Airborne Stability of Simian Virus 40

The influence of relative humidity on the airborne survival of simian virus 40 (SV40) was studied by allowing virus aerosols to age in rotating drums at 21 or 32 C and at a relative humidity (RH) value ranging from 22 to 88%. Airborne SV40 virus was stable at every RH tested at 21 C, but aerosols maintained at 32 C were inactivated within 60 min at mid-range RH values. The unusual stability at 21 C over a broad RH range indicates that potentially biohazardous situations may occur under laboratory conditions if this virus becomes accidentally airborne.     

Characterization of UVC light sensitivity of vaccinia virus

Interest in airborne smallpox transmission has been renewed because of concerns regarding the potential use of smallpox virus as a biothreat agent. Air disinfection via upper-room 254-nm germicidal UV (UVC) light in public buildings may reduce the impact of primary agent releases, prevent secondary airborne transmission, and be effective prior to the time when public health authorities are aware of a smallpox outbreak. We characterized the susceptibility of vaccinia virus aerosols, as a surrogate for smallpox, to UVC light by using a benchtop, one-pass aerosol chamber. We evaluated virus susceptibility to UVC doses ranging from 0.1 to 3.2 J/m(2), three relative humidity (RH) levels (20%, 60%, and 80%), and suspensions of virus in either water or synthetic respiratory fluid. Dose-response plots show that vaccinia virus susceptibility increased with decreasing RH. These plots also show a significant nonlinear component and a poor fit when using a first-order decay model but show a reasonable fit when we assume that virus susceptibility follows a log-normal distribution. The overall effects of RH (P < 0.0001) and the suspending medium (P = 0.014) were statistically significant. When controlling for the suspending medium, the RH remained a significant factor (P < 0.0001) and the effect of the suspending medium was significant overall (P < 0.0001) after controlling for RH. Virus susceptibility did not appear to be a function of virus particle size. This work provides an essential scientific basis for the design of effective upper-room UVC installations for the prevention of airborne infection transmission of smallpox virus by characterizing the susceptibility of an important orthopoxvirus to UVC exposure.     

Observations on the survival and infectivity of airborne rinderpest virus

The most frequent means of spread of rinderpest, a cause of major epizootics and of great economic losses among cattle in the tropics, remains undetermined. Studies on airborne virus in vitro demonstrated survival for at least 30 min at low RH and a somewhat inferior period of survival at high RH; viability was greatly reduced at 50–60% RH. The infectivity of 3 strains of airborne rinderpest virus for cattle was demonstrated by a technique employing direct inhalation of viral aerosols.     

Airborne Detection and Quantification of Swine Influenza A Virus in Air Samples Collected Inside,Outside and Downwind from Swine Barns

Airborne transmission of influenza A virus (IAV) in swine is speculated to be an important route of virus dissemination, but data are scarce. This study attempted to detect and quantify airborne IAV by virus isolation and RRT-PCR in air samples collected under field conditions. This was accomplished by collecting air samples from four acutely infected pig farms and locating air samplers inside the barns, at the external exhaust fans and downwind from the farms at distances up to 2.1 km. IAV was detected in air samples collected in 3 out of 4 farms included in the study. Isolation of IAV was possible from air samples collected inside the barn at two of the farms and in one farm from the exhausted air. Between 13% and 100% of samples collected inside the barns tested RRT-PCR positive with an average viral load of 3.20E+05 IAV RNA copies/m³ of air. Percentage of exhaust positive air samples also ranged between 13% and 100% with an average viral load of 1.79E+04 RNA copies/m³ of air. Influenza virus RNA was detected in air samples collected between 1.5 and 2.1 Km away from the farms with viral levels significantly lower at 4.65E+03 RNA copies/m³. H1N1, H1N2 and H3N2 subtypes were detected in the air samples and the hemagglutinin gene sequences identified in the swine samples matched those in aerosols providing evidence that the viruses detected in the aerosols originated from the pigs in the farms under study. Overall our results indicate that pigs can be a source of IAV infectious aerosols and that these aerosols can be exhausted from pig barns and be transported downwind. The results from this study provide evidence of the risk of aerosol transmission in pigs under field conditions.     

