Evaluation of two different swab transport systems in the detection of avian influenza virus excretion from infected Pekin ducks (Anas platyrhynchos)

The role of wild birds in the epidemiology and ecology of influenza A viruses has long been recognised (Alexander, 2007a). As a result of the emergence of a H5N1 highly pathogenic avian influenza (HPAI) virus and the apparent role of wild birds in its spread across Asia, Europe and Africa, avian influenza (AI) wild bird surveillance has been implemented in many countries including, since February 2006, a mandatory programme in the European Union (CEC, 2006a). In the present study the detection of virus excreted from Pekin ducks (Anas platyrhynchos) infected experimentally with A/mallard/England/2126/07 (H3N6) was investigated over a fourteen day period post-infection using cloacal and oropharyngeal swabs, with (wet) and without (dry) viral transport medium which were collected from each duck in alternating order. For influenza A virus matrix gene RNA detection, wet oropharyngeal swabs were significantly more sensitive than dry oropharyngeal on days 4-5 after infection. For cloacal samples, dry swabs were equivalent or superior to wet swabs throughout the study. Although differences in detection between dry and wet swabs were observed, the qualitative bird-level results were unaffected, meaning that the infection status of individual birds was correctly determined.     

The 2003 results of monitoring of influenza A virus in the populations of wild birds in the south of Western Siberia

The paper presents the results of isolation of influenza A virus from 97 cloacal swabs of 11 species of aquatic and semiaquatic wild birds collected on the Chany Lake (the south of Western Siberia, Ob-Irtysh interarea). Six strains with subtypes H2 (2 strains), H3 (3 strains), and H5 (1 strain) were isolated from mallard ducks (Anas platyrhynchos). The total infection rate in the examined birds was 6.2% and that in the ducks was 9.7%. The paper deals with the phylogenetic analysis of hemagglutinin of genes of isolates and with the comparison of the obtained results with the 2002 data in the same region. Analysis of H5 strain hemagglutinin proteolytic site permits one to regard this strain as non-pathogenic.     

Subtyping influenza A virus with monoclonal antibodies and an indirect immunofluorescence assay

The recent association of certain influenza A virus subtypes with clinically relevant phenotypes has led to the increasing importance of subtyping by clinical virology laboratories. To provide clinical laboratories with a definitive immunofluorescence assay for the subtyping of influenza A virus isolates, we generated a panel of monoclonal antibodies (MAbs) against the major circulating influenza A virus subtypes using multiple inactivated H1N1, H3N2, and 2009 H1N1 strains individually as immunogens. Eleven MAbs that target hemagglutinin (HA) of H1N1 and H3N2 subtypes were selected. These MAbs were combined into three subtype-specific reagents, one each for pan-H1 (seasonal and 2009 strains), H3, and 2009 H1, for the subtyping of influenza A virus-positive specimens by indirect immunofluorescence assay (IFA). Each subtype-specific reagent was tested on 21 prototype influenza A virus strains and confirmed to be specific for its intended subtype. In addition, the subtyping reagents did not cross-react with any of 40 other viruses. The clinical performance of the subtyping reagents was evaluated with 75 archived clinical samples collected between 2006 and 2009 using the D(3) Ultra DFA influenza A virus identification reagent (Diagnostic Hybrids, Inc., Athens, OH) and the influenza A virus subtyping reagents by IFA simultaneously. Sixty-four samples grew virus and were subtyped as follows: 30 as H3N2, 9 as seasonal H1N1, and 25 as 2009 H1N1. RT-PCR was used to confirm the influenza A virus subtyping of these samples, and there was 100% agreement with IFA. This subtyping IFA provides clinical laboratories with a cost-effective diagnostic tool for better management of influenza virus infection and surveillance of influenza virus activity.     

