Quick assessment of influenza a virus infectivity with a long-range reverse-transcription quantitative polymerase chain reaction assay.

BACKGROUND: The polymerase chain reaction (PCR) is commonly used to detect viral pathogens because of its high sensitivity and specificity. However, conventional PCR methods cannot determine virus infectivity. Virus infectivity is conventionally examined with methods such as the plaque assay, even though such assays require several days. Long-range reverse-transcription quantitative PCR (RT-qPCR) has previously been suggested for the rapid assessment of RNA virus infectivity where the loss of infectivity is attributable to genomic fragmentation. METHODS: IAV was irradiated with 253.7 nm ultraviolet (UV) rays to induce genomic strand breaks that were confirmed by a full-length RT-PCR assay. The IAV was then subjected to plaque assay, conventional RT-qPCR and long-range RT-qPCR to examine the relationship between infectious titer and copy number. A simple linear regression analysis was performed to examine the correlation between the results of these assays. RESULTS: A long-range RT-qPCR assay was developed and validated for influenza A virus (IAV). Although only a few minutes of UV irradiation was required to completely inactivate IAV, genomic RNA remained detectable by the conventional RT-qPCR and the full-length RT-PCR for NS of viral genome following inactivation. A long-range RT-qPCR assay was then designed using RT-priming at the 3' termini of each genomic segment and subsequent qPCR of the 5' regions. UV-mediated IAV inactivation was successfully analyzed by the long-range RT-qPCR assay especially when targeting PA of the viral genome. This was also supported by the regression analysis that the long-range RT-qPCR is highly correlated with plaque assay (Adjusted R2 = 0.931, P = 0.000066). CONCLUSIONS: This study suggests that IAV infectivity can be predicted without the infectivity assays. The rapid detection of pathogenic IAV has, therefore, been achieved with this sensing technology.

Environmental Stability of Swine and Human Pandemic Influenza Viruses in Water under Variable Conditions of Temperature, Salinity, and pH.

UNLABELLED: The movement of influenza A viruses (IAVs) from wild bird reservoirs to domestic animals and humans is well established, but the transmission mechanisms that facilitate efficient movement across and within these host populations are not fully defined. Although predominant routes of transmission vary between host populations, the extent of environmental stability needed for efficient IAV transmission also may vary. Because of this, we hypothesized that virus stability would differ in response to varied host-related transmission mechanisms; if correct, such phenotypic variation might represent a potential marker for the emergence of novel animal or human influenza viruses. Here, the objective was to evaluate the ability of eight swine and six human IAV isolates to remain infective under various pH, temperature, and salinity conditions using a preestablished distilled water system. Swine and human viruses persisted longest at near-neutral pH, at cold temperatures, or under "freshwater" conditions. Additionally, no significant differences in persistence were observed between pandemic and nonpandemic IAVs. Our results indicate that there have been no apparent changes in the environmental stability of the viruses related to host adaptation. IMPORTANCE: This study assessed the environmental stability of eight swine and six human influenza A viruses (IAVs), including viruses associated with the 2009 H1N1 pandemic, in a distilled water system. The important findings of this work are that IAV persistence can be affected by environmental variables and that no marked changes were noted between human and swine IAVs or between pandemic and nonpandemic IAVs.

The temperature-sensitive and attenuation phenotypes conferred by mutations in the influenza virus PB2, PB1, and NP genes are influenced by the species of origin of the PB2 gene in reassortant viruses derived from influenza A/California/07/2009 and A/WSN/33 viruses.