Relationship between Humidity and Influenza A Viability in Droplets and Implications for Influenza’s Seasonality

Humidity has been associated with influenza’s seasonality, but the mechanisms underlying the relationship remain unclear. There is no consistent explanation for influenza’s transmission patterns that applies to both temperate and tropical regions. This study aimed to determine the relationship between ambient humidity and viability of the influenza A virus (IAV) during transmission between hosts and to explain the mechanisms underlying it. We measured the viability of IAV in droplets consisting of various model media, chosen to isolate effects of salts and proteins found in respiratory fluid, and in human mucus, at relative humidities (RH) ranging from 17% to 100%. In all media and mucus, viability was highest when RH was either close to 100% or below ∼50%. When RH decreased from 84% to 50%, the relationship between viability and RH depended on droplet composition: viability decreased in saline solutions, did not change significantly in solutions supplemented with proteins, and increased dramatically in mucus. Additionally, viral decay increased linearly with salt concentration in saline solutions but not when they were supplemented with proteins. There appear to be three regimes of IAV viability in droplets, defined by humidity: physiological conditions (∼100% RH) with high viability, concentrated conditions (50% to near 100% RH) with lower viability depending on the composition of media, and dry conditions (<50% RH) with high viability. This paradigm could help resolve conflicting findings in the literature on the relationship between IAV viability in aerosols and humidity, and results in human mucus could help explain influenza’s seasonality in different regions.     

Airborne Stability of Tailless Bacterial Viruses S-13 and MS-2

The effect of relative humidity (RH) on the airborne stability of two small bacterial viruses, S-13 and MS-2, was studied. Poorest recovery of S-13 was obtained at 50% RH. Humidification prior to aerosol sampling significantly increased the recovery of S-13 at RH deleterious to the airborne virus. A commercial preparation of MS-2 suspended in a buffered saline solution showed a rapid loss of viability at RH above 30%, whereas a laboratory preparation containing 1.3% tryptone showed high recoveries at all RH studied. Dilution of the commercial MS-2 into tryptone broth conferred stability on the airborne virus. Humidification prior to sampling significantly reduced the viable recovery from aerosols of commercial MS-2, whereas the laboratory preparation was unaffected.     

Characterization of UVC Light Sensitivity of Vaccinia Virus

Interest in airborne smallpox transmission has been renewed because of concerns regarding the potential use of smallpox virus as a biothreat agent. Air disinfection via upper-room 254-nm germicidal UV (UVC) light in public buildings may reduce the impact of primary agent releases, prevent secondary airborne transmission, and be effective prior to the time when public health authorities are aware of a smallpox outbreak. We characterized the susceptibility of vaccinia virus aerosols, as a surrogate for smallpox, to UVC light by using a benchtop, one-pass aerosol chamber. We evaluated virus susceptibility to UVC doses ranging from 0.1 to 3.2 J/m², three relative humidity (RH) levels (20%, 60%, and 80%), and suspensions of virus in either water or synthetic respiratory fluid. Dose-response plots show that vaccinia virus susceptibility increased with decreasing RH. These plots also show a significant nonlinear component and a poor fit when using a first-order decay model but show a reasonable fit when we assume that virus susceptibility follows a log-normal distribution. The overall effects of RH (P < 0.0001) and the suspending medium (P = 0.014) were statistically significant. When controlling for the suspending medium, the RH remained a significant factor (P < 0.0001) and the effect of the suspending medium was significant overall (P < 0.0001) after controlling for RH. Virus susceptibility did not appear to be a function of virus particle size. This work provides an essential scientific basis for the design of effective upper-room UVC installations for the prevention of airborne infection transmission of smallpox virus by characterizing the susceptibility of an important orthopoxvirus to UVC exposure.     

Modified Spinning Top Homogeneous Spray Apparatus for Use in Experimental Respiratory Disease Studies

The May spinning top generator was adapted to a modified Henderson tube for producing large aerosol particles (>4 mum) to obtain almost exclusive upper respiratory tract deposition of infectious aerosols in exposed mice. The system was installed in a biological safety cabinet to permit experimentation with pathogens. A novel mechanism utilizing parts from a machinists micrometer and the mechanical stage from a light microscope was developed for the spinning top generator as a means for precisely positioning the liquid feed needle. Aerosol light-scatter properties were continuously analyzed to provide relative measures of particle size distribution and aerosol concentration. When mice were exposed to influenza virus aerosols in which none of the virus was contained in particles with aerodynamic diameters <4 mum, essentially all of the virus was deposited in the upper respiratory tract tissues.     

