Avian Influenza Virus Isolates Research Articles

Isolation and molecular characterization of reassortant H11N3 subtype avian influenza viruses isolated from domestic ducks in Zhejiang Province in China.

In July 2013, six H11N3 subtype avian influenza viruses (AIVs) were isolated from domestic ducks in Zhejiang Province in Eastern China. These strains were characterized by whole genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that these strains clustered in the AIV Eurasian lineage, and these strains received their genes from H11, H7, and H1 AIVs in Eastern China. These strains were found to be minimally pathogenic in mice, and were able to replicate in mice without prior adaptation. Continued surveillance is needed considering the important role of domestic ducks in AIV reassortment.

Isolation and Genetic Characterization of Avian Influenza Viruses Isolated from Wild Birds in the Azov-Black Sea Region of Ukraine (2001-2012).

Wild bird surveillance for avian influenza virus (AIV) was conducted from 2001 to 2012 in the Azov - Black Sea region of the Ukraine, considered part of the transcontinental wild bird migration routes from northern Asia and Europe to the Mediterranean, Africa, and southwest Asia. A total of 6281 samples were collected from wild birds representing 27 families and eight orders for virus isolation. From these samples, 69 AIVs belonging to 15 of the 16 known hemagglutinin (HA) subtypes and seven of nine known neuraminidase (NA) subtypes were isolated. No H14, N5, or N9 subtypes were identified. In total, nine H6, eight H1, nine H5, seven H7, six H11, six H4, five H3, five H10, four H8, three H2, three H9, one H12, one H13, one H15, and one H16 HA subtypes were isolated. As for the NA subtypes, twelve N2, nine N6, eight N8, seven N7, six N3, four N4, and one undetermined were isolated. There were 27 HA and NA antigen combinations. All isolates were low pathogenic AIV except for eight highly pathogenic (HP) AIVs that were isolated during the H5N1 HPAI outbreaks of 2006-08. Sequencing and phylogenetic analysis of the HA genes revealed epidemiological connections between the Azov-Black Sea regions and Europe, Russia, Mongolia, and Southeast Asia. H1, H2, H3, H7, H8, H6, H9, and H13 AIV subtypes were closely related to European, Russian, Mongolian, and Georgian AIV isolates. H10, H11, and H12 AIV subtypes were epidemiologically linked to viruses from Europe and Southeast Asia. Serology conducted on serum and egg yolk samples also demonstrated previous exposure of many wild bird species to different AIVs. Our results demonstrate the great genetic diversity of AIVs in wild birds in the Azov-Black Sea region as well as the importance of this region for monitoring and studying the ecology of influenza viruses. This information furthers our understanding of the ecology of avian influenza viruses in wild bird species.

Isolation and genetic characterization of novel reassortant H6N6 subtype avian influenza viruses isolated from chickens in eastern China.

H6 subtype avian influenza viruses (AIVs) possess the ability to cross the species barrier to infect mammals and pose a threat to human health. From June 2014 to July 2015, 12 H6N6 AIVs were isolated from chickens in live-poultry markets in Zhejiang Province, Eastern China. Phylogenetic analysis showed that these isolates received their genes from H6 and H9N2 subtype AIVs of poultry in China. These novel reassortant viruses showed moderate pathogenicity in mice and were able to replicate in mice without prior adaptation. Considering that novel reassorted H6N6 viruses were isolated from chickens in this study, it is possible that these chickens play an important role in the generation of novel reassorted H6N6 AIVs, and these results emphasize the need for continued surveillance of the H6N6 AIVs circulating in poultry.

HOST-RESTRICTED RANGE OF H5N1 AVIAN INFLUENZA VIRUSES ASSOCIATED WITH CHARACTERS OF POLYMERASE COMPLEX OF PB2 AND PB1-F2 PROTEINS

Epidemiological studies on H5N1 avian influenza viruses indi-cated that the viruses do not transmit efficiently from human to human. Transmissibility of viruses among human population is very complex and polygenic. Studies on molecular determinants facilitating interspecies transmission of the viruses suggested that two polymerase complex proteins such as PB2 and PB1-F2 are important. PB2 is critical in determining the host specificity, whereas mutations in PB1-F2 increase the viral virulence. The study aimed to characterize the polymerase complex of PB2 and PB1-F2 proteins of H5N1 avian influenza viruses isolated from Indonesia. The DNA samples encoding the PB2 and PB1-F2 complex proteins of several H5N1 isolates were sequenced and analyzed. Pathogenicity of the viruses was studied in both avian and mammal models. The sequencing results showed that there was no mutation in both proteins of PB2 and PB1-F2 of the avian influenza virus isolates. The molecular character for host specificity was consistent with the animal experiment results. The H5N1 virus isolates were only infectious and pathogenic in chickens, but not in BALB/C mice as the mammal model. The study suggests that host range of H5N1 virus isolates of Indonesia is restricted to poultry and not transmisible to mammal model used in this study.

