The declaration of the human influenza A pandemic (H1N1) 2009 (H1N1/09) raised important questions, including origin and host range [1], [2]. Two of the three pandemics in the last century resulted in the spread of virus to pigs (H1N1, 1918; H3N2, 1968) with subsequent independent establishment and evolution within swine worldwide [3]. A key public and veterinary health consideration in the context of the evolving pandemic is whether the H1N1/09 virus could become established in pig populations [4]. We performed an infection and transmission study in pigs with A/California/07/09. In combination, clinical, pathological, modified influenza A matrix gene real time RT-PCR and viral genomic analyses have shown that infection results in the induction of clinical signs, viral pathogenesis restricted to the respiratory tract, infection dynamics consistent with endemic strains of influenza A in pigs, virus transmissibility between pigs and virus-host adaptation events. Our results demonstrate that extant H1N1/09 is fully capable of becoming established in global pig populations. We also show the roles of viral receptor specificity in both transmission and tissue tropism. Remarkably, following direct inoculation of pigs with virus quasispecies differing by amino acid substitutions in the haemagglutinin receptor-binding site, only virus with aspartic acid at position 225 (225D) was detected in nasal secretions of contact infected pigs. In contrast, in lower respiratory tract samples from directly inoculated pigs, with clearly demonstrable pulmonary pathology, there was apparent selection of a virus variant with glycine (225G). These findings provide potential clues to the existence and biological significance of viral receptor-binding variants with 225D and 225G during the 1918 pandemic [5].

Real time reverse transcriptase (RRT)-polymerase chain reaction (PCR) for the detection of Eurasian H5 avian influenza virus (AIV) isolates was adapted from an existing protocol, optimized, and validated using a number of genetically diverse H5 isolates (n = 51). These included 34 "Asian lineage" H5N1 highly pathogenic avian influenza (HPAI) viruses (2004-2006), plus 12 other H5 isolates from poultry outbreaks and wild birds in the Eastern Hemisphere (1996-2005). All 51 were positive by H5 Eurasian RRT-PCR. Specificity was assessed by testing representative isolates from all other AL virus subtypes (n = 52), non-AI avian pathogens (n = 8), plus a negative population of clinical specimens derived from AI-uninfected wild birds and poultry (n = 604); all were negative by H5 Eurasian RRT-PCR. RNA was directly extracted from suspect HPAI H5N1 clinical specimens (Africa, Asia, and Europe; 2005-2006; n = 58) from dead poultry and wild birds, and 55 recorded as positive by H5 Eurasian RRT-PCR: Fifty-one of these 55 were in agreement with positive AIV isolation in embryonated chickens' eggs. H5 Eurasian RRT-PCR was invaluable in H5 outbreak diagnosis and management by virtue of its rapidity and high degree of sensitivity and specificity. This method provides a platform for automation that can be applied for large-scale intensive investigations, including surveillance.

Background  Avian influenza (AI) caused by H7 AI viruses (AIVs) of both low pathogenicity (LP) and high pathogenicity (HP) are notifiable poultry diseases. Objectives  Design and validate two RealTime reverse transcriptase polymerase chain reactions (RRT PCRs) for Eurasian H7 AIV detection and pathotyping. Methods  The H7 RRT PCRs amplified within the (i) HA2 and (ii) cleavage site CS regions of the haemagglutinin gene. Both were validated against 65 H7 AIVs, 57 non‐H7 AIVs and 259 poultry swabs in comparison to M gene (AI generic) RRT PCR and virus isolation (VI). An additional 38 swabs and 20 tissue specimens extended validation against M gene RRT PCR. Results  Both H7 RRT PCRs amplified all 61 Eurasian lineage H7 AIVs and none of 57 non‐H7 AIVs. A total of 297 poultry swabs were used to determine diagnostic sensitivity and specificity relative to M gene RRT PCR, sensitivity was 95·4% and 64·6% for the HA2 and CS RRT PCRs respectively, and specificity 97·9% and 99·6% respectively. The H7 HA2 RRT PCR was more sensitive than VI. This was emphasized by analysis of 37 swabs from turkeys infected experimentally with HPAI H7N1 virus sampled at 24 hours post‐inoculation and LPAI H7N1 chicken infections sampled at 40–64 hours. Although less sensitive, usefulness of the H7 CS RRT PCR was confirmed by the correct molecular pathotyping for all 61 Eurasian lineage H7 AIVs tested. Conclusions  The high sensitivity of H7 HA2 RRT PCR confirms its suitability for use in poultry surveillance and disease diagnosis. H7 CS RRT PCR provides an opportunity for rapid pathotyping of H7 AIVs.

