In Vitro Reassortment between Endemic H1N2 and 2009 H1N1 Pandemic Swine Influenza Viruses Generates Attenuated Viruses

The pandemic H1N1 (pH1N1) influenza virus was first reported in humans in the spring of 2009 and soon thereafter was identified in numerous species, including swine. Reassortant viruses, presumably arising from the co-infection of pH1N1 and endemic swine influenza virus (SIV), were subsequently identified from diagnostic samples collected from swine. In this study, co-infection of swine testicle (ST) cells with swine-derived endemic H1N2 (MN745) and pH1N1 (MN432) yielded two reassortant H1N2 viruses (R1 and R2), both possessing a matrix gene derived from pH1N1. In ST cells, the reassortant viruses had growth kinetics similar to the parental H1N2 virus and reached titers approximately 2 log10 TCID50/mL higher than the pH1N1 virus, while in A549 cells these viruses had similar growth kinetics. Intranasal challenge of pigs with H1N2, pH1N1, R1 or R2 found that all viruses were capable of infecting and transmitting between direct contact pigs as measured by real time reverse transcription PCR of nasal swabs. Lung samples were also PCR-positive for all challenge groups and influenza-associated microscopic lesions were detected by histology. Interestingly, infectious virus was detected in lung samples for pigs challenged with the parental H1N2 and pH1N1 at levels significantly higher than either reassortant virus despite similar levels of viral RNA. Results of our experiment suggested that the reassortant viruses generated through in vitro cell culture system were attenuated without gaining any selective growth advantage in pigs over the parental lineages. Thus, reassortant influenza viruses described in this study may provide a good system to study genetic basis of the attenuation and its mechanism

Computationally Efficient Direction Finding for a Mixture of Circular and Strictly Noncircular Sources with Uniform Rectangular Arrays

In this paper, a novel two-dimensional (2D) direction-of-arrival (DOA) estimation algorithm for the mixed circular and strictly noncircular sources is proposed. A general array model with a mixture of signals is firstly built based on uniform rectangular arrays (URAs), and then, the approach, which uses the rank-reduction-based ROOT-MUSIC, can solve 2D DOA estimation problem. Besides, the theoretical error of the proposed algorithm, a criterion of the performance for evaluation, is analyzed by the first-order Taylor expression using second-order statistics. As verified by the simulation results, a better DOA estimation performance and a lower computational complexity are achieved by the proposed algorithm than the existing methods resorting to the noncircularity of the incoming signals

Comparison of Porcine Airway and Intestinal Epithelial Cell Lines for the Susceptibility and Expression of Pattern Recognition Receptors upon Influenza Virus Infection

Influenza viruses infect the epithelial cells of the swine respiratory tract. Cell lines derived from the respiratory tract of pigs could serve as an excellent in vitro model for studying the pathogenesis of influenza viruses. In this study, we examined the replication of influenza viruses in the MK1-OSU cell line, which was clonally derived from pig airway epithelium. MK1-OSU cells expressed both cytokeratin and vimentin proteins and displayed several sugar moieties on the cell membrane. These cells also expressed both Sial2-3Gal and Sial2-6Gal receptors and were susceptible to swine influenza A, but not to human B and C viruses. Interestingly, these cells were also permissive to infection by influenza D virus that utilized 9-O-acetylated glycans. To study the differences in the expression of pattern recognition receptors (PRRs) upon influenza virus infection in the respiratory and digestive tract, we compared the protein expression of various PRRs in MK1-OSU cells with that in the SD-PJEC cell line, a clonally derived cell line from the porcine jejunal epithelium. Toll-like receptor 7 (TLR-7) and melanoma differentiation-associated protein 5 (MDA5) receptors showed decreased expression in influenza A infected MK1-OSU cells, while only TLR-7 expression decreased in SD-PJEC cells. Further research is warranted to study the mechanism behind the virus-mediated suppression of these proteins. Overall, this study shows that the porcine respiratory epithelial cell line, MK1-OSU, could serve as an in-vitro model for studying the pathogenesis and innate immune responses to porcine influenza viruses

Multicycle growth curves for pH1N1 and H1N2, both before (x+0) and after 10 passes (x+10) in A549 cells, as well as reassortant viruses R1 and R2.

<p>Data represent means of triplicate growth curves.</p

Single nucleotide polymorphisms identified in pH1N1 and H1N2 following 10 passages in ST cells as compared to genome sequences prior to cell passaging^a.

<p>For non-synonymous mutations, amino acid changes are shown in parentheses.</p>a<p>For reassortant viruses R1 and R2, single nucleotide polymorphisms are reported based on changes observed from the parent segment at x+0.</p

Four pigs were added to each of four rooms containing pigs challenged intranasally with 2 mL of 6.2 log10 TCID50/mL with H1N2, pH1N1, R1 or R2 at day one post infection.

<p>Nasal swabs were collected daily and analyzed by rt-RT-PCR for virus shedding. Data represent the mean Ct value for the four nasal swabs collected at each time point. Samples with different subscripts at a time point are statistically different (P<0.05).</p

Lung samples were collected on day five post infection and analyzed for TCID50 by virus replication assay in ST cells.

<p>Data represent the mean log₁₀ TCID₅₀/mL for the four lung samples per treatment group. Samples with different subscripts within the same treatment group are statistically different (P<0.05).</p

Domestic Pigs Are Susceptible to Infection with Influenza B Viruses

Influenza B virus (IBV) causes seasonal epidemics in humans. Although IBV has been isolated from seals, humans are considered the primary host and reservoir of this important pathogen. It is unclear whether other animal species can support the replication of IBV and serve as a reservoir. Swine are naturally infected with both influenza A and C viruses. To determine the susceptibility of pigs to IBV infection, we conducted a serological survey for U.S. Midwest domestic swine herds from 2010 to 2012. Results of this study showed that antibodies to IBVs were detected in 38.5% (20/52) of sampled farms, and 7.3% (41/560) of tested swine serum samples were positive for IBV antibodies. Furthermore, swine herds infected with porcine reproductive and respiratory syndrome virus (PRRSV) showed a higher prevalence of IBV antibodies in our 2014 survey. In addition, IBV was detected in 3 nasal swabs collected from PRRSV-seropositive pigs by real-time RT-PCR and sequencing. Finally, an experimental infection in pigs, via intranasal and intratracheal routes, was performed using one representative virus from each of the two genetically and antigenically distinct lineages of IBVs: B/Brisbane/60/2008 (Victoria lineage) and B/Yamagata/16/1988 (Yamagata lineage). Pigs developed influenza-like symptoms and lung lesions, and they seroconverted after virus inoculation. Pigs infected with B/Brisbane/60/2008 virus successfully transmitted the virus to sentinel animals. Taken together, our data demonstrate that pigs are susceptible to IBV infection; therefore, they warrant further surveillance and investigation of swine as a potential host for human IBV