Swine Influenza Isolates Research Articles

Isolation of Swine Influenza A Virus in Cell Cultures and Embryonated Chicken Eggs.

Influenza virus isolation is a procedure to obtain a live and infectious virus that can be used for antigenic characterization, pathogenesis investigation, vaccine production, and so on. Embryonated chicken egg inoculation is traditionally considered the "gold standard" method for influenza virus isolation and propagation. However, many primary cells and continuous cell lines have also been examined or developed for influenza virus isolation and replication. Specifically, influenza A virus in swine (IAV-S) isolation and propagation has been attempted and compared in embryonated chicken eggs, some primary porcine cells, and a number of continuous cell lines. Currently, Madin-Darby canine kidney (MDCK) cells remain the most commonly used cell line for the isolation, propagation, and titration of IAV-S. Virus isolation in embryonated chicken eggs or in different cell lines offers alternative approaches when IAV-S isolation in MDCK cells is unsuccessful. Optimal specimens for IAV-S isolation includes nasal swabs, nasopharyngeal swabs, oral fluids, bronchoalveolar lavage, lung tissues, and so on. In this chapter, we describe the procedures of sample processing, IAV-S isolation in MDCK cells and in embryonated chicken eggs, as well as the methods used for confirming the virus isolation results.

Complete genome sequencing of H1N1pdm09 swine influenza isolates from Nigeria reveals likely reverse zoonotic transmission at the human-animal interface in intensive piggery

ABSTRACTPrevailing agro-ecological conditions and intermingling of human and animals in intensive farms in urban and peri-urban areas in Africa favour cross species transmission of pathogens at the human-animal interface. However, molecular epidemiology studies of zoonotic swine influenza viruses in this region are limited. In this study, isolates of pandemic influenza virus (H1N1pdm09) obtained from pigs in Nigeria were fully sequenced. BLAST of swine influenza virus genes from Nigeria was carried out in GenBank and gene alignment was done using MEGA version 7. Maximum likelihood method (PhyML program) was used to determine gene evolutionary relationships with other viruses and phylogenetic trees were constructed to infer genomic clusters and relationship. Swine influenza viruses isolated and sequenced in this study were monophyletic and 99% congenetic with human isolates from Nigeria, Cameroon, Ghana and USA suggesting reverse zoonotic transmission from humans to pigs in intensive husbandry. A Q240R and S31N substitution among others were detected in the haemagglutinin and matrix genes, respectively, indicating potentials for mutations during interspecies co-mingling and transmission. The A/H1N1pdm09 viruses circulating in pigs that are also exposed to avian influenza in the same epidemiological zones could engender emergence of novel viruses with zoonotic or pandemic potential requiring enhanced surveillance and monitoring.

Detection and Isolation of Swine Influenza A Virus in Spiked Oral Fluid and Samples from Individually Housed, Experimentally Infected Pigs: Potential Role of Porcine Oral Fluid in Active Influenza A Virus Surveillance in Swine.

BackgroundThe lack of seasonality of swine influenza A virus (swIAV) in combination with the capacity of swine to harbor a large number of co-circulating IAV lineages, resulting in the risk for the emergence of influenza viruses with pandemic potential, stress the importance of swIAV surveillance. To date, active surveillance of swIAV worldwide is barely done because of the short detection period in nasal swab samples. Therefore, more sensitive diagnostic methods to monitor circulating virus strains are requisite.MethodsqRT-PCR and virus isolations were performed on oral fluid and nasal swabs collected from individually housed pigs that were infected sequentially with H1N1 and H3N2 swIAV strains. The same methods were also applied to oral fluid samples spiked with H1N1 to study the influence of conservation time and temperature on swIAV infectivity and detectability in porcine oral fluid.ResultsAll swIAV infected animals were found qRT-PCR positive in both nasal swabs and oral fluid. However, swIAV could be detected for a longer period in oral fluid than in nasal swabs. Despite the high detectability of swIAV in oral fluid, virus isolation from oral fluid collected from infected pigs was rare. These results are supported by laboratory studies showing that the PCR detectability of swIAV remains unaltered during a 24 h incubation period in oral fluid, while swIAV infectivity drops dramatically immediately upon contact with oral fluid (3 log titer reduction) and gets lost after 24 h conservation in oral fluid at ambient temperature.ConclusionsOur data indicate that porcine oral fluid has the potential to replace nasal swabs for molecular diagnostic purposes. The difficulty to isolate swIAV from oral fluid could pose a drawback for its use in active surveillance programs.