UNLABELLED: Live attenuated influenza vaccines in the United States are derived from a human virus that is temperature sensitive (ts), characterized by restricted (≥ 100-fold) replication at 39 °C. The ts genetic signature (ts sig) has been mapped to 5 loci in 3 genes: PB1 (391 E, 581 G, and 661 T), PB2 (265 S), and NP (34 G). However, when transferred into avian and swine influenza viruses, only partial ts and attenuation phenotypes occur. To investigate the reason for this, we introduced the ts sig into the human origin virus A/WSN/33 (WSN), the avian-origin virus A/Vietnam/1203/04 (VN04), and the swine origin triple-reassortant 2009 pandemic H1N1 virus A/California/07/2009 (CA07), which contains gene segments from human, avian, and swine viruses. The VN04(ts sig) and CA07(ts sig) viruses replicated efficiently in Madin-Darby canine kidney (MDCK) cells at 39 °C, but the replication of WSN(ts sig) was restricted ≥ 100-fold compared to that at 33 °C. Reassortant CA07(ts sig) viruses were generated with individual polymerase gene segments from WSN, and vice versa. Only ts sig viruses with a PB2 gene segment derived from WSN were restricted in replication ≥ 100-fold at 39 °C. In ferrets, the CA07(ts sig) virus replicated in the upper and lower respiratory tract, but the replication of a reassortant CA07(ts sig) virus with a WSN PB2 gene was severely restricted in the lungs. Taken together, these data suggest that the origin of the PB2 gene segment influences the ts phenotype in vitro and attenuation in vivo. This could have implications for the design of novel live vaccines against animal origin influenza viruses. IMPORTANCE: Live attenuated influenza vaccines (LAIVs) on temperature-sensitive (ts) backbones derived from animal origin influenza viruses are being sought for use in the poultry and swine industries and to protect people against animal origin influenza. However, inserting the ts genetic signature from a licensed LAIV backbone fails to fully attenuate these viruses. Our data indicate this is associated with the presence of a PB2 gene segment derived from an avian influenza virus. We show that a reassortant 2009 pandemic H1N1 virus with the ts signature from a licensed LAIV donor virus is ts in vitro and attenuated in vivo when the PB2 gene is derived from a human origin virus but not from an avian virus. Our study provides information that could benefit the rational design of alternative LAIV backbones against animal origin influenza viruses.

Inactivation of the infectivity of two highly pathogenic avian influenza viruses and a virulent Newcastle disease virus by ultraviolet radiation.

Exposure of a virulent isolate of Newcastle disease virus (NDV) and two highly pathogenic avian influenza (HPAI) viruses, one of H7N1 subtype and the other H5N1 subtype, to a continuous ultraviolet B flux of approximately 90µW/cm(2), which models solar ultraviolet radiation, resulted in an exponential decline in infectivity with time. The time taken for a reduction in titre of 1 log10 median tissue culture infectious dose for each virus was: NDV, 69 min; H7N1 HPAI virus, 158 min; and H5N1 HPAI, virus 167 min.

Return of inactivated whole-virus vaccine for superior efficacy.

The swine, influenza, H1N1 outbreak in 2009 highlighted the inadequacy of the currently used antibody-based vaccine strategies as a preventive measure for combating influenza pandemics. The ultimate goal for successful control of newly arising influenza outbreaks is to design a single-shot vaccine that will provide long-lasting immunity against all strains of influenza A virus. A large amount of data from animal studies has indicated that the cross-reactive cytotoxic T (Tc) cell response against conserved influenza virus epitopes may be the key immune response needed for a universal influenza vaccine. However, decades of research have shown that the development of safe T-cell-based vaccines for influenza is not an easy task. Here, I discuss the overlooked but potentially highly advantageous inactivation method, namely, γ-ray irradiation, as a mean to reach the Holy Grail of influenza vaccinology.

Thermostability of subpopulations of H2N3 influenza virus isolates from mallard ducks.

