2.0) treatment, or ultraviolet radiation treatment, although a small percentage (less than 2%) of the virus preparation was found to be resistant to these treatments. In addition, more than 99% of the infectivity of MVM was lost after storage at 4C for 10 weeks, although the virus was stable on storage in liquid nitrogen.     

Apobec 3G Efficiently Reduces Infectivity of the Human Exogenous Gammaretrovirus XMRV

Background

The human exogenous gammaretrovirus XMRV is thought to be implicated in prostate cancer and chronic fatigue syndrome. Besides pressing epidemiologic questions, the elucidation of the tissue and cell tropism of the virus, as well as its sensitivity to retroviral restriction factors is of fundamental importance. The Apobec3 (A3) proteins, a family of cytidine deaminases, are one important group of host proteins that control primary infection and efficient viral spread.

Methodology/Principal Findings

Here we demonstrate that XMRV is resistant to human Apobec 3B, 3C and 3F, while being highly susceptible to the human A3G protein, a factor which is known to confer antiviral activity against most retroviruses. We show that XMRV as well as MoMLV virions package Apobec proteins independent of their specific restriction activity. hA3G was found to be a potent inhibitor of XMRV as well as of MoMLV infectivity. In contrast to MoMLV, XMRV infection can also be partially reduced by low concentrations of mA3. Interestingly, established prostate cancer cell lines, which are highly susceptible to XMRV infection, do not or only weakly express hA3G.

Conclusions

Our findings confirm and extend recently published data that show restriction of XMRV infection by hA3G. The results will be of value to explore which cells are infected with XMRV and efficiently support viral spread in vivo. Furthermore, the observation that XMRV infection can be reduced by mA3 is of interest with regard to the current natural reservoir of XMRV infection.     

No evidence for XMRV in German CFS and MS patients with fatigue despite the ability of the virus to infect human blood cells in vitro

Background

Xenotropic murine leukemia virus-related virus (XMRV), a novel human retrovirus originally identified in prostate cancer tissues, has recently been associated with chronic fatigue syndrome (CFS), a disabling disease of unknown etiology affecting millions of people worldwide. However, several subsequent studies failed to detect the virus in patients suffering from these illnesses or in healthy subjects. Here we report the results of efforts to detect antibody responses and viral sequences in samples from a cohort of German CFS and relapsing remitting multiple sclerosis (MS) patients with fatigue symptoms.

Methodology

Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors. Fatigue severity in MS patients was assessed using the Fatigue Severity Scale (FSS). Validated Gag- and Env-ELISA assays were used to screen sera for XMRV antibodies. PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene. In addition, PBMC cultures were exposed to 22Rv1-derived XMRV to assess infectivity and virus production.

Conclusion

None of the screened sera from CFS and MS patients or healthy blood donors tested positive for XMRV specific antibodies and all PBMC (and PBMC plus LNCaP) cultures remained negative for XMRV sequences by nested PCR. These results argue against an association between XMRV infection and CFS and MS in Germany. However, we could confirm that PBMC cultures from healthy donors and from CFS patients can be experimentally infected by XMRV, resulting in the release of low levels of transmittable virus.     

Cholesterol Depletion Inactivates XMRV and Leads to Viral Envelope Protein Release from Virions: Evidence for Role of Cholesterol in XMRV Infection

Membrane cholesterol plays an important role in replication of HIV-1 and other retroviruses. Here, we report that the gammaretrovirus XMRV requires cholesterol and lipid rafts for infection and replication. We demonstrate that treatment of XMRV with a low concentration (10 mM) of 2-hydroxypropyl-β-cyclodextrin (2OHpβCD) partially depleted virion-associated cholesterol resulting in complete inactivation of the virus. This effect could not be reversed by adding cholesterol back to treated virions. Further analysis revealed that following cholesterol depletion, virus-associated Env protein was significantly reduced while the virions remained intact and retained core proteins. Increasing concentrations of 2OHpβCD (≥20 mM) resulted in loss of the majority of virion-associated cholesterol, causing disruption of membrane integrity and loss of internal Gag proteins and viral RNA. Depletion of cholesterol from XMRV-infected cells significantly reduced virus release, suggesting that cholesterol and intact lipid rafts are required for the budding process of XMRV. These results suggest that unlike glycoproteins of other retroviruses, the association of XMRV glycoprotein with virions is highly dependent on cholesterol and lipid rafts.     

High Humidity Leads to Loss of Infectious Influenza Virus from Simulated Coughs

Background

The role of relative humidity in the aerosol transmission of influenza was examined in a simulated examination room containing coughing and breathing manikins.

Methods

Nebulized influenza was coughed into the examination room and Bioaerosol samplers collected size-fractionated aerosols (<1 µM, 1–4 µM, and >4 µM aerodynamic diameters) adjacent to the breathing manikin’s mouth and also at other locations within the room. At constant temperature, the RH was varied from 7–73% and infectivity was assessed by the viral plaque assay.

Results

Total virus collected for 60 minutes retained 70.6–77.3% infectivity at relative humidity ≤23% but only 14.6–22.2% at relative humidity ≥43%. Analysis of the individual aerosol fractions showed a similar loss in infectivity among the fractions. Time interval analysis showed that most of the loss in infectivity within each aerosol fraction occurred 0–15 minutes after coughing. Thereafter, losses in infectivity continued up to 5 hours after coughing, however, the rate of decline at 45% relative humidity was not statistically different than that at 20% regardless of the aerosol fraction analyzed.

