Resistance of Aerosolized Bacterial Viruses to Relative Humidity and Temperature

The use of aerosolized bacteriophages as surrogates for hazardous viruses might simplify and accelerate the discovery of links between viral components and their persistence in the airborne state under diverse environmental conditions. In this study, four structurally distinct lytic phages, MS2 (single-stranded RNA [ssRNA]), ϕ6 (double-stranded RNA [dsRNA]), ϕX174 (single-stranded DNA [ssDNA]), and PR772 (double-stranded DNA [dsDNA]), were nebulized into a rotating chamber and exposed to various levels of relative humidity (RH) and temperature as well as to germicidal UV radiation. The aerosolized viral particles were allowed to remain airborne for up to 14 h before being sampled for analysis by plaque assays and quantitative PCRs. Phages ϕ6 and MS2 were the most resistant at low levels of relative humidity, while ϕX174 was more resistant at 80% RH. Phage ϕ6 lost its infectivity immediately after exposure to 30°C and 80% RH. The infectivity of all tested phages rapidly declined as a function of the exposure time to UVC radiation, phage MS2 being the most resistant. Taken altogether, our data indicate that these aerosolized phages behave differently under various environmental conditions and highlight the necessity of carefully selecting viral simulants in bioaerosol studies.     

Solar Disinfection of Viruses in Polyethylene Terephthalate Bottles

Solar disinfection (SODIS) of drinking water in polyethylene terephthalate (PET) bottles is a simple, efficient point-of-use technique for the inactivation of many bacterial pathogens. In contrast, the efficiency of SODIS against viruses is not well known. In this work, we studied the inactivation of bacteriophages (MS2 and ϕX174) and human viruses (echovirus 11 and adenovirus type 2) by SODIS. We conducted experiments in PET bottles exposed to (simulated) sunlight at different temperatures (15, 22, 26, and 40°C) and in water sources of diverse compositions and origins (India and Switzerland). Good inactivation of MS2 (>6-log inactivation after exposure to a total fluence of 1.34 kJ/cm²) was achieved in Swiss tap water at 22°C, while less-efficient inactivation was observed in Indian waters and for echovirus (1.5-log inactivation at the same fluence). The DNA viruses studied, ϕX174 and adenovirus, were resistant to SODIS, and the inactivation observed was equivalent to that occurring in the dark. High temperatures enhanced MS2 inactivation substantially; at 40°C, 3-log inactivation was achieved in Swiss tap water after exposure to a fluence of only 0.18 kJ/cm². Overall, our findings demonstrate that SODIS may reduce the load of single-stranded RNA (ssRNA) viruses, such as echoviruses, particularly at high temperatures and in photoreactive matrices. In contrast, complementary measures may be needed to ensure efficient inactivation during SODIS of DNA viruses resistant to oxidation.     

Evaluation of Murine Norovirus, Feline Calicivirus, Poliovirus, and MS2 as Surrogates for Human Norovirus in a Model of Viral Persistence in Surface Water and Groundwater

Human noroviruses (NoVs) are a significant cause of nonbacterial gastroenteritis worldwide, with contaminated drinking water a potential transmission route. The absence of a cell culture infectivity model for NoV necessitates the use of molecular methods and/or viral surrogate models amenable to cell culture to predict NoV inactivation. The NoV surrogates murine NoV (MNV), feline calicivirus (FCV), poliovirus (PV), and male-specific coliphage MS2, in conjunction with Norwalk virus (NV), were spiked into surface water samples (n = 9) and groundwater samples (n = 6). Viral persistence was monitored at 25°C and 4°C by periodically analyzing virus infectivity (for all surrogate viruses) and nucleic acid (NA) for all tested viruses. FCV infectivity reduction rates were significantly higher than those of the other surrogate viruses. Infectivity reduction rates were significantly higher than NA reduction rates at 25°C (0.18 and 0.09 log₁₀/day for FCV, 0.13 and 0.10 log₁₀/day for PV, 0.12 and 0.06 log₁₀/day for MS2, and 0.09 and 0.05 log₁₀/day for MNV) but not significant at 4°C. According to a multiple linear regression model, the NV NA reduction rates (0.04 ± 0.01 log₁₀/day) were not significantly different from the NA reduction rates of MS2 (0.05 ± 0.03 log₁₀/day) and MNV (0.04 ± 0.03 log₁₀/day) and were significantly different from those of FCV (0.08 ± 0.03 log₁₀/day) and PV (0.09 ± 0.03 log₁₀/day) at 25°C. In conclusion, MNV shows great promise as a human NoV surrogate due to its genetic similarity and environmental stability. FCV was much less stable and thus questionable as an adequate surrogate for human NoVs in surface water and groundwater.     