Maintenance of avian influenza virus in waterfowl populations requires that virions remain infectious while in the environment. Temperature has been shown to negatively correlate with persistence time, which is the duration for which virions are infectious. However, thermostability can vary between isolates regardless of subtype, and it is not known whether this variation occurs when host and geographic location of isolation are controlled. In this study, we analyzed the thermostabilities of 7 H2N3 viruses isolated from mallard ducks in Alberta, Canada. Virus samples were incubated at 37 degrees C and 55 degrees C, and infectivity titers were calculated at different time points. Based on the rate of infectivity inactivation at 37 degrees C, isolates could be grouped into either a thermosensitive or thermostable fraction for both egg- and MDCK-grown virus populations. Titers decreased more rapidly for isolates incubated at 55 degrees C, and this loss of infectivity occurred in a nonlinear, 2-step process, which is in contrast with the consensus on thermostability. This suggests that stock samples contain a mixture of subpopulations with different thermostabilities. The rate of decrease for the sensitive fraction was approximately 14 times higher than that for the stable fraction. The presence of subpopulations is further supported by selection experiments and plaque purification, both of which result in homogenous populations that exhibit linear decreases of infectivity titer. Therefore, variation of thermostability of influenza virus isolates begins at the level of the population. The presence of subpopulations with high thermostability suggests that avian viruses can persist in water longer than previously estimated, thus increasing the probability of transmission to susceptible hosts.

Kinetics of UV(254) inactivation of selected viral pathogens in a static system.

AIMS: The objective of this study was to estimate UV(254) inactivation constants for four viral pathogens: influenza virus type A, porcine respiratory and reproductive syndrome virus (PRRSV), bovine viral diarrhoea virus (BVDV) and reovirus. METHODS AND RESULTS: Viruses in culture medium were exposed to one of nine doses of UV(254) and then titrated for infectious virus. Analysis showed that viral inactivation by UV(254) was more accurately described by a two-stage inactivation model vs a standard one-stage inactivation model. CONCLUSIONS: The results provided evidence for the existence of two heterogeneous viral subpopulations among the viruses tested, one highly susceptible to UV(254) inactivation and the other more resistant. Importantly, inactivation constants based on the one-stage inactivation model would have underestimated the UV(254) dose required for the inactivation of these viruses under the conditions of the experiment. SIGNIFICANCE AND IMPACT OF THE STUDY: To improve the accuracy of estimates, it is recommended that research involving the inactivation of micro-organisms evaluates inactivation kinetics using both one-stage and two-stage models. These results will be of interest to persons responsible for microbial agents under laboratory or field conditions.

Role of viral infectivity in the induction of influenza virus-specific cytotoxic T cells.

This report examines the requirement for infectious virus in the induction of influenza virus-specific cytotoxic T cells. Infectious influenza virus was found to be highly efficient at generating both primary and secondary cytotoxic T-cell response in vivo. Inactivated influenza virus however, failed to stimulate a detectable cytotoxic T-cell response in vivo even at immunizing doses 10(5)-10(6)-fold higher than the minimum stimulatory dose of infectious virus. Likewise inactivated virus failed to sensitize target cells for T cell-mediated lysis in vitro but could stimulate a specific cytotoxic response from primed cells in vitro. Possible requirements for the induction of virus-specific cytotoxic T-cell responses are discussed in light of these observations and those of other investigators.

Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling.

Introduction of a new influenza virus in humans urges quick analysis of its virological and immunological characteristics to determine the impact on public health and to develop protective measures for the human population. At present, however, the necessity of executing pandemic influenza virus research under biosafety level 3 (BSL-3) high-containment conditions severely hampers timely characterization of such viruses. We tested heat, formalin, Triton X-100, and beta-propiolactone treatments for their potencies in inactivating human influenza A(H3N2) and avian A(H7N3) viruses, as well as seasonal and pandemic A(H1N1) virus isolates, while allowing the specimens to retain their virological and immunological properties. Successful heat inactivation coincided with the loss of hemagglutinin (HA) and neuraminidase (NA) characteristics, and beta-propiolactone inactivation reduced the hemagglutination titer and NA activity of the human influenza virus 10-fold or more. Although Triton X-100 treatment resulted in inconsistent HA activity, the NA activities in culture supernatants were enhanced consistently. Nonetheless, formalin treatment permitted the best retention of HA and NA properties. Triton X-100 treatment proved to be the easiest-to-use influenza virus inactivation protocol for application in combination with phenotypic NA inhibitor susceptibility assays, while formalin treatment preserved B-cell and T-cell epitope antigenicity, allowing the detection of both humoral and cellular immune responses. In conclusion, we demonstrated successful influenza virus characterization using formalin- and Triton X-100-inactivated virus samples. Application of these inactivation protocols limits work under BSL-3 conditions to virus culture, thus enabling more timely determination of public health impact and development of protective measures when a new influenza virus, e.g., pandemic A(H1N1)v virus, is introduced in humans.