HOST-RESTRICTED RANGE OF H5N1 AVIAN INFLUENZA VIRUSES ASSOCIATED WITH CHARACTERS OF POLYMERASE COMPLEX OF PB2 AND PB1-F2 PROTEINS

Epidemiological studies on H5N1 avian influenza viruses indi-cated that the viruses do not transmit efficiently from human to human. Transmissibility of viruses among human population is very complex and polygenic. Studies on molecular determinants facilitating interspecies transmission of the viruses suggested that two polymerase complex proteins such as PB2 and PB1-F2 are important. PB2 is critical in determining the host specificity, whereas mutations in PB1-F2 increase the viral virulence. The study aimed to characterize the polymerase complex of PB2 and PB1-F2 proteins of H5N1 avian influenza viruses isolated from Indonesia. The DNA samples encoding the PB2 and PB1-F2 complex proteins of several H5N1 isolates were sequenced and analyzed. Pathogenicity of the viruses was studied in both avian and mammal models. The sequencing results showed that there was no mutation in both proteins of PB2 and PB1-F2 of the avian influenza virus isolates. The molecular character for host specificity was consistent with the animal experiment results. The H5N1 virus isolates were only infectious and pathogenic in chickens, but not in BALB/C mice as the mammal model. The study suggests that host range of H5N1 virus isolates of Indonesia is restricted to poultry and not transmisible to mammal model used in this study.

Evidence for genetic variation of Eurasian avian influenza viruses of subtype H15: the first report of an H15N7 virus.

Since the first detection of H15 avian influenza viruses(AIVs) in Australia in 1979, only seven H15 strains have been reported. A new H15 AIV was detected in Ukraine in 2010, carrying the unique HA-NA subtype combination H15N7. This virus replicated efficiently in chicken eggs, and antisera against it reacted strongly with the homologous antigen, but with lower titers when using the reference Australian antigen. The amino acid motifs of the HA cleavage site and receptor-binding site were different from those in the Australian viruses. The new virus, together with an H15 virus from Siberia from 2008, constitutes a new clade of H15 AIV isolates.

Serial passage in ducks of a low-pathogenic avian influenza virus isolated from a chicken reveals a high mutation rate in the hemagglutinin that is likely due to selection in the host.

A comparative study of the ability of three low-pathogenic avian influenza virus (LPAIV) isolates to be transmitted from duck to duck was performed. Pekin ducks were inoculated with two LPAIV isolates from chickens (A/Ck/PA/13609/93 [H5N2], H5N2-Ck; A/Ck/TX/167280-4/02 [H5N3], H5N3-Ck) and one isolate from a wild bird (A/Mute Swan/ MI/451072/06 [H5N1], H5N1-WB). During the establishment of the passage model, only two viruses (H5N1, H5N2) were able to be transmitted from duck to duck. Transmission of these isolates was dependent on the inoculation dose and route of infection. Analysis of swab samples taken from ducks revealed that the wild-bird isolate, H5N1-WB, was primarily shed via the cloacal route. The chicken isolate, H5N2-Ck, was isolated from cloacal as well as oro-pharyngeal swabs. Analysis of the amino acid sequences of the viral surface glycoproteins showed that the hemagglutinin (HA) of the H5N2-Ck isolate was under a stronger evolutionary pressure than the HA of the H5N1-WB isolate, as indicated by the presence of a larger number of amino acid changes observed during passage. The neuraminidase (NA) of both viruses showed either no (in the case of H5N1-WB) or very few amino acid changes.

