Sensitivity of oral fluids for detecting influenza A virus in populations of vaccinated and non-vaccinated pigs

Please cite this paper as: Romagosa et al. (2011) Sensitivity of oral fluids for detecting influenza A virus in populations of vaccinated and non‐vaccinated pigs. Influenza and Other Respiratory Viruses. Background/objective  We evaluated the sensitivity of PCR on oral fluids in detecting influenza virus in vaccinated and non‐vaccinated pigs. Methods  Three‐week‐old influenza‐free pigs were divided into three groups: (i) control, non‐vaccinated, (ii) vaccinated with a commercial, heterologous vaccine, and (iii) vaccinated with an experimental, homologous vaccine. After vaccination, an influenza‐infected pig was placed in contact with each of the groups. Individual nasal swabs and pen oral fluids were collected daily. Viral RNA was tested for the presence of influenza by RRT‐PCR and virus isolation attempted from oral fluids. A pen was considered positive if at least one nasal swab was positive. Results  Based on nasal swab results, 43·8% of pens were detected positive but only 35% based on oral fluids. Overall sensitivity of oral fluids was 80%, and virus was isolated from 51% of RRT‐PCR‐positive oral fluids. The kappa coefficient for agreement (ĸ) between oral fluids and nasal swabs was 0·82. Among groups, ĸ was 1 (95% CI, 1–1), 0·74 (95% CI, 0·55–0·92), and 0·76 (95% CI, 0·5–1) for control, heterologous, and homologous‐vaccinated groups, respectively. There was less agreement when within pen prevalence was 10% or less. Probability of detecting influenza virus in oral fluids was 99% when within pen prevalence was higher than 18% and decreased to 69% when prevalence was 9%. Conclusions  Results indicated that pen‐based collection of oral fluids is a sensitive method to detect influenza even when within pen prevalence is low and when pigs have been vaccinated and highlight the potential use of oral fluids for influenza surveillance.     

Surveillance of feral cats for influenza A virus in north central Florida

Please cite this paper as: Gordy JT et al. (2012) Surveillance of feral cats for influenza A virus in North Central Florida. Influenza and Other Respiratory Viruses 6(5), 341–347. Background Transmission of highly pathogenic avian influenza and the recent pandemic H1N1 viruses to domestic cats and other felids creates concern because of the morbidity and mortality associated with human infections as well as disease in the infected animals. Experimental infections have demonstrated transmission of influenza viruses in cats. Objectives An epidemiologic survey of feral cats was conducted to determine their exposure to influenza A virus. Methods Feral cat sera and oropharyngeal and rectal swabs were collected from November 2008 through July 2010 in Alachua County, FL and were tested for evidence of influenza A virus infection by virus isolation, PCR, and serological assay. Results and conclusions No virus was isolated from any of 927 cats examined using MDCK cell or embryonated chicken egg culture methods, nor was viral RNA detected by RT‐PCR in 200 samples tested. However, 0.43% of cats tested antibody positive for influenza A by commercial ELISA. These results suggest feral cats in this region are at minimal risk for influenza A virus infection.     

Differentiated swine airway epithelial cell cultures for the investigation of influenza A virus infection and replication

Please cite this paper as: Bateman et al. (2013) Differentiated swine airway epithelial cell cultures for the investigation of influenza A virus infection and replication. Influenza and Other Respiratory Viruses 7(2) 139–150. Background Differentiated human airway epithelial cell cultures have been utilized to investigate cystic fibrosis, wound healing, and characteristics of viral infections. These cultures, grown at an air–liquid interface (ALI) in media with defined hormones and growth factors, recapitulate many aspects of the in vivo respiratory tract and allow for experimental studies at the cellular level. Objectives To optimize growth conditions for differentiated swine airway epithelial cultures and to use these cultures to examine influenza virus infection and replication. Methods Primary swine respiratory epithelial cells were grown at an air–liquid interface with varying amounts of retinoic acid and epidermal growth factor. Cells grown with optimized concentrations of these factors for 4 weeks differentiated into multilayer epithelial cell cultures resembling the lining of the swine respiratory tract. Influenza virus infection and replication were examined in these cultures. Results/Conclusions Retinoic acid promoted ciliogenesis, whereas epidermal growth factor controlled the thickness of the pseudoepithelium. The optimal concentrations for differentiated swine cell cultures were 1·5 ng/ml epidermal growth factor and 100 nm retinoic acid. Influenza A viruses infected and productively replicated in these cultures in the absence of exogenous trypsin, suggesting that the cultures express a protease capable of activating influenza virus hemagglutinin. Differences in virus infection and replication characteristics found previously in pigs in vivo were recapitulated in the swine cultures. This system could be a useful tool for a range of applications, including investigating influenza virus species specificity, defining cell tropism of influenza viruses in the swine respiratory epithelium, and studying other swine respiratory diseases.     

