Distinction of subtype-specific antibodies against European porcine influenza viruses by indirect ELISA based on recombinant hemagglutinin protein fragment-1

BACKGROUND: Serological investigations of swine influenza virus infections and epidemiological conclusions thereof are challenging due to the complex and regionally variable pattern of co-circulating viral subtypes and lineages and varying vaccination regimes. Detection of subtype-specific antibodies currently depends on hemagglutination inhibition (HI) assays which are difficult to standardize and unsuitable for large scale investigations. METHODS: The nucleocapsid protein (NP) and HA1 fragments of the hemagglutinin protein (HA) of five different lineages (H1N1av, H1N1pdm, H1pdmN2, H1N2, H3N2) of swine influenza viruses were bacterially expressed and used as diagnostic antigens in indirect ELISA. RESULTS: Proteins were co-translationally mono-biotinylated and refolded in vitro into an antigenically authentic conformation. Western blotting and indirect ELISA revealed highly subtype-specific antigenic characteristics of the recombinant HA1 proteins although some cross reactivity especially among antigens of the H1 subtype were evident. Discrimination of antibodies directed against four swine influenza virus subtypes co-circulating in Germany was feasible using the indirect ELISA format. CONCLUSIONS: Bacterially expressed recombinant NP and HA1 swine influenza virus proteins served as antigens in indirect ELISAs and provided an alternative to commercial blocking NP ELISA and HI assays concerning generic (NP-specific) and HA subtype-specific sero-diagnostics, respectively, on a herd basis

Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform

Influenza A viruses (IAVs) are important human and animal pathogens with high impact on human and animal health. In Denmark, a passive surveillance program for IAV in pigs has been performed since 2011, where screening tests and subsequent subtyping are performed by reverse transcription quantitative real-time PCR (RT-qPCR). A disadvantage of the current subtyping system is that several assays are needed to cover the wide range of circulating subtypes, which makes the system expensive and time-consuming. Therefore, the aim of the present study was to develop a high-throughput method, which could improve surveillance of swine influenza viruses (swIAVs) and lower the costs of virus subtyping. Twelve qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six assays specific for the internal genes of IAV were developed and optimized for the high-throughput qPCR platform BioMark (Fluidigm). The qPCR assays were validated and optimized to run under the same reaction conditions using a 48.48 dynamic array (48.48DA). The sensitivity and specificity was assessed by testing virus isolates and field samples with known subtypes. The results revealed a performance of the swIAV 48.48DA similar to conventional real-time analysis, and furthermore, the specificity of swIAV 48.48DA was very high and without cross reactions between the assays. This high-throughput system provides a cost-effective alternative for subtyping of swIAVs

Round table on morbilliviruses in marine mammals.

Since 1988 morbilliviruses have been increasingly recognized and held responsible for mass mortality amongst harbour seals (Phoca vitulina) and other seal species. Virus isolations and characterization proved that morbilliviruses from seals in Northwest Europe were genetically distinct from other known members of this group including canine distemper virus (CDV), rinderpest virus, peste des petits ruminants virus and measles virus. An epidemic in Baikal seals in 1987 was apparently caused by a morbillivirus closely related to CDV so that two morbilliviruses have now been identified in two geographically distant seal populations, with only the group of isolates from Northwest Europe forming a new member of the genus morbillivirus: phocid distemper virus (PDV). Because of distemper-like disease, the Baikal seal morbillivirus was tentatively named PDV-2 in spite of its possible identity with CDV. The appearance of morbilliviruses in the Mediterranean Sea causing high mortality amongst dolphins should further increase the research activities on protection strategies for endangered species of marine mammals

Simultaneous detection and differentiation by multiplex real time RT-PCR of highly pathogenic avian influenza subtype H5N1 classic (clade 2.2.1 proper) and escape mutant (clade 2.2.1 variant) lineages in Egypt

