Muthannan Andavar Ramakrishnan

The feasibility of using high resolution genome sequencing of influenza A viruses to detect mixed infections and quasispecies.

Background The rapidly expanding availability of de novo sequencing technologies can greatly facilitate efforts to monitor the relatively high mutation rates of influenza A viruses and the detection of quasispecies. Both the mutation rates and the lineages of influenza A viruses are likely to play an important role in the natural history of these viruses and the emergence of phenotypically and antigenically distinct strains. Methodology and Principal Findings We evaluated quasispecies and mixed infections by de novo sequencing the whole genomes of 10 virus isolates, including eight avian influenza viruses grown in embryonated chicken eggs (six waterfowl isolates - five H3N2 and one H4N6; an H7N3 turkey isolate; and a bald eagle isolate with H1N1/H2N1 mixed infection), and two tissue cultured H3N2 swine influenza viruses. Two waterfowl cloacal swabs were included in the analysis. Full-length sequences of all segments were obtained with 20 to 787-X coverage for the ten viruses and one cloacal swab. The second cloacal swab yielded 15 influenza reads of ∼230 bases, sufficient for bioinformatic inference of mixed infections or quasispecies. Genomic subpopulations or quasispecies of viruses were identified in four egg grown avian influenza isolates and one cell cultured swine virus. A bald eagle isolate and the second cloacal swab showed evidence of mixed infections with two (H1 and H2) and three (H1, H3, and H4) HA subtypes, respectively. Multiple sequence differences were identified between cloacal swab and the virus recovered using embryonated chicken eggs. Conclusions We describe a new approach to comprehensively identify mixed infections and quasispecies in low passage influenza A isolates and cloacal swabs and add to the understanding of the ecology of influenza A virus populations.

In-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus

Hand disinfection is considered important in preventing the transmission of viruses, including norovirus. We investigated the virucidal efficacy of nine hand sanitisers (four alcohol-based sanitisers, three non-alcoholic sanitisers and two triclosan-containing antimicrobial liquid soaps) against feline calicivirus, a surrogate for norovirus, on artificially contaminated fingertips for 30 s and 2 min contact periods. Among alcohol-based sanitisers, a product containing 99.5% ethanol was more effective than those containing 62% ethanol, 70% isopropanol or 91% isopropanol. A log(10) virus reduction factor of 1.00-1.30 was achieved with 99.5% ethanol but those containing a lower alcohol concentration only achieved a log(10) reduction factor of <or=0.67. Antiseptics containing 10% povidone-iodine (equivalent to 1% available iodine) reduced virus titre by a log(10) reduction factor of 2.67 within 30s contact time. This viral reduction rate was higher than that achieved with any of the alcohol-based sanitisers, non-alcoholic sanitisers or antimicrobial soaps. The two antimicrobial soaps tested showed minimal virus reduction (a log(10) reduction factor of 0.17-0.50), which is similar to that obtained by washing hands without any soap (a log(10) reduction factor of 0.33-0.42). These results indicate that triclosan-containing antimicrobial soaps or alcohol-based hand rubs may be inadequate for preventing norovirus transmission. Further research on alternative hand sanitisers should continue for effective control of norovirus infections.

Development of a method for bacteria and virus recovery from heating, ventilation, and air conditioning (HVAC) filters.

The aim of the work presented here is to study the effectiveness of building air handling units (AHUs) in serving as high volume sampling devices for airborne bacteria and viruses. An HVAC test facility constructed according to ASHRAE Standard 52.2-1999 was used for the controlled loading of HVAC filter media with aerosolized bacteria and virus. Nonpathogenic Bacillus subtilis var. niger was chosen as a surrogate for Bacillus anthracis. Three animal viruses; transmissible gastroenteritis virus (TGEV), avian pneumovirus (APV), and fowlpox virus were chosen as surrogates for three human viruses; SARS coronavirus, respiratory syncytial virus, and smallpox virus; respectively. These bacteria and viruses were nebulized in separate tests and injected into the test duct of the test facility upstream of a MERV 14 filter. SKC Biosamplers upstream and downstream of the test filter served as reference samplers. The collection efficiency of the filter media was calculated to be 96.5 +/- 1.5% for B. subtilis, however no collection efficiency was measured for the viruses as no live virus was ever recovered from the downstream samplers. Filter samples were cut from the test filter and eluted by hand-shaking. An extraction efficiency of 105 +/- 19% was calculated for B. subtilis. The viruses were extracted at much lower efficiencies (0.7-20%). Our results indicate that the airborne concentration of spore-forming bacteria in building AHUs may be determined by analyzing the material collected on HVAC filter media, however culture-based analytical techniques are impractical for virus recovery. Molecular-based identification techniques such as PCR could be used.