Effect of Temperature and Relative Humidity on the Survival of Foodborne Viruses during Food Storage

Millions of people suffer from foodborne diseases throughout the world every year, and the importance of food safety has grown worldwide in recent years. The aim of this study was to investigate the survival of hepatitis A virus (HAV) and viral surrogates of human norovirus (HuNoV) (bacteriophage MS2 and murine norovirus [MNV]) in food over time. HAV, MNV, and MS2 were inoculated onto either the digestive gland of oysters or the surface of fresh peppers, and their survival on these food matrices was measured under various temperature (4°C, 15°C, 25°C, and 40°C) and relative humidity (RH) (50% and 70%) conditions. Inoculated viruses were recovered from food samples and quantified by a plaque assay at predetermined time points over 2 weeks (0, 1, 3, 7, 10, and 14 days). Virus survival was influenced primarily by temperature. On peppers at 40°C and at 50% RH, >4- and 6-log reductions of MNV and HAV, respectively, occurred within 1 day. All three viruses survived better on oysters. In addition, HAV survived better at 70% RH than at 50% RH. The survival data for HAV, MS2, and MNV were fit to three different mathematical models (linear, Weibull, and biphasic models). Among them, the biphasic model was optimum in terms of goodness of fit. The results of this study suggest that major foodborne viruses such as HAV and HuNoV can survive over prolonged periods of time with a limited reduction in numbers. Because a persistence of foodborne virus on contaminated foods was observed, precautionary preventive measures should be performed.     

Feline Calicivirus,Murine Norovirus,Porcine Sapovirus,and Tulane Virus Survival on Postharvest Lettuce

Human norovirus (HuNoV) is the leading cause of foodborne illnesses, with an increasing number of outbreaks associated with leafy greens. Because HuNoV cannot be routinely cultured, culturable feline calicivirus (FCV), murine norovirus (MNV), porcine sapovirus (SaV), and Tulane virus (TV) have been used as surrogates. These viruses are generated in different cell lines as infected cell lysates, which may differentially affect their stability. Our objective was to uniformly compare the survival of these viruses on postharvest lettuce while evaluating the effects of cell lysates on their survival. Viruses were semipurified from cell lysates by ultrafiltration or ultracentrifugation followed by resuspension in sterile water. Virus survival was examined before and after semipurification: in suspension at room temperature (RT) until day 28 and on lettuce leaves stored at RT for 3 days or at 4°C for 7 and 14 days. In suspension, both methods significantly enhanced the survival of all viruses. On lettuce, the survival of MNV in cell lysates was similar to that in water, under all storage conditions. In contrast, the survival of FCV, SaV, and TV was differentially enhanced, under different storage conditions, by removing cell lysates. Following semipurification, viruses showed similar persistence to each other on lettuce stored under all conditions, with the exception of ultracentrifugation-purified FCV, which showed a higher inactivation rate than MNV at 4°C for 14 days. In conclusion, the presence of cell lysates in viral suspensions underestimated the survivability of these surrogate viruses, while viral semipurification revealed similar survivabilities on postharvest lettuce leaves.     

Survival of Murine Norovirus,Tulane Virus,and Hepatitis A Virus on Alfalfa Seeds and Sprouts during Storage and Germination