<p>Abstract</p> <p>Background</p> <p>The endemic status of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 in Egypt continues to devastate the local poultry industry and poses a permanent threat for human health. Several genetically and antigenically distinct H5N1 lineages co-circulate in Egypt: Strains of clade 2.2.1 proper replicate mainly in backyard birds causing the bulk of human infections, while a variant lineage within 2.2.1 (2.2.1v) appears to be perpetuated mainly in commercial poultry farms in Egypt. Viruses of the 2.2.1v lineage represent drift variants escaping from conventional vaccine-induced immunity and some of these strains also escaped detection by commercial real time reverse transcriptase PCR (RT-qPCR) protocols due to mismatches in the primers/probe binding sites.</p> <p>Results</p> <p>We developed therefore a versatile, sensitive and lineage-specific multiplex RT-qPCR for detection and typing of H5N1 viruses in Egypt. Analytical characterization was carried out using 50 Egyptian HPAIV H5N1 strains isolated since 2006 and 45 other avian influenza viruses (AIV). A detection limit of 400 cRNA copies per ml sample matrix was found. Higher diagnostic sensitivity of the multiplex assay in comparison to other generic H5 or M-gene based RT-qPCR assays were found by examination of 63 swab samples from experimentally infected chickens and 50 AIV-positive swab samples from different host species in the field in Egypt.</p> <p>Conclusions</p> <p>The new multiplex RT-qPCR assay could be useful for rapid high-throughput monitoring for the presence of HPAIV H5N1 in commercial poultry in Egypt. It may also aid in prospective epidemiological studies to further delineate and better control spread of HPAIV H5N1 in Egypt.</p

Pathogenicity of Highly Pathogenic Avian Influenza Virus (H5N1) in Adult Mute Swans

Adult, healthy mute swans were experimentally infected with highly pathogenic avian influenza virus A/Cygnus cygnus/Germany/R65/2006 subtype H5N1. Immunologically naive birds died, whereas animals with preexisting, naturally acquired avian influenza virus–specific antibodies became infected asymptomatically and shed virus. Adult mute swans are highly susceptible, excrete virus, and can be clinically protected by preexposure immunity

Limited Susceptibility of Chickens, Turkeys, and Mice to Pandemic (H1N1) 2009 Virus

To determine susceptibility of chickens, turkeys, and mice to pandemic (H1N1) 2009 virus, we conducted contact exposure and inoculation experiments. We demonstrated that chickens were refractory to infection. However, oculo-oronasally inoculated turkeys and intranasally inoculated mice seroconverted without clinical signs of infection

Experimental Infection and Natural Contact Exposure of Dogs with Avian Influenza Virus (H5N1)

Experiments that exposed influenza virus (H5N1)–infected cats to susceptible dogs did not result in intraspecies or interspecies transmission. Infected dogs showed increased body temperatures, viral RNA in pharyngeal swabs, and seroconversion but not fatal disease

Rapid detection and subtyping of European swine influenza viruses in porcine clinical samples by haemagglutinin- and neuraminidase-specific tetra- and triplex real-time RT-PCRs

BACKGROUND: A diversifying pool of mammalian‐adapted influenza A viruses (IAV) with largely unknown zoonotic potential is maintained in domestic swine populations worldwide. The most recent human influenza pandemic in 2009 was caused by a virus with genes originating from IAV isolated from swine. Swine influenza viruses (SIV) are widespread in European domestic pig populations and evolve dynamically. Knowledge regarding occurrence, spread and evolution of potentially zoonotic SIV in Europe is poorly understood. OBJECTIVES: Efficient SIV surveillance programmes depend on sensitive and specific diagnostic methods which allow for cost‐effective large‐scale analysis. METHODS: New SIV haemagglutinin (HA) and neuraminidase (NA) subtype‐ and lineage‐specific multiplex real‐time RT‐PCRs (RT‐qPCR) have been developed and validated with reference virus isolates and clinical samples. RESULTS: A diagnostic algorithm is proposed for the combined detection in clinical samples and subtyping of SIV strains currently circulating in Europe that is based on a generic, M‐gene‐specific influenza A virus RT‐qPCR. In a second step, positive samples are examined by tetraplex HA‐ and triplex NA‐specific RT‐qPCRs to differentiate the porcine subtypes H1, H3, N1 and N2. Within the HA subtype H1, lineages “av” (European avian‐derived), “hu” (European human‐derived) and “pdm” (human pandemic A/H1N1, 2009) are distinguished by RT‐qPCRs, and within the NA subtype N1, lineage “pdm” is differentiated. An RT‐PCR amplicon Sanger sequencing method of small fragments of the HA and NA genes is also proposed to safeguard against failure of multiplex RT‐qPCR subtyping. CONCLUSIONS: These new multiplex RT‐qPCR assays provide adequate tools for sustained SIV monitoring programmes in Europe