Using RRT-PCR analysis and virus isolation to determine the prevalence of avian influenza virus infections in ducks at Minto Flats State Game Refuge, Alaska, during August 2005

This study describes surveillance for avian influenza viruses (AIV) in the Minto Flats State Game Refuge, high-density waterfowl breeding grounds in Alaska. Five hundred paired cloacal samples from dabbling ducks (Northern Pintail, Mallard, Green Wing Teal, and Widgeon) were placed into ethanol and viral transport medium (VTM). Additional ethanol-preserved samples were taken. Of the ethanol-preserved samples, 25.6% were AIV RNA-positive by real-time RT-PCR. The hemagglutinin (HA) and neuraminidase (NA) subtypes were determined for 38 of the first-passage isolates, and four first-passage isolates could not be definitively subtyped. Five influenza A virus HA-NA combinations were identified: H3N6, H3N8, H4N6, H8N4, and H12N5. Differences in the prevalence of AIV infections by sex and by age classes of Northern Pintail and Mallard ducks were detected, but the significance of these differences is undefined. In the 500 paired samples, molecular screening detected positive birds at a higher rate than viral isolation (chi(2) = 8.35, p = 0.0035, df = 1); however, 20 AIV isolates were recovered from PCR-negative ducks. Further research is warranted to compare the two screening protocols' potential for estimating true prevalence in wild birds. Our success during 2005 indicates Minto Flats will be a valuable study site for a longitudinal research project designed to gain further insight into the natural history, evolution, and ecology of AIV in wild birds.     

Type- and subtype-specific detection of influenza viruses in clinical specimens by rapid culture assay.

A rapid culture assay which allows for the simultaneous typing and subtyping of currently circulating influenza A(H1N1), A(H3N2), and B viruses in clinical specimens was developed. Pools of monoclonal antibodies (MAbs) against influenza A and B viruses and MAbs HA1-71 and HA2-76, obtained by immunizing mice with the denatured hemagglutinin subfragments HA1 and HA2 of influenza virus A/Victoria/3/75, were used for immunoperoxidase staining of antigens in infected MDCK cells. MAb HA1-71 reacted exclusively with influenza A viruses of the H3 subtype, while MAb HA2-76 reacted with subtypes H1, H3, H4, H6, H8, H9, H10, H11, and H12, as determined with 78 human, 4 swine, and 10 avian influenza virus reference strains subtyped by the hemagglutination inhibition test. To determine if the technique can be used as a rapid diagnostic test, 263 known influenza virus-positive frozen nasal or throat swabs were inoculated into MDCK cells. After an overnight incubation, the cells were fixed and viral antigens were detected by immunoperoxidase staining. Influenza A viruses of the H1 and H3 subtypes were detected in 31 and 113 specimens, respectively. The subtypes of 10 influenza A virus-positive specimens could not be determined because they contained too little virus. Influenza B viruses were detected in 84 specimens, and 25 specimens were negative. We conclude that this assay is a rapid, convenient, non-labor-intensive, and relatively inexpensive test for detecting, typing, and subtyping influenza viruses in clinical specimens.     

Avian influenza virus isolates from wild birds replicate and cause disease in a mouse model of infection

The direct transmission of highly pathogenic avian influenza (HPAI) viruses to humans in Eurasia and subsequent disease has sparked research efforts leading to better understanding of HPAI virus transmission and pathogenicity in mammals. There has been minimal focus on examining the capacity of circulating low pathogenic wild bird avian influenza viruses to infect mammals. We have utilized a mouse model for influenza virus infection to examine 28 North American wild bird avian influenza virus isolates that include the hemagglutinin subtypes H2, H3, H4, H6, H7, and H11. We demonstrate that many wild bird avian influenza viruses of several different hemagglutinin types replicate in this mouse model without adaptation and induce histopathologic lesions similar to other influenza virus infections but cause minimal morbidity. These findings demonstrate the potential of wild avian influenza viruses to directly infect mice without prior adaptation and support their potential role in emergence of pandemic influenza.     

Novel influenza A viruses isolated from Canadian feral ducks: including strains antigenically related to swine influenza (Hsw1N1) viruses.