Isolation and characterization of a novel H10N2 avian influenza virus from a domestic duck in Eastern China

During the surveillance for avian influenza viruses (AIVs) in live poultry markets (LPMs) in Eastern China, in 2013, an H10N2 AIV was isolated from a domestic duck. Phylogenetic analysis showed that this strain received its genes from H10, H1 and H7 AIVs of wild birds in China. The virulence of this strain was examined in chickens and mice, and was found to be low pathogenic in chickens but demonstrated moderate pathogenicity in mice. These results suggest that active surveillance of AIVs in LPMs should be used in an early warning system for avian influenza outbreaks.

Variation in protection of four divergent avian influenza virus vaccine seed strains against eight clade 2.2.1 and 2.2.1.1. Egyptian H5N1 high pathogenicity variants in poultry.

BackgroundHighly pathogenic (HP) H5N1 avian influenza virus (AIV) was introduced to Egyptian poultry in 2006 and has since become enzootic. Vaccination has been utilized as a control tool combined with other control methods, but for a variety of reasons, the disease has not been eradicated. In 2007, an antigenically divergent hemagglutinin subclade, 2.2.1.1, emerged from the original clade 2.2.1 viruses.ObjectivesThe objective was to evaluate four diverse AIV isolates for use as vaccines in chickens, including two commercial vaccines and two additional contemporary isolates, against challenge with numerous clade 2.2.1 and clade 2.2.1.1 H5N1 HPAIV Egyptian isolates to assess the variation in protection among different vaccine and challenge virus combinations.MethodsVaccination-challenge studies with four vaccines and up to eight challenge strains with each vaccine for a total of 25 vaccination-challenge groups were conducted with chickens. An additional eight groups served as sham-vaccinated controls. Mortality, mean death time, morbidity, virus, and pre-challenge antibodies were evaluated as metrics of protection. Hemagglutination inhibition data were used to visualize the antigenic relatedness of the isolates.Results and conclusionsAlthough all but one vaccine-challenge virus combination significantly reduced shed and mortality as compared to sham vaccinates, there were differences in protection among the vaccines relative to one another based on challenge virus. This emphasizes the difficulty in vaccinating against diverse, evolving virus populations, and the importance of selecting optimal vaccine seed strains for successful HPAIV control.

Improved methods for isolation of avian influenza virus

Isolation of viruses using chick embryos is a classical virological method. Inoculation of the allantoic cavity and use of allantoic fluid is a common method of passaging isolated avian influenza viruses. In the present study, 2490 fresh fecal samples and 4967 old fecal samples were investigated and subjected to conventional passaging (allantoic fluid method). Two newly developed methods-the allantochorion and allantoic fluid mixed method and the chick embryo and allantoic fluid mixed method-were also examined. The rates of influenza virus isolation for these three methods were compared. There appeared to be little difference among these methods when fresh fecal samples were used. However, for the old fecal samples, isolation rates for influenza virus were significantly higher for the chick embryo and allantoic fluid mixed method compared with the conventional allantoic fluid method. All viruses isolated using the conventional allantoic fluid method were isolated successfully using the two newly developed methods. These results suggest that using chick embryos in conjunction with allantoic fluid is effective for early virus isolation, especially for fecal samples that are not fresh. Additionally, practical chick embryo passage methods are described that improve significantly the rate of isolation of influenza viruses from fecal samples of migratory birds in a complex wild ecological environment.

Evaluation of direct antiviral activity of the Deva-5 herb formulation and extracts of five Asian plants against influenza A virus H3N8.