甲型（H1N1）（2009）流感病毒三重RT-PCR快速检测方法的建立

目的建立一种早期快速检测甲型（H1N1）（2009）流感流行病毒株的方法。方法将2009年分离的甲型（H1N1）流感流行病毒株基因与以前的流感病毒株进行序列比对,找出其特异的基因序列,设计针对甲型（H1N1）（2009）流行株的血凝素（HA）、神经氨酸酶（NA）、核蛋白（NP）基因的三对特异引物,采用RT-PCR同时扩增三条目的片段,通过琼脂糖凝胶电泳进行检测。结果此方法对临床标本的阳性检出率为71%。结论采用三重PCR同时扩增甲型（H1N1）（2009）流感流行病毒的三段特异序列既缩短检测时间又提高了检测特异性,无交叉反应,是一种有效可行的快速检测甲型（H1N1）（2009）流感流行病毒株的方法。     

Isolation of novel triple-reassortant swine H3N2 influenza viruses possessing the hemagglutinin and neuraminidase genes of a seasonal influenza virus in Vietnam in 2010

Surveillance of swine influenza viruses (SIVs) in 31 pig farms in northern and southern parts of Vietnam was conducted. Six H3N2 influenza A viruses were isolated from a pig farm in southern Vietnam. They were novel genetic reassortants between a triple-reassortant SIV and a human seasonal H3N2 virus. Their hemagglutinin and neuraminidase genes were derived from a human virus circulating around 2004-2006 and the remaining genes from a triple-reassortant SIV that originated in North America. This is the first report describing the isolation of a novel triple-reassortant SIV in Vietnam.     

流感病毒蛋白糖基化及其功能研究进展

甲型流感病毒容易发生变异,可以引发局部或全球范围的流行。每次流感病毒新型别的出现都对人类健康和社会经济产生极大的危害,其一直是研究的热点。本文对甲型流感病毒血凝素(hemagglutinin,HA)和神经氨酸酶(neuraminidase,NA)两种蛋白糖基化及其功能、研究方法等方面进行综述。     

Serologic evidence of human influenza virus infections in swine populations,Cambodia

Background This study was conducted from 2006 to 2010 and investigated the seroprevalence of influenza A viruses in Cambodian pigs, including human H1N1, H3N2, 2009 pandemic H1N1 (A(H1N1)pdm09), and highly pathogenic avian H5N1 influenza A viruses. Methods A total of 1147 sera obtained from pigs in Cambodia were tested by haemagglutination inhibition (HI) assays for antibody to human influenza A viruses along with both HI and microneutralization (MN) tests to assess immunological responses to H5N1 virus. The results were compared by year, age, and province. Results Antibodies against a human influenza A virus were detected in 14·9% of samples. A(H1N1)pdm09 virus were dominant over the study period (23·1%), followed by those to human H1N1 (17·3%) and H3N2 subtypes (9·9%). No pigs were serologically positive for avian H5 influenza viruses. The seroprevalence of human H1N1 and H3N2 influenza viruses peaked in 2008, while that of A(H1N1)pdm09 reached a peak in 2010. No significant differences in seroprevalence to human influenza subtypes were observed in different age groups. Conclusions Cambodian pigs were exposed to human strains of influenza A viruses either prior to or during this study. The implications of these high prevalence rates imply human‐to‐swine influenza virus transmission in Cambodia. Although pigs are mostly raised in small non‐commercial farms, our preliminary results provide evidence of sustained human influenza virus circulation in pig populations in Cambodia.     