Conclusion

At low relative humidity, influenza retains maximal infectivity and inactivation of the virus at higher relative humidity occurs rapidly after coughing. Although virus carried on aerosol particles <4 µM have the potential for remaining suspended in air currents longer and traveling further distances than those on larger particles, their rapid inactivation at high humidity tempers this concern. Maintaining indoor relative humidity >40% will significantly reduce the infectivity of aerosolized virus.     

Evolution of the HIV-1 nef gene in HLA-B*57 Positive Elite Suppressors

During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4% identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6%) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6%) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests.     

Construction and applications of SARS-CoV-2 pseudoviruses: a mini review

The ongoing coronavirus disease 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed a serious threat to global public health and social stability. There is an urgent need for understanding the nature and infection mechanism of the virus. Owing to its high infectivity and pathogenicity and lack of effective treatments, live SARS-CoV-2 has to be handled in biosafety level 3 laboratories, which has impeded research into SARS-CoV-2 and the development of vaccines and therapeutics. Pseudotyped viruses that lack certain gene sequences of the virulent virus are safer and can be investigated in biosafety level 2 laboratories, providing a useful virological tool for the study of SARS-CoV-2. In this review, we will discuss the construction of SARS-CoV-2 pseudoviruses based on different packaging systems, current applications, limitations, and further explorations.     

The Prostate Cancer-Associated Human Retrovirus XMRV Lacks Direct Transforming Activity but Can Induce Low Rates of Transformation in Cultured Cells

The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, but a causal relationship has not been established. Here, we have used cultured fibroblast and epithelial cell lines to test the hypothesis that XMRV might have direct transforming activity but found only rare transformation events, suggestive of indirect transformation, even when the target cells expressed the human Xpr1 cell entry receptor for XMRV. Characterization of cells from three transformed foci showed that all were infected with and produced XMRV, and one produced a highly active transforming virus, presumably generated by recombination between XMRV and host cell nucleic acids. Given the sequence similarity of XMRV to mink cell focus-forming (MCF) viruses and the enhanced leukemogenic activity of the latter, we tested XMRV for related MCF-like cytopathic activities in cultured mink cells but found none. These results indicate that XMRV has no direct transforming activity but can activate endogenous oncogenes, resulting in cell transformation. As part of these experiments, we show that XMRV can infect and be produced at a high titer from human HT-1080 fibrosarcoma cells that express TRIM5α (Ref1), showing that XMRV is resistant to TRIM5α restriction. In addition, XMRV poorly infects NIH 3T3 cells expressing human Xpr1 but relatively efficiently infects BALB 3T3 cells expressing human Xpr1, showing that XMRV is a B-tropic virus and that its infectivity is regulated by the Fv1 mouse locus.The association of human prostate cancer with mutations that impair the function of the antiviral defense protein RNase L suggested a role for virus in prostate cancer. Indeed, analysis of cDNA from prostate tumors by use of a DNA microarray (Virochip) containing conserved DNA sequences from all known virus families indicated the presence of a novel gammaretrovirus in 40% of prostate cancer patients having homozygous R462Q mutations in RNase L (35). Cloning and sequencing of the virus revealed a close similarity to mouse xenotropic retroviruses; thus, the new virus was named XMRV (xenotropic murine leukemia virus-related virus) (35). Importantly, XMRV has been found integrated into human genomic DNA from tumor-bearing prostatic tissue samples of 11 patients, showing that XMRV can indeed infect humans and is not a laboratory contaminant (7, 13). Although an initial study found XMRV only in tumor stromal cells (35), recent studies have found XMRV in the prostate carcinoma cell line 22Rv1 (14) and in malignant epithelial cells in prostate tumors (34).XMRV lacks a host cell-derived oncogene, but examples of oncogenic activity in Env proteins from other retroviruses (1, 6, 16, 24) raise the possibility that the Env protein of XMRV might also be oncogenic. Such activity could be a result of interaction of the XMRV Env protein with the virus entry receptor Xpr1 (7, 14), which shows similarity to a yeast protein involved in G protein-coupled signal transduction (2), or interaction with other cellular proteins that do not function as virus entry receptors, as is the case for jaagsiekte sheep retrovirus (JSRV) Env (interacting protein unknown) (16) and the Env protein of spleen focus-forming virus, which interacts with and activates the erythropoietin receptor and the receptor tyrosine kinase Stk (24). Detection of XMRV oncogenic activity would strengthen the argument for a role for XMRV in prostate cancer.In addition, while XMRV shows the highest sequence similarity to the mouse xenotropic retroviruses, it is also similar to the mink cell focus-forming (MCF) retroviruses of mice, which are highly leukemogenic due to their ability to multiply reinfect cells, leading to more-frequent activation of cellular oncogenes (36). MCF viruses were first defined by their ability to induce foci of altered cells in mink cell layers (11). Initially, it was unclear whether these foci were the result of cell transformation or cytopathic effects of the virus (11), but it is clear now that these foci result from cytopathic effects related to the ability of MCF viruses to multiply reinfect cells in what can be a receptor-independent manner, leading to cell apoptosis (23, 36, 37). It was thus important to determine if XMRV has similar properties and might be able to more frequently activate cellular oncogenes.Here, we have found that while XMRV lacks direct transforming activity in the fibroblast and epithelial cell lines tested and does not induce cytopathic effects typical of multiple reinfection by MCF viruses, it is able to induce rare transformed foci in a rat fibroblast cell line. Interestingly, in one case, transformation led to the production of a highly active oncogenic retrovirus.