Overview: Replication of porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes significant losses in the pig industry, is one of the most important animal pathogens of global significance. Since the discovery of the virus, significant progress has been made in understanding its epidemiology and transmission, but no adequate control measures are yet available to eliminate infection with this pathogen. The genome replication of PRRSV is required to reproduce, within a few hours of infection, the millions of progeny virions that establish, disseminate, and maintain infection. Replication of the viral RNA genome is a multistep process involving a replication complex that is formed not only from components of viral and cellular origin but also from the viral genomic RNA template; this replication complex is embedded within particular virus-induced membrane vesicles. PRRSV RNA replication is directed by at least 14 replicase proteins that have both common enzymatic activities, including viral RNA polymerase, and also unusual and poorly understood RNA-processing functions. In this review, we summarize our current understanding of PRRSV replication, which is important for developing a successful strategy for the prevention and control of this pathogen.     

Effect of Relative Humidity on the Survival of Airborne Unicellular Algae

A method is described which is suitable for assessing the effects of relative humidity (RH) on the viability of two unicellular algae in experimental aerosols. Viable cells of Nannochloris atomus collected from the airborne state were detected by plating onto agar surfaces of an appropriate growth medium, whereas viable airborne cells of Synechococcus sp., because of unreliable growth on solid media, were determined by a liquid assay system. The assays were performed at intervals during short-term and prolonged storage of algal aerosols in chambers preconditioned to a selected RH and temperature. Both species showed the greatest loss in viability during the first minute after atomization, and the extent of this inactivation, as a function of RH, reflected the subsequent long-term survival. The airborne eukaryotic alga was unable to survive at an RH below 91%, whereas the airborne prokaryotic alga was comparatively stable over a wide humidity range. Initial inactivation was least and long-term survival best, for both species, at 94% RH.     

Microbiological Identification and Analysis of Swine Lungs Collected from Carcasses in Swine Farms, China

The primary objective of this 3 years study was to determine the prevalence of porcine pathogens of the lungs of swine in swine farms in southern China. A total of 5,420 samples were collected from 200 swine farms. The bacterium that was most commonly isolated was Streptococcus suis, with 10.24 % of the samples being positive, 114 lungs (2.1 %) were positive for pseudorabies virus and 263 (4.85 %) were positive for classical swine fever virus; much lower than positive for PRRSV (15.1 %, p = 0.023) and PCV2 (13.8 %, p = 0.038). lungs that were positive for PRRSV and/or PCV-2 have significantly increased odds of being positive for any of the S. suis (9.79 vs. 0.44 %, p = 0.003).     

Surface-displayed porcine reproductive and respiratory syndrome virus from cell culture onto gram-positive enhancer matrix particles

Vaccine immunization is now one of the most effective ways to control porcine reproductive and respiratory syndrome virus (PRRSV) infection. Impurity is one of the main factors affecting vaccine safety and efficacy. Here we present a novel innovative PRRSV purification approach based on surface display technology. First, a bifunctional protein PA-GRFT (protein anchor-griffithsin), the crucial factor in the purification process, was successfully produced in Escherichia coli yielding 80 mg/L of broth culture. Then PRRSV purification was performed by incubation of PA-GRFT with PRRSV and gram-positive enhancer matrix (GEM) particles, followed by centrifugation to collect virions loaded onto GEM particles. Our results showed that most of the bulk impurities had been removed, and PA-GRFT could capture PRRSV onto GEM particles. Our lactic acid bacteria-based purification method, which is promising as ease of operation, low cost and easy to scale-up, may represent a candidate method for the large-scale purification of this virus for vaccine production.     

“Broadbeam” irradiation of mammalian cells using a vertical microbeam facility

A “broadbeam” facility is demonstrated for the vertical microbeam at Surrey’s Ion Beam Centre, validating the new technique used by Barazzuol et al. (Radiat Res 177:651–662, 2012). Here, droplets with a diameter of about 4 mm of 15,000 mammalian cells in suspension were pipetted onto defined locations on a 42-mm-diameter cell dish with each droplet individually irradiated in “broadbeam” mode with 2 MeV protons and 4 MeV alpha particles and assayed for clonogenicity. This method enables multiple experimental data points to be rapidly collected from the same cell dish. Initially, the Surrey vertical beamline was designed for the targeted irradiation of single cells with single counted ions. Here, the benefits of both targeted single-cell and broadbeam irradiations being available at the same facility are discussed: in particular, high-throughput cell irradiation experiments can be conducted on the same system as time-intensive focused-beam experiments with the added benefits of fluorescent microscopy, cell recognition and time-lapse capabilities. The limitations of the system based on a 2 MV tandem accelerator are also discussed, including the uncertainties associated with particle Poisson counting statistics, spread of linear energy transfer in the nucleus and a timed dose delivery. These uncertainties are calculated with Monte Carlo methods. An analysis of how this uncertainty affects relative biological effect measurements is made and discussed.     