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Please cite this paper as: Slomka et al. (2010) Real time reverse transcription (RRT)‐polymerase chain reaction (PCR) methods for detection of pandemic (H1N1) 2009 influenza virus and European swine influenza A virus infections in pigs. Influenza and Other Respiratory Viruses 4(5), 277–293. Background  There is a requirement to detect and differentiate pandemic (H1N1) 2009 (H1N1v) and established swine influenza A viruses (SIVs) by real time reverse transcription (RRT) PCR methods. Objectives  First, modify an existing matrix (M) gene RRT PCR for sensitive generic detection of H1N1v and other European SIVs. Second, design an H1 RRT PCR to specifically detect H1N1v infections. Methods  RRT PCR assays were used to test laboratory isolates of SIV (n = 51; 37 European and 14 North American), H1N1v (n = 5) and avian influenza virus (AIV; n = 43). Diagnostic sensitivity and specificity were calculated for swabs (n = 133) and tissues (n = 116) collected from field cases and pigs infected experimentally with SIVs and H1N1v. Results  The “perfect match” M gene RRT PCR was the most sensitive variant of this test for detection of established European SIVs and H1N1v. H1 RRT PCR specifically detected H1N1v but not European SIVs. Validation with clinical specimens included comparison with virus isolation (VI) as a “gold standard”, while field infection with H1N1v in swine was independently confirmed by sequencing H1N1v amplified by conventional RT PCR. “Perfect match” M gene RRT PCR had 100% sensitivity and 95·2% specificity for swabs, 93·6% and 98·6% for tissues. H1 RRT PCR demonstrated sensitivity and specificity of 100% and 99·1%, respectively, for the swabs, and 100% and 100% for the tissues. Conclusions  Two RRT PCRs for the purposes of (i) generic detection of SIV and H1N1v infection in European pigs, and for (ii) specific detection of H1N1v (pandemic influenza) infection were validated.

BackgroundOne requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes.ResultsThe relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells.ConclusionsBased on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation.

Wild ducks and gulls are the major reservoirs for avian influenza A viruses (AIVs). The mechanisms that drive AIV evolution are complex at sites where various duck and gull species from multiple flyways breed, winter, or stage. The Republic of Georgia is located at the intersection of three migratory flyways: the Central Asian flyway, the East Africa/West Asia flyway, and the Black Sea/Mediterranean flyway. For six complete study years (2010 to 2016), we collected AIV samples from various duck and gull species that breed, migrate, and overwinter in Georgia. We found a substantial subtype diversity of viruses that varied in prevalence from year to year. Low-pathogenic AIV (LPAIV) subtypes included H1N1, H2N3, H2N5, H2N7, H3N8, H4N2, H6N2, H7N3, H7N7, H9N1, H9N3, H10N4, H10N7, H11N1, H13N2, H13N6, H13N8, and H16N3, and two highly pathogenic AIVs (HPAIVs) belonging to clade 2.3.4.4, H5N5 and H5N8, were found. Whole-genome phylogenetic trees showed significant host species lineage restriction for nearly all gene segments and significant differences in observed reassortment rates, as defined by quantification of phylogenetic incongruence, and in nucleotide sequence diversity for LPAIVs among different host species. Hemagglutinin clade 2.3.4.4 H5N8 viruses, which circulated in Eurasia during 2014 and 2015, did not reassort, but analysis after their subsequent dissemination during 2016 and 2017 revealed reassortment in all gene segments except NP and NS. Some virus lineages appeared to be unrelated to AIVs in wild bird populations in other regions, with maintenance of local AIVs in Georgia, whereas other lineages showed considerable genetic interrelationships with viruses circulating in other parts of Eurasia and Africa, despite relative undersampling in the area.IMPORTANCE Waterbirds (e.g., gulls and ducks) are natural reservoirs of avian influenza viruses (AIVs) and have been shown to mediate the dispersal of AIVs at intercontinental scales during seasonal migration. The segmented genome of influenza viruses enables viral RNA from different lineages to mix or reassort when two viruses infect the same host. Such reassortant viruses have been identified in most major human influenza pandemics and several poultry outbreaks. Despite their importance, we have only recently begun to understand AIV evolution and reassortment in their natural host reservoirs. This comprehensive study illustrates AIV evolutionary dynamics within a multihost ecosystem at a stopover site where three major migratory flyways intersect. Our analysis of this ecosystem over a 6-year period provides a snapshot of how these viruses are linked to global AIV populations. Understanding the evolution of AIVs in the natural host is imperative to mitigating both the risk of incursion into domestic poultry and the potential risk to mammalian hosts, including humans.

Commercial heat treatment procedures for molluscan shellfish are based on data obtained for the inactivation of hepatitis A virus (HAV) in cockles. However, the most frequently reported illness associated with consumption of bivalve molluscs is gastroenteritis caused by small round structured viruses (SRSVs) of the Norwalk group. Conditions for inactivation of SRSVs are unknown. In this study a feline calicivirus was used as a model for the SRSV group and conditions for its heat inactivation determined. Experiments showed that feline calicivirus is more readily inactivated in shellfish than HAV, and confirmed that current heating recommendations to the UK shellfish industry are adequate. A reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of calicivirus in shellfish was developed and results compared with isolation in cell culture. The RT-PCR detected virus in some samples that failed to yield virus on culture. This has important implications if molecular virology techniques are to be used in the design and monitoring of shellfish treatment procedures and for routine testing of food samples.