Human norovirus (huNoV) and hepatitis A virus (HAV) have been involved in several produce-associated outbreaks and identified as major food-borne viral etiologies. In this study, the survival of huNoV surrogates (murine norovirus [MNV] and Tulane virus [TV]) and HAV was investigated on alfalfa seeds during storage and postgermination. Alfalfa seeds were inoculated with MNV, TV, or HAV with titers of 6.46 ± 0.06 log PFU/g, 3.87 ± 0.38 log PFU/g, or 7.01 ± 0.07 log 50% tissue culture infectious doses (TCID₅₀)/g, respectively. Inoculated seeds were stored for up to 50 days at 22°C and sampled during that storage period on days 0, 2, 5, 10, and 15. Following storage, virus presence was monitored over a 1-week germination period. Viruses remained infectious after 50 days, with titers of 1.61 ± 0.19 log PFU/g, 0.85 ± 0.21 log PFU/g, and 3.43 ± 0.21 log TCID₅₀/g for MNV, TV, and HAV, respectively. HAV demonstrated greater persistence than MNV and TV, without a statistically significant reduction over 20 days (<1 log TCID₅₀/g); however, relatively high levels of genomic copies of all viruses persisted over the testing time period. Low titers of viruses were found on sprouts and were located in all tissues as well as in sprout-spent water sampled on days 1, 3, and 6 following seed planting. Results revealed the persistence of viruses in seeds for a prolonged period of time, and perhaps of greater importance these data suggest the ease of which virus may transfer from seeds to sprouts and spent water during germination. These findings highlight the importance of sanitation and prevention procedures before and during germination.     

Comparison of Five Bacteriophages as Models for Viral Aerosol Studies

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols.     

The Fitness Effects of Random Mutations in Single-Stranded DNA and RNA Bacteriophages

Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment in which we subjected three ssDNA (ΦX174, G4, F1) and three ssRNA phages (Qβ, MS2, and SP) to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups. Second, we used site-directed mutagenesis to obtain 45 clones of ΦX174 and 42 clones of Qβ carrying random single-nucleotide substitutions and assayed them for fitness. In ΦX174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Qβ, these figures were 29% and 10%, respectively. It seems therefore that high mutational sensitivity is a general property of viruses with small genomes, including those infecting animals, plants, and bacteria. Mutational fitness effects are important for understanding processes of fitness decline, but also of neutral evolution and adaptation. As such, these findings can contribute to explain the evolution of ssDNA and ssRNA viruses.     

Effect of Weak Acid Hypochlorous Solution on Selected Viruses and Bacteria of Laboratory Rodents

Weak acid hypochlorous solution (WAHS) is known to have efficacy for inactivating pathogens and to be relatively safe with respect to the live body. Based on these advantages, many animal facilities have recently been introducing WAHS for daily cleaning of animal houses. In this study, we determined the effect of WAHS in inactivating specific pathogens of laboratory rodents and pathogens of opportunistic infection. WAHS with an actual chloride concentration of 60 ppm and a pH value of 6.0 was generated using purpose-built equipment. One volume of mouse hepatitis virus (MHV), Sendai virus, lymphocytic choriomeningitis virus, Bordetella bronchiseptica, Pasteurella pneumotropica, Corynebacterium kutscheri, Staphylococcus aureus, and Pseudomonas aeruginosa was mixed with 9 or 99 volumes of WAHS (×10 and ×100 reaction) for various periods (0.5, 1, and 5 min) at 25°C. After incubation, the remaining infectious viruses and live bacteria were determined by plaque assay or culture. In the ×100 reaction mixture, infectious viruses and live bacteria could not be detected for any of the pathogens examined even with the 0.5-min incubation. However, the effects for MHV, B. bronchiseptica, and P. aeruginosa were variable in the ×10 reaction mixture with the 0.5- and 1-min incubations. Sufficient effects were obtained by elongation of the reaction time to 5 min. In the case of MHV, reducing organic substances in the virus stock resulted in the WAHS being completely effective. WAHS is recommended for daily cleaning in animal facilities but should be used properly in order to obtain a sufficient effect, which includes such things as using a large enough volume to reduce effects of organic substances.     

An Inexpensive,Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 10⁷ viruses ml⁻¹. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 10⁶ to 1.37 × 10⁸ viruses ml⁻¹ when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 10⁶ viruses ml⁻¹) encountered in field and laboratory samples.     

The Adsorption of Bacteriophage ϕX174 to its Host

The adsorption of purified ϕX174 to E. coli C and to E. coli C cell walls was investigated. Adsorption was measured by assaying for unadsorbed plaque formers. The amount of irreversible and reversible adsorption depends upon pH and divalent ion concentration. Maximum irreversible adsorption occurs in 0.1 M CaCl₂ at 36°C. There is no detectable reversible adsorption at conditions of pH and CaCl₂ concentration optimum for irreversible adsorption. Under these optimum conditions, diffusion is not the rate-limiting factor, and the encounter efficiency appears to be low. The rate constant is 1.0 × 10^-10 ml/sec. Phages adsorbed irreversibly to live cells cause infection and to the isolated cell walls apparently cause release of DNA. There is a specific ϕX174 receptor site on the mucocomplex portion of the cell wall.     