Twelve influenza A viruses, antigenically related to the Ho, H1 and Hsw1 subtypes, were isolated from cloacal samples of feral ducks in Canada. Antigenic comparisons showed that these viruses were most closely related to the recent HSW1N1 isolates from man and pigs, whereas in vivo pathogenicity tests revealed differences between the Hsw1N1 viruses from the ducks and those from humans and pigs. Antigenic characterization of 94 additional influenza A viruses from the ducks showed four haemagglutinin subtypes (Hav1, Hav4, Hav5 and Hav7), an unclassified haemagglutinin, and six neuraminidase subtypes (N1, N2, Neq2, Nav1, Nav2 and Nav5) in various combinations, some of which are novel and have not previously been reported. Three of these duck influenza viruses possessed a haemagglutinin antigenically related to that of classical fowl plaque virus. A much higher percentage of virus isolations were from juvenile ducks (18.5%) than from adults (5%). All of the ducks, from which viruses were isolated, appeared healthy at the time of sampling. Serological studies on a limited number of humans and domestic birds living in close proximity to the Canadian ducks revealed no evidence of interspecies transmission. Our findings suggest that these birds serve as a substantial reservoir of antigenically diverse influenza viruses, including isolates antigenically related to the current human and animal influenza viruses. This reservoir in nature may be perpetuated by a cycle involving annual infection of juvenile birds followed by transmission to the remaining susceptible birds until the next congregation during the breeding season.     

Potency of an inactivated influenza vaccine prepared from A/duck/Hong Kong/960/1980 (H6N2) against a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice

H6 influenza viruses are prevalent in domestic and wild birds in Eurasian countries and have been isolated from pigs and a human. To prepare for an influenza pandemic, we have established an influenza virus library consisting of more than 1,300 influenza virus strains, including 144 combinations of 16 hemagglutinin and 9 neuraminidase subtypes. H6 viruses in the library were classified into Early, Group II, Group III, and W312 sublineages and the North America lineage on the basis of their phylogenetic features. Chicken antisera to A/duck/Hong Kong/960/1980 (H6N2) of the Early sublineage broadly reacted with viruses of different sublineages in a hemagglutinin inhibition test. A whole inactivated virus particle vaccine was prepared from A/duck/Hong Kong/960/1980 (H6N2) which was stocked in the influenza virus library. The potency of this vaccine against A/duck/Vietnam/OIE-0033/2012 (H6N2), which belongs to a different sublineage, was evaluated in mice. The test vaccine was sufficiently potent to induce an immune response that reduced the impact of disease caused by a challenge with A/duck/Vietnam/OIE-0033/2012 (H6N2) in mice. The present results indicate that the whole inactivated virus particle vaccine prepared from a virus strain in the influenza virus library is useful as a vaccine against pandemic influenza.     

Genetic variety of influenza A virus in the populations of wild birds in the south of Western Siberia

Influenza A virus variants belonging to H3 and H4 subtypes were isolated from wild ducks inhabiting in the south of Western Siberia. Phylogenetic analysis of hemagglutinin (HA) gene of these viruses has revealed that H3 isolates are closely related to those isolated from the bird inhabiting in West Europe (A/Teal/Germany/wv01r/01, A/Duck/Ukraine/1/63) and China (A/Aquatic bird/Hong Kong/399/99); and those isolated from the birds inhabiting in Germany (A/Garganey/Germany/wv157k/01, A/Teal/Germany/wv153k/01). Thus, closely related influenza A virus variants circulate in the populations of the wild birds inhabiting in greatly spaced regions of Eurasia.     

Variation in viral shedding patterns between different wild bird species infected experimentally with low-pathogenicity avian influenza viruses that originated from wild birds