Experimental inoculation of pigs with pandemic H1N1 2009 virus and HI cross-reactivity with contemporary swine influenza virus antisera

Please cite this paper as: Vincent et al. (2010) Experimental inoculation of pigs with pandemic H1N1 2009 virus and HI cross-reactivity with contemporary swine influenza virus antisera. Influenza and Other Respiratory Viruses 4(2), 53–60 Background A novel A/H1N1 was identified in the human population in North America in April 2009. The gene constellation of the virus was a combination from swine influenza A viruses (SIV) of North American and Eurasian lineages that had never before been identified in swine or other species. Objectives The objectives were to (i) evaluate the clinical response of swine following experimental inoculation with pandemic H1N1 2009; (ii) assess serologic cross-reactivity between H1N1 2009 and contemporary SIV antisera; and (iii) develop a molecular assay to differentiate North American-lineage SIV from H1N1 2009. Methods Experiment 1: Weaned pigs were experimentally infected with A/California/04/2009 (H1N1). Experiment 2: The cross-reactivity of a panel of US SIV H1N1 or H1N2 antisera with three isolates of pandemic A/H1N1 was evaluated. Experiment 3: A polymerase chain reaction (PCR)-based diagnostic test was developed and validated on samples from experimentally infected pigs. Results and Conclusions In experiment 1, all inoculated pigs demonstrated clinical signs and lesions similar to those induced by endemic SIV. Viable virus and antigen were only detected in the respiratory tract. In experiment 2, serologic cross-reactivity was limited against H1N1 2009 isolates, notably among virus antisera from the same HA phylogenetic cluster. The limited cross-reactivity suggests North American pigs may not be fully protected against H1N1 2009 from previous exposure or vaccination and novel tests are needed to rapidly diagnose the introduction of H1N1 2009. In experiment 3, an RT–PCR test that discriminates between H1N1 2009 and endemic North American SIV was developed and validated on clinical samples.     

Seasonal and pandemic influenza surveillance considerations for constructing multicomponent systems

Abstract Surveillance for influenza is essential for the selection of influenza vaccine components and detection of human infections with novel influenza A viruses that may signal the start of a pandemic. Virologic surveillance provides the foundation from which this information can be obtained. However, morbidity and mortality data are needed to better understand the burden of disease, which, in turn, can provide useful information for policy makers relevant to the allocation of resources for prevention and control efforts. Data on the impact of influenza can be used to identify groups at increased risk for severe influenza‐related complications, develop prevention and control policies, and monitor the effect of these policies. Influenza surveillance systems frequently monitor outpatient illness, hospitalizations, and deaths, but selection of influenza surveillance components should be based on the surveillance goals and objectives of the jurisdiction.     

Detection of oseltamivir‐resistant zoonotic and animal influenza A viruses using the rapid influenza antiviral resistance test

Mutations in the influenza virus neuraminidase (NA) that cause reduced susceptibility to the NA inhibitor (NAI) oseltamivir may occur naturally or following antiviral treatment. Currently, detection uses either a traditional NA inhibition assay or gene sequencing to identify known markers associated with reduced inhibition by oseltamivir. Both methods are laborious and require trained personnel. The influenza antiviral resistance test (iART), a prototype system developed by Becton, Dickinson and Company for research use only, offers a rapid and simple method to identify such viruses. This study investigated application of iART to influenza A viruses isolated from non‐human hosts with a variety of NA subtypes (N1‐N9).     

Assessment of seasonality of influenza in swine using field submissions to a diagnostic laboratory in Ontario between 2007 and 2012

Background

Seasonality of any infectious disease is important for its control and monitoring. While influenza seasonality in people has been evaluated extensively, this question has not been studied well in swine populations.

Objective

The goal of this study was to investigate seasonality of influenza in swine, using diagnostic submissions to a diagnostic laboratory.

Methods

Two thousand seven hundred and eleven virological tests within 685 submissions and 5471 serological tests within 193 submissions in Ontario swine between 2007 and 2012 were included in the study and converted to total monthly number of virological and serological submissions, and the number of positive submissions. Data were analyzed by time-series decomposition, fixed-effect Poisson, random-effect Poisson regression with month as uncorrelated and correlated random effects.