Effect of Prehumidification on Sampling of Selected Airborne Viruses

Studies were undertaken to determine if a prewetting device (humidifier bulb) used in combination with an all glass impinger (AGI-30) would increase the recovery of airborne mengovirus-37A, vesicular stomatitis virus (VSV), and the S-13 coliphage. Suspensions of T3 coliphage with mengovirus-37A, VSV, or S-13 were aerosolized and collected by using the AGI-30-humidifier bulb combination to sample the aerosols before and after shifts in relative humidities (RH). These studies revealed the following. (i) At low RH values there was a 3 to 4 log increase in recovery of airborne T3 phage; (ii) concomitantly, the recovery of mengovirus-37A and VSV decreased; and (iii) only at the mid-range RH values was the recovery of S-13 enhanced. The prehumidification technique significantly increased the recovery of airborne T3 phage but decreased the recovery of the two animal viruses tested.     

Single-walled carbon nanotubes modulate pulmonary immune responses and increase pandemic influenza a virus titers in mice

Background

Numerous toxicological studies have focused on injury caused by exposure to single types of nanoparticles, but few have investigated how such exposures impact a host’s immune response to pathogen challenge. Few studies have shown that nanoparticles can alter a host’s response to pathogens (chiefly bacteria) but there is even less knowledge of the impact of such particles on viral infections. In this study, we performed experiments to investigate if exposure of mice to single-walled carbon nanotubes (SWCNT) alters immune mechanisms and viral titers following subsequent influenza A virus (IAV) infection.

Methods

Male C57BL/6 mice were exposed to 20 μg of SWCNT or control vehicle by intratracheal instillation followed by intranasal exposure to 3.2?×?10⁴ TCID₅₀ IAV or PBS after 3 days. On day 7 mice were euthanized and near-infrared fluorescence (NIRF) imaging was used to track SWCNT in lung tissues. Viral titers, histopathology, and mRNA expression of antiviral and inflammatory genes were measured in lung tissue. Differential cell counts and cytokine levels were quantified in bronchoalveolar lavage fluid (BALF).

Results

Viral titers showed a 63-fold increase in IAV in SWCNT + IAV exposed lungs compared to the IAV only exposure. Quantitation of immune cells in BALF indicated an increase of neutrophils in the IAV group and a mixed profile of lymphocytes and neutrophils in SWCNT + IAV treated mice. NIRF indicated SWCNT remained in the lung throughout the experiment and localized in the junctions of terminal bronchioles, alveolar ducts, and surrounding alveoli. The dual exposure exacerbated pulmonary inflammation and tissue lesions compared to SWCNT or IAV single exposures. IAV exposure increased several cytokine and chemokine levels in BALF, but greater levels of IL-4, IL-12 (P70), IP-10, MIP-1, MIP-1α, MIP-1β, and RANTES were evident in the SWCNT?+?IAV group. The expression of tlr3, ifnβ1, rantes, ifit2, ifit3, and il8 was induced by IAV alone but several anti-viral targets showed a repressed trend (ifits) with pre-exposure to SWCNT.

Conclusions

These findings reveal a pronounced effect of SWCNT on IAV infection in vivo as evidenced by exacerbated lung injury, increased viral titers and several cytokines/chemokines levels, and reduction of anti-viral gene expression. These results imply that SWCNT can increase susceptibility to respiratory viral infections as a novel mechanism of toxicity.

     

Identification of putative interactions between swine and human influenza A virus nucleoprotein and human host proteins

Background

Influenza A viruses (IAVs) are important pathogens that affect the health of humans and many additional animal species. IAVs are enveloped, negative single-stranded RNA viruses whose genome encodes at least ten proteins. The IAV nucleoprotein (NP) is a structural protein that associates with the viral RNA and is essential for virus replication. Understanding how IAVs interact with host proteins is essential for elucidating all of the required processes for viral replication, restrictions in species host range, and potential targets for antiviral therapies.

Methods

In this study, the NP from a swine IAV was cloned into a yeast two-hybrid “bait” vector for expression of a yeast Gal4 binding domain (BD)-NP fusion protein. This “bait” was used to screen a Y2H human HeLa cell “prey” library which consisted of human proteins fused to the Gal4 protein’s activation domain (AD). The interaction of “bait” and “prey” proteins resulted in activation of reporter genes.