The prevalence of infection with avian influenza (AI) virus varies significantly between taxonomic Orders and even between species within the same Order. The current understanding of AI infection and virus shedding parameters in wild birds is limited and largely based on trials conducted in mallards (Anas platyrhynchos). The objective of the present study was to provide experimental data to examine species-related differences in susceptibility and viral shedding associated with wild bird-origin low-pathogenicity avian influenza (LPAI) viruses in multiple duck species and gulls. Thus mallards, redheads (Aythya americana), wood ducks (Aix sponsa), and laughing gulls (Leucophaeus atricilla) were inoculated experimentally with three wild mallard-origin LPAI viruses representing multiple subtypes. Variation in susceptibility and patterns of viral shedding associated with LPAI virus infection was evident between the duck and gull species. Consistent with the literature, mallards excreted virus predominantly via the gastrointestinal tract. In wood ducks, redheads, and laughing gulls, AI virus was detected more often in oropharyngeal swabs than cloacal swabs. The results of this study suggest that LPAI shedding varies between taxonomically related avian species. Such differences may be important for understanding the potential role of individual species in the transmission and maintenance of LPAI viruses and may have implications for improving sampling strategies for LPAI detection. Additional comparative studies, which include LPAI viruses originating from non-mallard species, are necessary to further characterize these infections in wild avian species other than mallards and provide a mechanism to explain these differences in viral excretion.     

The influenza virus gene pool in a poultry market in South central china

We surveyed influenza activity in a live poultry market in Central China for 16 months, isolating viruses from 1% of 6360 fecal samples. We obtained multiple H3N6, H9N2, H2N9, H3N3, and H4N6 isolates and single H1N1 and H3N2 isolates. Two distinct H3 molecules were identified; other hemagglutinin subtypes were phylogenetically homogeneous. The H3N6 viruses (9 genotypes) and H9N2 viruses (4 genotypes) were genetically heterogeneous, whereas the H2N9, H3N3 and H4N6 viruses had single genotypes. Thirteen representative viruses were tested for their ability to replicate in quail and chickens. All tested viruses replicated in the respiratory tract of quail. Only nine of the viruses were shed in detectable levels in infected chickens, and four of these were detected in less than 50% of infected birds. A single H4N6 isolate caused disease and systemic spread in chickens. These findings show that quail are broadly susceptible to different subtypes of influenza A virus.     

Development of polymerase chain reaction-based test systems for influenza A virus isolation and typing

Influenza viruses are spread worldwide and cause the disease in birds and mammals, including human beings. Moreover, birds are the natural reservoir of influenza A viruses. Early detection of newly emerging influenza viruses requires permanent monitoring. Traditional viral shedding methods using cell cultures and chicken embryos are time-consuming and laborious analysis when a large number of samples are examined. The paper describes the use of polymerase chain reaction-based test systems to detect influenza A virus and to differentiate its two subtypes: H5 and H7. The developed test systems were evaluated on 19 reference influenza A virus strains. Thereafter, more than 1500 samples from wild and domestic birds, collected in the Russian Federation in 2004-2006, were studied. The data of these test systems were compared with the techniques of viral shedding in the cell cultures and chicken embryos.     

Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples

A fluorogenic PCR-based method (TaqMan-PCR) was developed for typing and subtyping of influenza virus genomes in clinical specimens. The TaqMan-PCR employs a probe technology that exploits the endogenous 5'-3' nuclease activity of the Taq DNA polymerase to allow direct detection of the amplicon by release of a fluorescent reporter during the PCR. Therefore, post-PCR analysis is avoided since hybridization with the fluorogenic probe and quantification of the amplified product is performed simultaneously during PCR cycling. The specificity of the method was evaluated on 86 influenza A (25 H1N1 and 61 H3N2) and 49 influenza B virus reference strains and isolates. The sensitivity of the technique was found to be at the level of 0.1 50% tissue culture infective dose. This TaqMan-PCR was applied prospectively to surveillance work by community-based sampling in Germany during the last two influenza seasons. Seven hundred five throat swabs were analyzed during the winter of 1997-1998. A total of 195 of 705 samples (28%) were positive by PCR. Influenza viruses could be isolated from 125 specimens (18%). During the 1998-1999 season, 1,840 respiratory samples were received. Influenza viruses were isolated from 281 specimens (15%) out of 525 throat swabs (29%) which were positive for influenza A or B virus by TaqMan-PCR. Further differentiation of influenza A virus-positive swabs revealed an intensive circulation of the subtype H3N2 during both seasons, 1997-1998 and 1998-1999. The TaqMan-PCR was much more sensitive than culture and revealed an excellent correlation for typing and subtyping of influenza viruses when samples were positive by both methods.