Role of absolute humidity in the inactivation of influenza viruses on stainless steel surfaces at elevated temperatures.

Influenza virus has been found to persist in the environment for hours to days, allowing for secondary transmission of influenza via inanimate objects known as fomites. We evaluated the efficacy of heat and moisture for the decontamination of surfaces for the purpose of preventing of the spread of influenza. Aqueous suspensions of influenza A virus were deposited onto stainless steel coupons, allowed to dry under ambient conditions, and exposed to temperatures of 55 degrees C, 60 degrees C, or 65 degrees C and relative humidity (RH) of 25%, 50%, or 75% for up to 1 h. Quantitative virus assays were performed on the solution used to wash the viruses from these coupons, and results were compared with the solution used to wash coupons treated similarly but left under ambient conditions. Inactivation of influenza virus on surfaces increased with increasing temperature, RH, and exposure time. Reductions of greater than 5 logs of influenza virus on surfaces were achieved at temperatures of 60 and 65 degrees C, exposure times of 30 and 60 min, and RH of 50 and 75%. Our data also suggest that absolute humidity is a better predictor of surface inactivation than RH and allows the prediction of survival using two parameters rather than three. Modest amounts of heat and adequate moisture can provide effective disinfection of surfaces while not harming surfaces, electrical systems, or mechanical components, leaving no harmful residues behind after treatment and requiring a relatively short amount of time.

Cells process exogenous proteins for recognition by cytotoxic T lymphocytes.

Cells exposed to intact, noninfectious influenza virus were shown to be recognized by class I-restricted anti-influenza cytotoxic T lymphocytes (CTLs). Both internal and external proteins derived from virions were processed by cells for CTL recognition. Sensitization required the inactivation of viral neuraminidase activity and could be inhibited by preventing fusion of viral and cellular membranes. These findings are important in designing vaccines to elicit CTL responses, since they demonstrate that cells can process intact, exogenous proteins for recognition by CTLs and suggest that such processing depends on introduction of exogenous proteins into the cytoplasm.

Preliminary study for evaluation of avian influenza virus inactivation in contaminated poultry products using electron beam irradiation.

Spread of avian influenza is associated with movement of infected poultry and poultry products, and, as a result, strict international trade restrictions are in place. In addition, the possibility of transmission of avian influenza virus (AIV) strains to mammalian species by the consumption or handling of infected poultry, although a rare occurrence, is an important trade concern. Traditional methods for poultry product decontamination, such as thermal processing, are effective in inactivating AIV. However, alternative technologies such as electron beam (E-beam) irradiation offer some advantages in maintaining organoleptic properties of fresh poultry products. This study was designed to evaluate the applicability of high-energy (10 MeV) E-beam irradiation to inactivate AIV and reduce virus loads in egg products and poultry meat. Commercially available egg-white and ground turkey meat samples were spiked with the low-pathogenic A/chicken/TX/2002 H5N3 AIV and exposed to varying doses of high-energy E-beam irradiation in increments ranging from 0 kGy to 8 kGy. The viral titres in irradiated samples showed a linear dose-dependent reduction. The dose required to achieve 90% reduction (i.e. the D (10) value) of viable AIV loads was 2.3 kGy in phosphate buffer, 1.6 kGy in egg-white and 2.6 kGy in ground turkey meat samples. The effectiveness of E-beam irradiation to inactivate AIV was similar to the previously reported effectiveness of the E-beam against poliovirus and rotavirus. These results illustrate the potential applicability of high-energy (10 MeV) E-beam irradiation as a poultry product decontamination technology to inactivate AIV.