Possible Role of a Cell Surface Carbohydrate in Evolution of Resistance to Viral Infections in Old World Primates

Due to inactivation of the α1,3-galactosyltransferase gene (GGTA1, or the α1,3GT gene) approximately 28 million years ago, the carbohydrate αGal (Galα1,3Galβ1,4GlcNAc) is not expressed on the cells of Old World monkeys and apes (including humans) but is expressed in all other mammals. The proposed selective advantage of this mutation for these primates is the ability to produce anti-Gal antibodies, which may be an effective immune component in neutralizing αGal-expressing pathogens. However, loss of α1,3GT expression may have been advantageous by providing natural resistance against viral pathogens that exploited the α1,3GT pathway or cell surface αGal for infection. Infections of paired cell lines with differential expression of α1,3GT showed that Sindbis viruses (SINV) preferentially replicate in α1,3GT-positive cells, whereas herpes simplex viruses type 1 and type 2 (HSV-1 and HSV-2) preferentially grow in cells lacking α1,3GT. Viral growth and spread correlated with the ability of the different viruses to successfully initiate infection in the presence or absence of α1,3GT expression. GT knockout (KO) suckling mice infected with SINV strains (AR339 and S.A.AR86) experienced significant delay in onset of disease symptoms and mortality compared to wild-type (WT) B6 suckling mice. In contrast, HSV-2-infected GT KO mice had higher viral titers in spleen and liver and exhibited significantly more focal hepatic necrosis than WT B6 mice. This study demonstrates that α1,3GT activity plays a role in the course of infections for certain viruses. Furthermore, this study has implications for the evolution of resistance to viral infections in primates.     

Kinetics of Transmission, Infectivity, and Genome Stability of Two Novel Mouse Norovirus Isolates in Breeding Mice

Murine noroviruses are a recently discovered group of viruses found within mouse research colonies in many animal facilities worldwide. In this study, we used 2 novel mouse norovirus (MNV) wildtype isolates to examine the kinetics of transmission and tissue distribution in breeding units of NOD.CB17-Prkdc^scid/J and backcrossed NOD.CB17-Prkdc^scid/J × NOD/ShiLtJ (N1) mice. Viral shedding in feces and dissemination to tissues of infected offspring mice were monitored by RT-PCR over a 6-wk period postpartum. Histologic sections of tissues from mice exposed to MNV were examined for lesions and their sera monitored for the presence of antibodies to MNV. Viruses shed in feces of parental and offspring mice were compared for sequence homology of the Orf2 gene. Studies showed that the wildtype viruses MNV5 and MNV6 behaved differently in terms of the kinetics of transmission and distribution to tissues of offspring mice. For MNV5, virus transmission from parents to offspring was not seen before 3 wk after birth, and neither isolate was transmitted between cages of infected and control mice. Susceptibility to infection was statistically different between the 2 mouse strains used in the study. Both immunodeficient NOD.CB17-Prkdc^scid/J mice and NOD.CB17-Prkdc^scid/J × NOD/ShiLtJ offspring capable of mounting an immune response shed virus in their feces throughout the 6-wk study period, but no gross or histologic lesions were present in infected tissues. Progeny viruses isolated from the feces of infected offspring showed numerous mutations in the Orf2 gene for MNV5 but not MNV6. These results confirm previous studies demonstrating that the biology of MNV in mice varies substantially with each virus isolate and mouse strain infected.Abbreviations: MNV, murine norovirus; MLN, mesenteric lymph nodes; NOD-scid, NOD.CB17-Prkdc^scid/J; VP1, viral protein 1The recent discovery of murine-specific noroviruses¹⁵ has stimulated concern in the laboratory animal health community regarding the potential for this group of viruses to cause disease in breeding colonies of mice or to negatively impact research with mice from norovirus infected colonies. Current knowledge of the biology of noroviruses in mice (MNV) is constrained by the limited number of virus isolates and mouse strains studied. One study¹⁵described the biologic and physicochemical properties of the original MNV1 isolated from mice deficient in a specific innate immune function. More recently, this innate immune deficiency has been mapped to STAT1 regulation of IFNαβ secretion.²¹Previous work¹⁵ demonstrated that inoculation of MNV1 into mouse strains deficient in the acquired immune response (129 RAG 2^−/−, B6 RAG1^−/−) resulted in the development of persistent infections with no evidence of disease, whereas inoculation of fully immunocompetent mice (129S6/SvEvTac) resulted in rapid elimination of MNV1, with viral RNA undetectable in the viscera by 3 d after inoculation. More recently, infections of outbred immunocompetent mouse strains with 3 wildtype isolates of MNV obtained from different geographic areas of the United States have been described.¹¹ Virus was detected in the feces and tissue of infected mice throughout the 8-wk study, suggesting that some isolates of MNV may persistently infect immunocompetent mice.The purpose of the present investigation was to extend the current knowledge of MNV by using 2 isolates of the virus in mouse strains that have not been previously used as infection models for MNV. We examined natural virus transmission from infected breeders to offspring, kinetics of infection within litters of infected breeding mice, and the pathogenesis of infection in breeding colonies of mice. In addition, we examined the effect of virus passage from parents to offspring on genomic stability of these 2 viral isolates. Exposure of offspring of immunodeficient mice and immunocompetent mice to the 2 different isolates of MNV resulted in different patterns of virus transmission, susceptibility to infection and kinetics of infection as shown by the progressive spread of virus within litters and in intestinal and extraintestinal tissues. MNV was shed persistently in the feces of all mice tested regardless of immune status, and viral progeny isolated from offspring mice contained genome sequence differences from the parent virus in the Orf2 gene, an area of the MNV genome known to be susceptible to mutations.     