Imported pigs may have introduced the first classical swine influenza viruses into Mainland China

ObjectivesThe first classical swine influenza A H1N1 viruses were isolated in Mainland China in 1991. To aid surveillance of swine influenza viruses as part of pandemic preparedness, we sought to identify their origin. MethodsWe sequenced and phylogenically analyzed 19 swine influenza viruses isolated in 1991 and 1992 in China and compared them with viruses isolated from other regions during the same period. ResultsAll 19 swine influenza viruses analyzed in our study shared the highest similarity with the classical swine influenza virus A/Swine/Maryland/23239/1991 (H1N1). Phylogenetic trees of eight segmented genes exhibited similar topology, with all segments in the cluster of classical swine influenza viruses. In addition, antigenic analysis also indicated that the tested isolated were related to classical swine influenza isolates. ConclusionsClassical swine H1N1 influenza viruses were predominant in Beijing pig herds during this period. Since both antibody and virus detections did not indicate the presence of CS H1N1 before 1991 in Mainland China, we combined with the data on pigs imported to and exported from China and concluded that these viruses might spread to China via pigs imported from North America and that they could affect the genetic evolution and transmission dynamics of swine influenza viruses in Hong Kong.

Human Infection from Avian-like Influenza A (H1N1) Viruses in Pigs, China

In investigating influenza in an immunodeficient child in China, in December 2010, we found that the influenza virus showed high sequence identity to that of swine. Serologic evidence indicated that viral persistence in pigs was the source of infection. Continued surveillance of pigs and systemic analysis of swine influenza isolates are needed.

Comparison of the receptor binding properties of contemporary swine isolates and early human pandemic H1N1 isolates (Novel 2009 H1N1)

We have utilized glycan microarray technology to determine the receptor binding properties of early isolates from the recent 2009 H1N1 human pandemic (pdmH1N1), and compared them to North American swine influenza isolates from the same year, as well as past seasonal H1N1 human isolates. We showed that the pdmH1N1 strains, as well as the swine influenza isolates examined, bound almost exclusively to glycans with α2,6-linked sialic acid with little binding detected for α2,3-linked species. This is highlighted by pair-wise comparisons between compounds with identical glycan backbones, differing only in the chemistry of their terminal linkages. The overall similarities in receptor binding profiles displayed by pdmH1N1 strains and swine isolates indicate that little or no adaptation appeared to be necessary in the binding component of HA for transmission from pig to human, and subsequent human to human spread.

GiRaF: robust, computational identification of influenza reassortments via graph mining

Reassortments in the influenza virus—a process where strains exchange genetic segments—have been implicated in two out of three pandemics of the 20th century as well as the 2009 H1N1 outbreak. While advances in sequencing have led to an explosion in the number of whole-genome sequences that are available, an understanding of the rate and distribution of reassortments and their role in viral evolution is still lacking. An important factor in this is the paucity of automated tools for confident identification of reassortments from sequence data due to the challenges of analyzing large, uncertain viral phylogenies. We describe here a novel computational method, called GiRaF (Graph-incompatibility-based Reassortment Finder), that robustly identifies reassortments in a fully automated fashion while accounting for uncertainties in the inferred phylogenies. The algorithms behind GiRaF search large collections of Markov chain Monte Carlo (MCMC)-sampled trees for groups of incompatible splits using a fast biclique enumeration algorithm coupled with several statistical tests to identify sets of taxa with differential phylogenetic placement. GiRaF correctly finds known reassortments in human, avian, and swine influenza populations, including the evolutionary events that led to the recent ‘swine flu’ outbreak. GiRaF also identifies several previously unreported reassortments via whole-genome studies to catalog events in H5N1 and swine influenza isolates.