A note on the inactivation of influenza A viruses by solar radiation, relative humidity and temperature.

We critically investigate the claim put forward by Sagripanti and Lytle ([2007] Photochem. Photobiol. 83, 1278-1282) that inactivation of influenza A virus by solar radiation can explain the seasonality of influenza epidemics. We correct an error in the Sagripanti and Lytle paper and show that changes in relative humidity and temperature affect influenza virus inactivation as strongly as variation in solar radiation. Furthermore, it appears unlikely that transmission in outdoor settings plays an important role during influenza outbreaks, because influenza A virus is sensitive to a wide range of environmental factors.

Irradiation by ultraviolet light-emitting diodes inactivates influenza a viruses by inhibiting replication and transcription of viral RNA in host cells.

Influenza A viruses (IAVs) pose a serious global threat to humans and their livestock, especially poultry and pigs. This study aimed to investigate how to inactivate IAVs by using different ultraviolet-light-emitting diodes (UV-LEDs). We developed sterilization equipment with light-emitting diodes (LEDs) those peak wavelengths were 365 nm (UVA-LED), 310 nm (UVB-LED), and 280 nm (UVC-LED). These UV-LED irradiations decreased dose fluence-dependent plaque-forming units of IAV H1N1 subtype (A/Puerto Rico/8/1934) infected Madin-Darby canine kidney (MDCK) cells, but the inactivation efficiency of UVA-LED was significantly lower than UVB- and UVC-LED. UV-LED irradiations did not alter hemagglutination titer, but decreased accumulation of intracellular total viral RNA in infected MDCK cells was observed. Additionally, UV-LED irradiations suppressed the accumulation of intracellular mRNA (messenger RNA), vRNA (viral RNA), and cRNA (complementary RNA), as measured by strand-specific RT-PCR. These results suggest that UV-LEDs inhibit host cell replication and transcription of viral RNA. Both UVB- and UVC-LED irradiation decreased focus-forming unit (FFU) of H5N1 subtype (A/Crow/Kyoto/53/2004), a highly pathogenic avian IAV (HPAI), in infected MDCK cells, and the amount of FFU were lower than the H1N1 subtype. From these results, it appears that IAVs may have different sensitivity among the subtypes, and UVB- and UVC-LED may be suitable for HPAI virus inactivation.

Inactivation of influenza virus by solar radiation.

Influenza virus is readily transmitted by aerosols and its inactivation in the environment could play a role in limiting the spread of influenza epidemics. Ultraviolet radiation in sunlight is the primary virucidal agent in the environment but the time that influenza virus remains infectious outside its infected host remains to be established. In this study, we calculated the expected inactivation of influenza A virus by solar ultraviolet radiation in several cities of the world during different times of the year. The inactivation rates reported here indicate that influenza A virions should remain infectious after release from the host for several days during the winter "flu season" in many temperate-zone cities, with continued risk for reaerosolization and human infection. The correlation between low and high solar virucidal radiation and high and low disease prevalence, respectively, suggest that inactivation of viruses in the environment by solar UV radiation plays a role in the seasonal occurrence of influenza pandemics.

Efficient Structure Resonance Energy Transfer from Microwaves to Confined Acoustic Vibrations in Viruses.