Microfluidic Quantitative PCR for Simultaneous Quantification of Multiple Viruses in Environmental Water Samples

To secure food and water safety, quantitative information on multiple pathogens is important. In this study, we developed a microfluidic quantitative PCR (MFQPCR) system to simultaneously quantify 11 major human viral pathogens, including adenovirus, Aichi virus, astrovirus, enterovirus, human norovirus, rotavirus, sapovirus, and hepatitis A and E viruses. Murine norovirus and mengovirus were also quantified in our MFQPCR system as a sample processing control and an internal amplification control, respectively. River water contaminated with effluents from a wastewater treatment plant in Sapporo, Japan, was collected and used to validate our MFQPCR system for multiple viruses. High-throughput quantitative information was obtained with a quantification limit of 2 copies/μl of cDNA/DNA. Using this MFQPCR system, we could simultaneously quantify multiple viral pathogens in environmental water samples. The viral quantities obtained using MFQPCR were similar to those determined by conventional quantitative PCR. Thus, the MFQPCR system developed in this study can provide direct and quantitative information for viral pathogens, which is essential for risk assessments.     

Silver Nanoparticles Decrease the Viability of Cryptosporidium parvum Oocysts

Oocysts of the waterborne protozoan parasite Cryptosporidium parvum are highly resistant to chlorine disinfection. We show here that both silver nanoparticles (AgNPs) and silver ions significantly decrease oocyst viability, in a dose-dependent manner, between concentrations of 0.005 and 500 μg/ml, as assessed by an excystation assay and the shell/sporozoite ratio. For percent excystation, the results are statistically significant for 500 μg/ml of AgNPs, with reductions from 83% for the control to 33% with AgNPs. For Ag ions, the results were statistically significant at 500 and 5,000 μg/ml, but the percent excystation values were reduced only to 66 and 62%, respectively, from 86% for the control. The sporozoite/shell ratio was affected to a greater extent following AgNP exposure, presumably because sporozoites are destroyed by interaction with NPs. We also demonstrated via hyperspectral imaging that there is a dual mode of interaction, with Ag ions entering the oocyst and destroying the sporozoites while AgNPs interact with the cell wall and, at high concentrations, are able to fully break the oocyst wall.     