Determination of 50% endpoint titer using a simple formula

Two commonly used methods for calculating 50% endpoint using serial dilutions are Spearman-Karber method and Reed and Muench method. To understand/apply the above formulas, moderate statistical/mathematical skills are necessary. In this paper, a simple formula/method for calculating 50% endpoints has been proposed. The formula yields essentially similar results as those of the Spearman-Karber method. The formula has been rigorously evaluated with several samples.

Reassortant influenza A viruses in wild duck populations: effects on viral shedding and persistence in water

Wild ducks of the genus Anas represent the natural hosts for a large genetic diversity of influenza A viruses. In these hosts, co-infections with different virus genotypes are frequent and result in high rates of genetic reassortment. Recent genomic data have provided information regarding the pattern and frequency of these reassortant viruses in duck populations; however, potential consequences on viral shedding and maintenance in the environment have not been investigated. On the basis of full-genome sequencing, we identified five virus genotypes, in a wild duck population in northwestern Minnesota (USA), that naturally arose from genetic reassortments. We investigated the effects of influenza A virus genotype on the viral shedding pattern in Mallards (Anas platyrhynchos) and the duration of infectivity in water, under different temperature regimens. Overall, we found that variation in the viral genome composition of these isolates had limited effects on duration, extent and pattern of viral shedding, as well as on the reduction of infectivity in water over time. These results support that, in wild ducks, functionally equivalent gene segments could be maintained in virus populations with no fitness costs when genetic reassortments occur.

Background culturable bacteria aerosol in two large public buildings using HVAC filters as long term, passive, high-volume air samplers

Background culturable bacteria aerosols were collected and identified in two large public buildings located in Minneapolis, Minnesota and Seattle, Washington over a period of 5 months and 3 months, respectively. The installed particulate air filters in the ventilation systems were used as the aerosol sampling devices at each location. Both pre and final filters were collected from four air handing units at each site to determine the influence of location within the building, time of year, geographical location and difference between indoor and outdoor air. Sections of each loaded filter were eluted with 10 ml of phosphate buffered saline (PBS). The resulting solutions were cultured on blood agar plates and incubated for 24 h at 36 degrees C. Various types of growth media were then used for subculturing, followed by categorization using a BioLog MicroStation (Biolog, Hayward, CA, USA) and manual observation. Environmental parameters were gathered near each filter by the embedded on-site environmental monitoring systems to determine the effect of temperature, humidity and air flow. Thirty nine different species of bacteria were identified, 17 found only in Minneapolis and 5 only in Seattle. The hardy spore-forming genus Bacillus was the most commonly identified and showed the highest concentrations. A significant decrease in the number of species and their concentration occurred in the Minneapolis air handling unit supplying 100% outdoor air in winter, however no significant correlations between bacteria concentration and environmental parameters were found.