Virus is known to resonate in the confined-acoustic dipolar mode with microwave of the same frequency. However this effect was not considered in previous virus-microwave interaction studies and microwave-based virus epidemic prevention. Here we show that this structure-resonant energy transfer effect from microwaves to virus can be efficient enough so that airborne virus was inactivated with reasonable microwave power density safe for the open public. We demonstrate this effect by measuring the residual viral infectivity of influenza A virus after illuminating microwaves with different frequencies and powers. We also established a theoretical model to estimate the microwaves power threshold for virus inactivation and good agreement with experiments was obtained. Such structure-resonant energy transfer induced inactivation is mainly through physically fracturing the virus structure, which was confirmed by real-time reverse transcription polymerase chain reaction. These results provide a pathway toward establishing a new epidemic prevention strategy in open public for airborne virus.

Comparison of gamma and neutron radiation inactivation of influenza A virus.

Radiation inactivation of viral pathogens has potential application in sterilization and in the manufacture of biological reagents, including the production of non-infectious viral antigens. Viral inactivation by gamma radiation has been extensively investigated, but few direct comparisons to other qualities of radiation have been explored. Experiments were designed to examine direct radiation damage by both gamma photons (gamma) and neutrons (n) while minimizing methodological differences. Frozen samples of influenza A X31/H3N2 and PR8/H1N1 were exposed to gamma and n at doses between 0 and 15.6 kGy. Other experimental parameters, including dose-rate, were not varied. Virus titers were determined by tissue culture infectious dose (TCID(50)) and plaque forming unit (PFU) assays. D(10) values, kGy per log reduction, were calculated from these assays. PR8 D(10) values based on PFU assays were approximately 2 and 5 kGy for gamma and n exposures, respectively, and those based on TCID(50) were approximately 6 and 14 kGy. Similar results were obtained for the A/X31 strain. The data demonstrate that gamma was 2-3-fold more effective than n, with a relative biological effectiveness (RBE) range of 0.43-0.65. These neutron results are likely the first reported for a medically relevant virus. PAGE analysis of viral proteins and RNAs failed to show macromolecular damage. D(10) values were found to be similar to a broad summary of previously reported gamma inactivation values for other virus types. The dependence of the magnitudes of D(10) on titer assay in this study suggests that more than one titer method should be used to determine if complete inactivation has occurred.

Sensitivity of Burkitt lymphoma Daudi cells to inactive influenza virus.

Interaction of UV-inactivated influenza A/X47 virus at high multiplicity caused a rapid inhibition in cellular protein and DNA synthesis, thus arresting Burkitt-lymphoma-derived Daudi cell multiplication, and eventually killing the cells. The mechanism of the cytolytic effect is presumably, linked to the increase in cell membrane permeability indicated by elevation in 51Cr release. This might be the consequence of the mass adsorption and/or penetration of viral particles.

Improvements in methods for calculating virus titer estimates from TCID50 and plaque assays.

Calculation of titer estimates and use of titer reduction assays are fundamental approaches used by virologists. Titer assays being biological assays and based on limiting dilution methods require good error control, both methodologically and analytically. The need for good statistical analysis is likely to become even greater as in clinical, manufacturing, as well as the research settings, improved analytical criteria, quality control, and assurance standards are adopted. Furthermore, increasingly, virus titer assays are based on high throughput methods, which generate continuous rather than traditional quantal data. Described here are two different weighted linear regression methods to determine TCID50 and PFU titers from CPE assays. The TCID50 analysis makes use of a generalized least squares approach using continuous colorimetric data. The plaque analysis makes use of weighted least squares forced through the origin using quantal plaque data generated by serial dilutions. Both methods are improvements in titer and error estimation compared to simpler calculation methods. These methods may have greatest value when lack of experimental material or costs of analysis precludes extensive replicate titer determinations but good estimates of titers and/or treatment differences are essential.

Synthesis of interferon in human leukocyte cells stimulated with influenza virus.

Strains of influenza A virus were tested for their ability to induce interferon synthesis in suspension of human leukocytes. Both, active as well as UV-inactivated strains were shown to be weak interferon inducers as compared to Newcastle virus. It appeared that interferon synthesis in human leukocytes was conditioned not by infectivity of the virus but by the presence of its surface antigens--hemagglutinin and neuraminidase.