BackgroundThe herb formulation Deva-5 is used in traditional medicine to treat acute infectious diseases. Deva-5 is composed of five herbs: Gentiana decumbens L., Momordica cochinchinensis L., Hypecoum erectum L., Polygonum bistorta L., and Terminalia chebula Retz. Deva-5 and its five components were investigated for in vitro antiviral activity against avian influenza A virus subtype H3N8.MethodsThe water extracts of the herbal parts of G. decumbens, H. erectum and P. bistorta, the seeds of T. chebula and M. cochinchinensis and Deva-5 were prepared by boiling and clarified by low-speed centrifugation and filtration. To assess the antiviral properties, avian influenza virus isolate A/Teal/Tunka/7/2010(H3N8) was incubated at 37°C for 30 min in the presence and absence of the extracts of five plants and DEVA-5 in various concentrations. Subsequently, the concentration of infectious virus in each sample was determined by plaque assays. Neutralisation indexes and 90% plaque reduction concentrations were estimated for each extract, and the significance of the data was evaluated using statistical methods.ResultsThe extracts of G. decumbens, H. erectum, P. bistorta and Deva-5 demonstrated no significant toxicity at concentrations up to 2%, whereas extracts of T. chebula and M. cochinchinensis were well-tolerated by Madin-Darby canine kidney cells at concentrations up to 1%. The extracts of H. erectum, M. cochinchinensis and T. chebula reduced the titre of A/Teal/Tunka/7/2010 (H3N8) by approximately five-fold (p ≤ 0.05). The other three extracts did not significantly reduce the infectivity of the virus. The plaque reduction neutralisation tests revealed that none of the extracts tested were able to inhibit formation of plaques by 90%. However, three extracts, H. erectum, T. chebula and M. cochinchinensis, were able to inhibit formation of plaques by more than 50% at low dilutions from 1:3 to 1:14. The T. chebula extract had a concentration-dependent inhibitory effect.ConclusionsFor the first time, the consistent direct antiviral action of the extracts of H. erectum, T. chebula and M. cochinchinensis was detected. These extracts significantly reduced the infectivity of influenza A virus H3N8 in vitro when used at high concentrations (0.5–1%). However, Deva-5 itself and the remainder of its components did not exhibit significant antiviral action. The results suggest that H. erectum, T. chebula and M. cochinchinensis plants contain substances with direct antiviral activity and could be promising sources of new antiviral drugs.

Cleavage site stability of Egyptian highly pathogenic avian influenza viruses in backyard chickens during 2009–2011

Two distinguishable subclades of H5N1 (classic and variant strains) are cocirculating among the poultry populations in Egypt despite the intensive vaccination programs. A study to investigate the genetic relationship between avian influenza virus (AIV) isolates from backyard chickens in Sharkia (2009-2011), subclades, and commercially available vaccines was carried out. Forty-eight suspected AIV infected birds were clinically examined and used for virus isolation followed by reverse transcription-polymerase chain reaction. Four H5N1 virus isolates were sequenced and analyzed. The intravenous pathogenicity index (IVPI) of three AIV isolates was determined. Thirty-four hemagglutinating viral agents (30 AIV subtype H5N1 and 4 Newcastle disease virus) were detected. Both the nucleotide and amino acid sequence identities of four H5N1 virus isolates (SHZA-0412/2009, SHZA-0801/2010, SHMK-1903/2010, and SHAH-1403/2011) were high--98.4-99.7% and 100%, respectively--indicative of their genetic homogeneity. The hemagglutinin cleavage site characterization revealed the presence of multiple basic amino acids (-PQRERRRKKR/GL-) of the highly pathogenic phenotype. These results were supported by IVPI in chickens of 2.69-2.90. The similarity of our isolates with H5N1 AIV vaccine strains (93.9-95.1%) was higher than that with H5N2 strains (77.8-91.9%). The divergence of four sequences with classic and variant lineages is 2-2.7% and 2.3-3%, respectively, with two amino acid substitutions (A249P and N251Y). Genetic characterization and IVPI data of backyard H5N1 isolates are indicative of a highly pathogenic avian influenza virus with hemagglutinin cleavage site constancy and two amino acids substitutions with Egyptian classic and variant lineages, suggesting a beginning of antigenic drift.

Avian influenza virus isolation, propagation, and titration in embryonated chicken eggs.

Avian influenza virus and some mammalian influenza A viruses are usually isolated, propagated, and titrated in embryonated chicken eggs (ECE). Most any sample type can be accommodated for culture with appropriate processing. Isolation may also be accomplished in cell culture particularly if mammalian lineage isolates are suspected, for example, swine influenza in turkey specimens. Culture is highly sensitive, but is not specific for influenza A, which may be an advantage because a sample may be screened for several agents at once. Once an agent is isolated in culture, the presence of influenza viruses is confirmed with any of several assays. The methods for propagating virus isolates in ECE are described.