Results

Seventeen positive bait-prey interactions were isolated in yeast. All of the “prey” isolated also interact in yeast with a NP “bait” cloned from a human IAV strain. Isolation and sequence analysis of the cDNAs encoding the human prey proteins revealed ten different human proteins. These host proteins are involved in various host cell processes and structures, including purine biosynthesis (PAICS), metabolism (ACOT13), proteasome (PA28B), DNA-binding (MSANTD3), cytoskeleton (CKAP5), potassium channel formation (KCTD9), zinc transporter function (SLC30A9), Na+/K+ ATPase function (ATP1B1), and RNA splicing (TRA2B).

Conclusions

Ten human proteins were identified as interacting with IAV NP in a Y2H screen. Some of these human proteins were reported in previous screens aimed at elucidating host proteins relevant to specific viral life cycle processes such as replication. This study extends previous findings by suggesting a mechanism by which these host proteins associate with the IAV, i.e., physical interaction with NP. Furthermore, this study revealed novel host protein-NP interactions in yeast.

     

Dynamics of airborne influenza A viruses indoors and dependence on humidity

There is mounting evidence that the aerosol transmission route plays a significant role in the spread of influenza in temperate regions and that the efficiency of this route depends on humidity. Nevertheless, the precise mechanisms by which humidity might influence transmissibility via the aerosol route have not been elucidated. We hypothesize that airborne concentrations of infectious influenza A viruses (IAVs) vary with humidity through its influence on virus inactivation rate and respiratory droplet size. To gain insight into the mechanisms by which humidity might influence aerosol transmission, we modeled the size distribution and dynamics of IAVs emitted from a cough in typical residential and public settings over a relative humidity (RH) range of 10-90%. The model incorporates the size transformation of virus-containing droplets due to evaporation and then removal by gravitational settling, ventilation, and virus inactivation. The predicted concentration of infectious IAVs in air is 2.4 times higher at 10% RH than at 90% RH after 10 min in a residential setting, and this ratio grows over time. Settling is important for removal of large droplets containing large amounts of IAVs, while ventilation and inactivation are relatively more important for removal of IAVs associated with droplets <5 μm. The inactivation rate increases linearly with RH; at the highest RH, inactivation can remove up to 28% of IAVs in 10 min. Humidity is an important variable in aerosol transmission of IAVs because it both induces droplet size transformation and affects IAV inactivation rates. Our model advances a mechanistic understanding of the aerosol transmission route, and results complement recent studies on the relationship between humidity and influenza's seasonality. Maintaining a high indoor RH and ventilation rate may help reduce chances of IAV infection.     

Comparative analysis of airborne bacteria and fungi in two salt mines in Poland

The aim of this work was to determine the genera or species composition and the number of colony forming units of airborne bacteria and fungi, respectively, in two salt mines in Poland “Wieliczka” (Lesser Poland) and “Polkowice–Sieroszowice” (Lower Silesia). Both of them are working environments characterized by extreme conditions, and additionally “Wieliczka,” officially placed on the UNESCO World Heritage Sites’ list, plays a role of tourist attraction. There are also some curative chambers located in this mine. Air samples were taken once in December 2015, between 6:00 a.m. and 9:00 a.m. There were nine measurement points located about 200 m underground in “Wieliczka” and six measurement points located in the working shafts about 400 m underground in “Polkowice–Sieroszowice.” The total volume of each air sample was 150 L. Air samples, collected in individual measurement points of both salt mines, were inoculated on two microbiological media: potato dextrose agar and tryptic soy agar using the impact method. We identified 10 and 3 fungal genera in the “Wieliczka” Salt Mine and in “Polkowice–Sieroszowice,” respectively. The most common were fungi of the Penicillium genus. In both mines, the Gram-positive bacteria of genus Micrococcus were detected most frequently. Among identified microorganisms, there were neither pathogenic fungi nor bacteria. The most prevalent microorganisms detected in indoor air were Gram-positive cocci, which constituted up to 80% of airborne microflora. Our results showed that microorganisms recorded in the air samples are not a threat to workers, tourists or patients. Neither pathogens nor potentially pathogenic microorganisms, listed as BSL-2, BSL-3 or BSL-4, were detected. The microbes identified during our analysis commonly occur in such environments as the soil, water and air. Some of the detected bacteria are component of natural microflora of human skin and mucous membranes, and they can cause only opportunistic infections in individuals depending on their health condition.