Three-way interactions between mosquito population,viral strain and temperature underlying chikungunya virus transmission potential

Interactions between pathogens and their insect vectors in nature are under the control of both genetic and non-genetic factors, yet most studies on mosquito vector competence for human pathogens are conducted in laboratory systems that do not consider genetic and/or environmental variability. Evaluating the risk of emergence of arthropod-borne viruses (arboviruses) of public health importance such as chikungunya virus (CHIKV) requires a more realistic appraisal of genetic and environmental contributions to vector competence. In particular, sources of variation do not necessarily act independently and may combine in the form of interactions. Here, we measured CHIKV transmission potential by the mosquito Aedes albopictus in all combinations of six worldwide vector populations, two virus strains and two ambient temperatures (20°C and 28°C). Overall, CHIKV transmission potential by Ae. albopictus strongly depended on the three-way combination of mosquito population, virus strain and temperature. Such genotype-by-genotype-by-environment (G × G × E) interactions question the relevance of vector competence studies conducted with a simpler set of conditions. Our results highlight the need to account for the complex interplay between vectors, pathogens and environmental factors to accurately assess the potential of vector-borne diseases to emerge.     

Horizontal and Vertical Distribution of Marine Virioplankton: A Basin Scale Investigation Based on a Global Cruise

Despite the fact that marine viruses have been increasingly studied in the last decade, there is little information on viral abundance and distribution on a global scale. In this study, we report on a global-scale survey covering the Pacific, Atlantic, and Indian Oceans on viral distribution using flow cytometry. Viruses were stained with the SYBR Green I, which targets only dsDNA viruses. The average viral abundance was 1.10±0.73×10⁷ ml⁻¹ in global surface oceans and decreased from the areas with high chlorophyll concentration (on average, 1.47±0.78×10⁷ ml⁻¹) to the oligotrophic subtropical gyres (on average, 6.34±2.18×10⁶ ml⁻¹). On a large-spatial-scale, viruses displayed significant relationships with both heterotrophic and autotrophic picoplankton abundance, suggesting that viral distribution is dependent on their host cell abundance. Our study provided a basin scale pattern of marine viral distributions and their relationship with major host cells, indicating that viruses play a significant role in the global marine ecosystem.     

The μ1 72-96 Loop Controls Conformational Transitions during Reovirus Cell Entry

The reovirus outer capsid protein μ1 forms a lattice surrounding the viral core. In the native state, μ1 determines the environmental stability of the viral capsid. Additionally, during cell entry, μ1 undergoes structural rearrangements that facilitate delivery of the viral cores across the membrane. To determine how the capsid-stabilizing functions of μ1 impinge on the capacity of μ1 to undergo conformational changes required for cell entry, we characterized viruses with mutations engineered at charged residues within the μ1 loop formed by residues 72 to 96 (72-96 loop). This loop is proposed to stabilize the capsid by mediating interactions between neighboring μ1 trimers and between trimers and the core. We found that mutations at Glu89 (E89) within this loop produced viruses with compromised efficiency for completing their replication cycle. ISVPs of E89 mutants converted to ISVP*s more readily than those of wild-type viruses. The E89 mutants yielded revertants with second-site substitutions within regions that mediate interaction between μ1 trimers at a site distinct from the 72-96 loop. These viruses also contained changes in regions that control interactions within μ1 trimers. Viruses containing these second-site changes displayed restored plaque phenotypes and were capable of undergoing ISVP-to-ISVP* conversion in a regulated manner. These findings highlight regions of μ1 that stabilize the reovirus capsid and demonstrate that an enhanced propensity to form ISVP*s in an unregulated manner compromises viral fitness.     

Murine Norovirus Infection Has No Significant Effect on Adaptive Immunity to Vaccinia Virus or Influenza A Virus