Protocol: Transmission and prevention of influenza in Hutterites: Zoonotic transmission of influenza A: swine & swine workers

BackgroundAmong swine, reassortment of influenza virus genes from birds, pigs, and humans could generate influenza viruses with pandemic potential. Humans with acute infection might also be a source of infection for swine production units. This article describes the study design and methods being used to assess influenza A transmission between swine workers and pigs. We hypothesize that transmission of swine influenza viruses to humans, transmission of human influenza viruses to swine, and reassortment of human and swine influenza A viruses is occurring. The project is part of a Team Grant; all Team Grant studies include active surveillance for influenza among Hutterite swine farmers in Alberta, Canada. This project also includes non-Hutterite swine farms that are experiencing swine respiratory illness.Methods/DesignNurses conduct active surveillance for influenza-like-illness (ILI), visiting participating communally owned and operated Hutterite swine farms twice weekly. Nasopharyngeal swabs and acute and convalescent sera are obtained from persons with any two such symptoms. Swabs are tested for influenza A and B by a real time RT-PCR (reverse transcriptase polymerase chain reaction) at the Alberta Provincial Laboratory for Public Health (ProvLab). Test-positive participants are advised that they have influenza. The occurrence of test-positive swine workers triggers sampling (swabbing, acute and convalescent serology) of the swine herd by veterinarians. Specimens obtained from swine are couriered to St. Jude Children's Research Hospital, Memphis, TN for testing. Veterinarians and herd owners are notified if animal specimens are test-positive for influenza. If swine ILI occurs, veterinarians obtain samples from the pigs; test-positives from the animals trigger nurses to obtain specimens (swabbing, acute and convalescent serology) from the swine workers. ProvLab cultures influenza virus from human specimens, freezes these cultures and human sera, and ships them to St. Jude where sera will be examined for antibodies to swine and human influenza virus strains or reassortants. Full length sequencing of all eight genes from the human and swine influenza isolates will be performed so that detailed comparisons can be performed between them.DiscussionThe declaration of pandemic influenza in June 2009, caused by a novel H1N1 virus that includes avian, swine and human genes, highlights the importance of investigations of human/swine influenza transmission.

Comparison of the usefulness of the CACO-2 cell line with standard substrates for isolation of swine influenza A viruses

Influenza A virus isolation is undertaken routinely in embryonated chicken eggs, but to improve virus detection various cell lines can be used. The CACO-2 cell line was compared to the MDCK cell line and embryonated chicken eggs for the isolation of H1N1, H1N2, H3N2 swine influenza A virus subtypes from clinical specimens. From 2006 to 2008, 104 influenza A samples found positive by PCR from 42 respiratory outbreaks in Italian swine farms were examined by virus isolation. Sixty swine influenza A viruses were isolated (16 H1N1, 28 H1N2 and 16 H3N2) and their growth behaviour on the different substrates was examined. 16/16 H1N1, 28/28 H1N2 and 8/16 of H3N2 viruses were isolated from the CACO-2 cell line, while 7/16 H1N1, 3/28 H1N2 and 16/16 H3N2 viruses were isolated using embryonated chicken eggs. Only 9/16 H1N1, 1/28 H1N2 and 6/16 H3N2 viruses replicated in MDCK cells. A link was found between viral hemagglutinin and the isolation rate on the various substrates. The CACO-2 line was statistically more sensitive (Fisher's exact test, p < 0.01) compared to the MDCK cells and embryonated chicken eggs for the isolation of H1N1 and H1N2 subtypes. In contrast influenza A H3N2 virus was isolated more readily in embryonated chicken eggs than in cultured cells (Fisher's exact test, p < 0.01).

2009 Swine-Origin Influenza A (H1N1) Resembles Previous Influenza Isolates

BackgroundIn April 2009, novel swine-origin influenza viruses (S-OIV) were identified in patients from Mexico and the United States. The viruses were genetically characterized as a novel influenza A (H1N1) strain originating in swine, and within a very short time the S-OIV strain spread across the globe via human-to-human contact.MethodologyWe conducted a comprehensive computational search of all available sequences of the surface proteins of H1N1 swine influenza isolates and found that a similar strain to S-OIV appeared in Thailand in 2000. The earlier isolates caused infections in pigs but only one sequenced human case, A/Thailand/271/2005 (H1N1).SignificanceDifferences between the Thai cases and S-OIV may help shed light on the ability of the current outbreak strain to spread rapidly among humans.

Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic

In March and early April 2009, a new swine-origin influenza A (H1N1) virus (S-OIV) emerged in Mexico and the United States. During the first few weeks of surveillance, the virus spread worldwide to 30 countries (as of May 11) by human-to-human transmission, causing the World Health Organization to raise its pandemic alert to level 5 of 6. This virus has the potential to develop into the first influenza pandemic of the twenty-first century. Here we use evolutionary analysis to estimate the timescale of the origins and the early development of the S-OIV epidemic. We show that it was derived from several viruses circulating in swine, and that the initial transmission to humans occurred several months before recognition of the outbreak. A phylogenetic estimate of the gaps in genetic surveillance indicates a long period of unsampled ancestry before the S-OIV outbreak, suggesting that the reassortment of swine lineages may have occurred years before emergence in humans, and that the multiple genetic ancestry of S-OIV is not indicative of an artificial origin. Furthermore, the unsampled history of the epidemic means that the nature and location of the genetically closest swine viruses reveal little about the immediate origin of the epidemic, despite the fact that we included a panel of closely related and previously unpublished swine influenza isolates. Our results highlight the need for systematic surveillance of influenza in swine, and provide evidence that the mixing of new genetic elements in swine can result in the emergence of viruses with pandemic potential in humans.

Swine Influenza A Evolution via Recombination - Genetic Drift Reservoir

AbstractThe looming influenza pandemic has focused attention^1-4^ on the rapid evolution of H5N1 and other human and avian serotypes. The basic tenets of influenza genetics^5^ define gradual changes as drifts caused by point mutations created by a polymerase that lacks a proof reading function. More abrupt changes have been linked to reassortment, which shuffles the eight sub-genomic segments of the influenza genome in dually infected host. The complex evolution of these viruses has created a challenge in vaccine development. Swine influenza isolates from 2003 and 2004 have been identified^6^ that have acquired a human influenza gene, PB1. My analysis of the eight gene segments found large portions of two genes, PB2 and PA, which were identical matches with 1977 swine isolates^7,8^. Additional regions were exact matches with 1998 and 2002 isolates,^9,10^ demonstrating homologous recombination between earlier genomes. The absolute fidelity discounts the role of point mutations in gene drift. Moreover the human PB1 gene represented a reservoir for acquisition of polymorphisms in human seasonal flu. These observations challenge the basic tenets of influenza genetics and provide a method for predicting the changes in seasonal and pandemic influenza, as well as other rapidly evolving genomes.

The nucleotide sequence of the HA1 of the haemagglutinin of an HI avian influenza virus isolate from turkeys in Germany provides additional evidence suggesting recent transmission from pigs

Summary The nucleotide sequence encoding the HA1 portion of the haemagglutinin gene of the influenza virus A/turkey/Germany/2482/90j isolated from birds kept in an area of many pig farms, was determined and compared with those of recent avian and swine influenza isolates. It was found to be closest to the ‘avian‐like’ swine H1N1 influenza viruses that have been reported in Europe since the early 1980s and may represent good evidence for transmission of these viruses back to birds after they have become established in pigs.

Recent H3N2 swine influenza virus with haemagglutinin and nucleoprotein genes similar to 1975 human strains

Of the four pandemic strains of human influenza A virus observed this century, the 1977 virus strain was very similar in all genes to a 1950 isolate. Since mammalian influenza A viruses change annually by genetic drift, this reappearance could only be attributed at that time to conservation of the virus in a frozen state. We report here the isolation of swine influenza A viruses with haemagglutinin and nucleoprotein genes which are virtually identical to those of the human virus that circulated in 1975. We have also found serological evidence that this virus is circulating extensively in Quebec swine herds. We propose that human-like H3N2 influenza A strains may remain invariant for long periods in swine, which may serve as a reservoir for human pandemics.

Correlation of influenza A virus nucleoprotein genes with host species

The RNAs coding for the nucleoproteins of a panel of influenza isolates from human and nonhuman hosts were compared by RNA-RNA hybridization to determine the extent of genetic diversity of this protein and to determine if related nucleoproteins (NP) are consistenly found in viruses from certain hosts. Five nucleoprotein groups were defined. Group 1 contains nearly all of the avian influenza viruses, group 2 includes only certain viruses isolated from gulls, group 3 includes all recent equine influenza strains, group 4 contains only equine/Prague/1/56, and group 5 contains all human and swine influenza isolates. The maintenance of specific nucleoproteins in viruses from certain species suggests that these proteins have evolved functionally significant differences that favor their replication in a specific host.