Detection of viruses in used ventilation filters from two large public buildings

Background Viral and bacterial pathogens may be present in the air after being released from infected individuals and animals. Filters are installed in the heating, ventilation, and air-conditioning (HVAC) systems of buildings to protect ventilation equipment and maintain healthy indoor air quality. These filters process enormous volumes of air. This study was undertaken to determine the utility of sampling used ventilation filters to assess the types and concentrations of virus aerosols present in buildings. Methods The HVAC filters from 2 large public buildings in Minneapolis and Seattle were sampled to determine the presence of human respiratory viruses and viruses with bioterrorism potential. Four air-handling units were selected from each building, and a total of 64 prefilters and final filters were tested for the presence of influenza A, influenza B, respiratory syncytial, corona, parainfluenza 1-3, adeno, orthopox, entero, Ebola, Marburg, Lassa fever, Machupo, eastern equine encephalitis, western equine encephalitis, and Venezuelan equine encephalitis viruses. Representative pieces of each filter were cut and eluted with a buffer solution. Results Attempts were made to detect viruses by inoculation of these eluates in cell cultures (Vero, MDCK, and RK-13) and specific pathogen-free embryonated chicken eggs. Two passages of eluates in cell cultures or these eggs did not reveal the presence of any live virus. The eluates were also examined by polymerase chain reaction or reverse-transcription polymerase chain reaction to detect the presence of viral DNA or RNA, respectively. Nine of the 64 filters tested were positive for influenza A virus, 2 filters were positive for influenza B virus, and 1 filter was positive for parainfluenza virus 1. Conclusion These findings indicate that existing building HVAC filters may be used as a method of detection for airborne viruses. As integrated long-term bioaerosol sampling devices, they may yield valuable information on the epidemiology and aerobiology of viruses in air that can inform the development of methods to prevent airborne transmission of viruses and possible deterrents against the spread of bioterrorism agents.

Immune responses and protective efficacy of binary ethylenimine (BEI)-inactivated bluetongue virus vaccines in sheep

Abbreviations: Al(OH)3, aluminium hydroxide; BEI, binary ethylenimine; BTV, bluetongue virus; CMI, cellmediated immune response; CPE, cytopathic effect; CRI, clinical reaction index; d.p.i., days post inoculation; DTH, delayed-type hypersensitivity; EHDV, epizootic haemorrhagic disease virus of deer; FIA, Freund’s incomplete adjuvant; PBS, phosphate-buffered saline; pfu, plaque-forming unit; PHA, phytohaemagglutinin; RBC, red blood cell

Zika virus - emergence, evolution, pathology, diagnosis, and control: current global scenario and future perspectives - a comprehensive review.

ABSTRACT This review converses the Zika virus which has attained global concern due to its rapid pandemic potential and impact on humans. Though Zika virus was first isolated in 1947, till the recent large-scale outbreak which occurred in Micronesia, in 2007, the virus was placed into the innocuous pathogen category. The World Health Organization on 1 February 2016 declared it as a ‘Public Health Emergency of International Concern.’ Of the note, American as well as Pacific Island strains/isolates is relatively closer to Asian lineage strains. The African and American strains share more than 87.5% and 95% homologies with Asian strains/isolates, respectively. Asian strains form independent clusters, except those isolated from China, suggesting relatively more diversity than African strains. Prevention and control are mainly aimed at the vector population (mosquitoes) with Aedes aegypti being the main species. Surveys in Africa and Asia indicated seropositivity in various animal species. However, so far its natural reservoir is unknown. There is an urgent need to understand why Zika virus has shifted from being a virus that caused mild illness to unforeseen birth defects as well as autoimmune-neurological problems. Unfortunately, an effective vaccine is not available yet. Availability of cryo-electron microscopy based on 3.8 Å resolution revealing mature Zika virus structure and the probable virus attachment site to host cell would provide critical insights into the development of antiviral treatments and vaccines.

Triple reassortant swine influenza A (H3N2) virus in waterfowl.

To the Editor: In 1998, a new lineage of triple reassortant influenza A (H3N2) virus (TR-H3N2) with genes from humans (hemmaglutinin [HA], neuraminidase [NA], and polymerase basic 1 [PB1]), swine (matrix [M], nonstructural [NS], and nucleoprotein [NP]), and birds (polymerase acidic [PA] and PB2) emerged in the U.S. swine population. Subsequently, similar viruses were isolated from turkeys (1,2), minks, and humans in the United States and Canada (3,4). In 2007, our national influenza surveillance resulted in isolation of 4 swine-like TR-H3N2 viruses from migratory waterfowl (3 from mallards [Anas platyrrhynchos] and 1 from a northern pintail [Anas acuta] of 266 birds sampled) in north-central South Dakota. We report on the characterization of these TR-H3N2 viruses and hypothesize about their potential for interspecies transmission.