Murine norovirus (MNV) is endemic in many research mouse colonies. Although MNV infections are typically asymptomatic in immunocompetent mice, the effects of MNV infection on subsequent experimental viral infections are poorly documented. Here, we infected C57BL/6 mice with MNV and then with either vaccinia virus or influenza A virus. MNV infection had no effect on CD8⁺ T-cell or antibody responses to secondary viruses or to secondary virus-induced morbidity or mortality. While our findings suggest that MNV has little influence on host immunity in immunocompetent mice, we would urge caution regarding the potential effects of MNV on immune responses to viruses and other pathogens, which must be determined on a system-by-system basis.Human norovirus (NoV) infections cause greater than 90% of nonbacterial gastroenteritis cases (4, 5) and are an important public health concern. Murine noroviruses (MNV) were recently identified (7) as highly pathogenic agents in immunocompromised mice, and serological studies indicate that over 20% of mice in research colonies are exposed to MNV (6). As with NoV, MNV is spread through the fecal-oral route. While NoV rapidly causes gastrointestinal symptoms and fever in healthy individuals, MNV is typically asymptomatic in immunocompetent mice.MNV isolates are both genetically and biologically diverse (13). In wild-type (wt) mice, some strains of MNV are rapidly cleared, while others persist (13). Controlling MNV infections requires elements of both innate and adaptive immunity. Mice with defects in interferon (IFN) signaling pathways demonstrate increased MNV lethality (7, 9). CD4⁺ and CD8⁺ T cells and B cells are all needed for complete MNV clearance (1, 2). Natural exposure of immunocompromised mice to MNV leads to inflammation of the liver, lungs, and peritoneal and pleural cavities (14).It is well established that infection with natural mouse viruses can greatly impact immune responses to infections with other viruses. The prevalence of MNV in research mouse colonies might therefore lead to irreproducible and variable results that significantly impact research efforts. Indeed, MNV was recently reported to alter disease progression in a mouse model of bacterium-induced inflammatory bowel disease (8). Concern over the potential effects of MNV on viral immunology research prompted a dedicated workshop at the 2008 Keystone Viral Immunity meeting (http://www.keystonesymposia.org). In the present study, we examined the effect of MNV infection on adaptive immune responses in wt mice to influenza A virus (IAV) and vaccinia virus (VV).We infected C57BL/6 mice perorally with a high dose (3 × 10⁷ PFU/mouse) of a plaque-purified MNV stock derived from MNV-CR6p2 (13). The capacity of this plaque-purified virus to persist in wt mice has been confirmed by quantitative PCR analysis and a plaque assay (D. Strong, L. Thackray, and H. Virgin, unpublished observation). We confirmed that the mice were infected by measuring anti-MNV antibodies (Abs) by using an enzyme-linked immunosorbent assay (ELISA) (data not shown). For all experiments, mice were infected with MNV at Washington University and shipped 4 to 5 days later to NIAID for further study. To contain MNV, infected mice were housed in microisolator cages in a quarantine room. In some experiments, control mice were housed in the same room as MNV-infected mice. Sera collected from control mice did not contain anti-MNV Abs as determined by ELISA (data not shown), confirming that transmission of MNV between mice housed in microisolator cages can be prevented by proper cage changing and aseptic handling of samples from infected mice.Upon intraperitoneal (i.p.) infection with either VV or IAV, mice mount robust CD8⁺ T-cell responses that peak, respectively, on day 6 or 7. Anti-VV and anti-IAV CD8⁺ T-cell responses in C57BL/6 mice conform to a well-established immunodominance hierarchy (3, 10). To determine to what extent MNV infection alters the magnitude and/or immunodominance hierarchy of CD8⁺ T-cell responses, we infected C57BL/6 mice i.p. with either VV or IAV 19 days following MNV infection. As controls, naïve mice (MNV negative) were infected with either virus. Lymphocytes were isolated from mice 6 days postinfection with VV and 7 days postinfection with IAV. The fraction of antigen-specific CD8⁺ T cells present in spleen and peritoneal exudate cells (PEC) was determined by intracellular IFN-γ staining after stimulation with synthetic peptides. MNV infection had little effect on the magnitude of splenic or PEC CD8⁺ T cells responding to VV (Fig. 1A and B) or IAV (Fig. 1C and D) infection. Regardless of MNV exposure history, splenic and PEC responses were dominated by B8R- and A8R-specific CD8⁺ T cells following VV infection (Fig. 1A and B) and by PA-specific and NP-specific CD8⁺ T cells following IAV infection (Fig. 1C and D).Open in a separate windowFIG. 