Results

All approaches identified seasonality in virological submissions (P < 0·02) with peak in January and April, and a trough in July, but were not able to detect seasonality of influenza-positive virological submissions (P > 0·13). Seasonality of positive serological submissions was identified only if independence between months was assumed (P < 0·03). Almost 50% of serological submissions had evidence of exposure to H3N2 and H1N1.

Conclusions

Thus, this study identified evidence of seasonality in influenza-like disease in swine herds, but not in circulation of influenza virus. Evidence of seasonality in exposure to influenza was dependent on assumptions of between-month correlation. High exposure to H3N2 and H1N1 subtypes warrants more detailed investigation of within-herd influenza virus circulation. The study provides initial insight into seasonality of influenza in swine and should be followed with herd-level studies.     

Transmission of influenza A(H1N1) 2009 pandemic viruses in Australian swine

Please cite this paper as: Deng et al. (2012). Transmission of influenza A(H1N1) 2009 pandemic viruses in Australian swine. Influenza and Other Respiratory Viruses 6(3), e42–e47. Background Swine have receptors for both human and avian influenza viruses and are a natural host for influenza A viruses. The 2009 influenza A(H1N1) pandemic (H1N1pdm) virus that was derived from avian, human and swine influenza viruses has infected pigs in various countries. Objectives To investigate the relationship between the H1N1pdm viruses isolated from piggery outbreaks in Australia and human samples associated with one of the outbreaks by phylogenetic analysis, and to determine whether there was any reassortment event occurring during the human‐pig interspecies transmission. Methods Real‐time RT‐PCR and full genome sequencing were carried out on RNA isolated from nasal swabs and/or virus cultures. Phylogenetic analysis was performed using the Geneious package. Results The influenza H1N1pdm outbreaks were detected in three pig farms located in three different states in Australia. Further analysis of the Queensland outbreak led to the identification of two distinct virus strains in the pigs. Two staff working in the same piggery were also infected with the same two strains found in the pigs. Full genome sequence analysis on the viruses isolated from pigs and humans did not identify any reassortment of these H1N1pdm viruses with seasonal or avian influenza A viruses. Conclusions This is the first report of swine infected with influenza in Australia and marked the end of the influenza‐free era for the Australian swine industry. Although no reassortment was detected in these cases, the ability of these viruses to cross between pigs and humans highlights the importance of monitoring swine for novel influenza infections.     

Influenza Research Database: an integrated bioinformatics resource for influenza research and surveillance

Please cite this paper as: Squires et al. (2012) Influenza research database: an integrated bioinformatics resource for influenza research and surveillance. Influenza and Other Respiratory Viruses 6(6), 404–416. Background The recent emergence of the 2009 pandemic influenza A/H1N1 virus has highlighted the value of free and open access to influenza virus genome sequence data integrated with information about other important virus characteristics. Design The Influenza Research Database (IRD, http://www.fludb.org ) is a free, open, publicly‐accessible resource funded by the U.S. National Institute of Allergy and Infectious Diseases through the Bioinformatics Resource Centers program. IRD provides a comprehensive, integrated database and analysis resource for influenza sequence, surveillance, and research data, including user‐friendly interfaces for data retrieval, visualization and comparative genomics analysis, together with personal log in‐protected ‘workbench’ spaces for saving data sets and analysis results. IRD integrates genomic, proteomic, immune epitope, and surveillance data from a variety of sources, including public databases, computational algorithms, external research groups, and the scientific literature. Results To demonstrate the utility of the data and analysis tools available in IRD, two scientific use cases are presented. A comparison of hemagglutinin sequence conservation and epitope coverage information revealed highly conserved protein regions that can be recognized by the human adaptive immune system as possible targets for inducing cross‐protective immunity. Phylogenetic and geospatial analysis of sequences from wild bird surveillance samples revealed a possible evolutionary connection between influenza virus from Delaware Bay shorebirds and Alberta ducks. Conclusions The IRD provides a wealth of integrated data and information about influenza virus to support research of the genetic determinants dictating virus pathogenicity, host range restriction and transmission, and to facilitate development of vaccines, diagnostics, and therapeutics.