1.MNV exposure does not alter CD8⁺ T-cell responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.p. with ∼1 × 10⁶ PFU of VV (A and B) or ∼1 × 10⁷ 50% tissue culture infective dose units of IAV (C and D), and specific CD8⁺ T cells were determined by intracellular IFN-γ staining after restimulating lymphocytes with peptides. Lymphocytes isolated from the spleen (A and C) and peritoneal cavity (B and D) were tested. MNV infections were completed 19 days prior to VV or IAV infections. Means and SEM are shown in panels A and C. A two-way analysis of variance and Bonferroni statistical analysis were completed for these experiments. Cells were pooled for peritoneal lavage samples as shown in panels B and D. Four to five mice/group were used for each experiment; data are representative of two independent experiments.To examine the effect of MNV infection on antiviral Ab responses, MNV-infected and control C57BL/6 mice were infected intranasally (i.n.) with a sublethal dose of either VV or IAV. Three weeks later, levels of anti-VV and anti-IAV Abs were determined by ELISA and hemagglutination inhibition assays, respectively. MNV infection did not significantly modify the magnitude of Ab responses to VV (Fig. ​(Fig.2A)2A) or IAV (Fig. ​(Fig.2B).2B). Next, we determined the effect of MNV infection on heavy chain class switching of anti-VV or anti-IAV Ab responses. Anti-VV and anti-IAV Ab responses exhibited similar heavy chain profiles dominated by immunoglobulin G2b (IgG2b) Abs regardless of MNV status (Fig. 2C and D). Thus, the CD8⁺ T-cell and Ab response to both VV and IAV appears to be essentially unaffected by chronic MNV infection. Since IgG anti-VV or anti-IAV Ab responses are entirely dependent on CD4⁺ T-cell help (11, 12), we can also infer that MNV also does not significantly affect CD4⁺ T-cell responses to VV or IAV.Open in a separate windowFIG. 2.MNV exposure does not alter Ab responses to VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with ∼1 × 10³ PFU of VV (A and C) or ∼50 50% tissue culture infective dose units of IAV (B and D), and virus-specific Abs were determined by ELISA (A, C, and D) or hemagglutination inhibition (B). The ELISA results shown in panel A measured the total IgG, while the ELISA results shown in panels C and D measured the individual isotype indicated. MNV infections were completed 19 days prior to VV or IAV infections. Means and standard errors of the means are shown in panels A, C, and D. Means are shown as lines in panel B. A two-way analysis of variance and Bonferroni statistical analysis were completed for experiments shown in panels A, C, and D, and t tests were completed for the experiment shown in panel B. Four to five mice/group were used for each experiment. O.D., optical density; HAI, hemagglutination inhibition.T-cell and Ab responses, together with innate immune mechanisms, collaborate to control viral replication and limit pathogenesis. To examine the effect of chronic MNV infection on VV-induced or IAV-induced pathogenesis, we infected C57BL/6 mice i.n. with a lethal or sublethal dose of VV or IAV and monitored body weight over a 16-day period. MNV-CR6p2 infection had no significant effect on morbidity or mortality from either virus (Fig. ​(Fig.33 and ​and4).4). Since MNV isolates are highly diverse, we decided to examine the effects of a second strain of MNV (MNV-CW3) which is fully cleared in immunocompetent mice. Mice that cleared MNV-CW3 (19 days post-MNV infection) were infected i.n. with VV or IAV. Once again, this strain of MNV had no effect on VV-induced or IAV-induced morbidity or mortality (Fig. ​(Fig.33 and ​and4).4). Future studies should address the extent to which other MNV strains affect the generation of adaptive immune responses to secondary viral infections.Open in a separate windowFIG. 3.MNV does not increase morbidity following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with a sublethal dose of VV (∼1 × 10³ PFU) (A) or IAV (∼50 50% tissue culture infective dose units) (B), and weight loss was recorded for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. A two-way analysis of variance and Bonferroni statistical analysis were completed. Four to five mice/group were used for each experiment.Open in a separate windowFIG. 4.MNV does not increase mortality following subsequent i.n. infection with VV or IAV. MNV-infected and naïve C57BL/6 mice were infected i.n. with VV (∼1 × 10⁴ PFU) (A) or IAV (∼500 50% tissue culture infective dose units) (B), and survival was monitored for 16 days postinfection. MNV infections were completed 19 days prior to VV or IAV infections. Eight to 10 mice/group were used for each experiment.Taken together, these data demonstrate that MNV infection has no significant effects on the measured immune response to VV or IAV. Our results cannot, however, be simply extrapolated to other viruses or microorganisms. Rather, the effect of MNV infection on host immunity in mouse model disease systems needs to be established on a system-by-system basis. Without this knowledge, the possible confounding effects of MNV infection will continue to undermine the confidence in results obtained using mice in colonies in which MNV infections are endemic.