A novel pyrosequencing assay for the detection of neuraminidase inhibitor resistance-conferring mutations among clinical isolates of avian H7N9 influenza virus

A novel reassortant avian influenza A virus (H7N9) emerged in humans in Eastern China in late February 2013. All virus strains were resistant to adamantanes (amantadine and rimantadine), but susceptible to neuraminidase inhibitors (NAIs) (oseltamivir and zanamivir). One strain (A/shanghai/1/2013) contained the R294K substitution in the neuraminidase (NA) gene, indicating resistance to oseltamivir. Pyrosequencing has proven to be a useful tool in the surveillance of drug resistance in influenza A viruses. Here, we describe a reverse transcription (RT)-PCR assay coupled with pyrosequencing to identify the NA residues E120, H276, and R294 (N9 numbering) of H7N9 viruses. A total of 43 specimens (26 clinical samples and 17 isolates) were tested. Only one isolate containing the E120V heterogenic mutation was detected by pyrosequencing and confirmed by Sanger sequencing. However, this mutation was not detected in the original clinical specimen. Since virus isolation might lead to the selection of variants that might not fully represent the virus population in the clinical specimens, we suggest that using pyrosequencing to detect NAI resistance in H7N9 viruses directly from clinical specimens rather than from cultured isolates. No cross-reactions with other types of influenza virus and respiratory tract viruses were found, and this assay has a sensitivity of 100 copies of synthetic RNA for all three codons. The high sensitivity and specificity of the assay should be sufficient for the detection of positive clinical specimens. In this study, we provide a rapid and reliable method for the characterization of NAI resistance in H7N9 viruses.

Replication Efficiency of Influenza A Virus H9N2: A Comparative Analysis Between Different Origin Cell Types

Background: Efficient isolation and detection of low pathogenic avian influenza viruses from surveillance samples continues to be a high priority. Currently, the new cell lines are considered for supporting the replication to high virus strains titers. Objectives: The replication efficiency of a low pathogenic avian influenza virus in different origin cells was evaluated under different conditions. Materials and Methods: Chicken embryo fibroblast (CEF) cell and human alveolar epithelial cell line (A549) were infected with H9N2 at a multiplicity of infection of 0.1. The amount of infectious virus released into the cell culture supernatants at various post-infection time intervals were tittered by tissue culture infectious dose (TCID50) assay. The impact of these cells adaptation was investigated by determination the virus genes nucleotide sequences. Results: The influenza virus infectivity was not significant difference in these cells in the presence of trypsin. The results of fusion assay and determination of cellular protease confirmed that A549 cells support virus entry with or without supplemental trypsin. However, the H9N2 virus showed lower titer and infectivity in the trypsin–free infected A549 cells within longer time. The comparative sequence analysis indicated several simultaneously nucleotide substitutions were occurred in NA of the virus replicated in A549 cells resulted in two fixed amino acid changes at positions G320 to A and G414 to A up to the fifth passage. Conclusions: After seven consecutive passages of both cell cultures, the H9N2 virus showed similar antigenicity, also no change on viral titer level and virus replication behavior in adaptation was found. The results highlighted the use of A549 cells for efficient virus isolation.

Effect of infectious bursal disease virus on pathogenicity of avian influenza virus subtype H9N2 in broiler chicks

In this experiment, pathogenesis of H9N2 avian influenza virus (AIV), experimentally infected with infectious bursal disease virus (IBDV) in broiler chicks was examined. Three groups of twenty were randomly selected. Day old chickens in group 1, were infected by 103 CID50 of IBDV intrabursaly, and in thirty days of age groups 1 and 2 were challenged with 106 EID50 H9N2, intranasaly-intraoculary. Chickens in group 3 remained as control (uninfected with neither IBDV or AIV). Tracheal and cloacal swabs, and tissue samples, were collected at 3, 7, and 11 days postinoculation (PI). Serum samples examined for antibodies against avian influenza virus (AIV) by hemagglutination inhibition test (HI). IBD caused lower H9N2 antibody level. IBDV infected chickens (g1) shed AI virus for a longer period than AIV infected birds (g2), from both trachea and cloac. IBDV was related with AIV in brain and liver. Isolation of AIV from trachea, conjunctiva, bursa and lung in IBDV infected group (1), prolonged till 11 days PI. Our study provides evidence that a previous history of IBDV infection in chickens may cause them to be more susceptible to H9N2 low pathogenic avian influenza (LPAI) virus infection and may alter its tissue tropism. Key words: Infectious bursal disease, avian influenza, virus shedding, broiler chicks.

Rapid molecular haemagglutinin subtyping of avian influenza isolates by specific real-time RT-PCR tests

Sixteen haemagglutinin (HA) subtypes of avian influenza viruses (AIV) have been described to date. Rapid subtype identification of any AIV is of major interest because of the possible serious consequences for the poultry industry and even public health. Molecular techniques currently allow immediate accurate subtype characterisation prior to virus isolation. In this study, a set of fourteen specific real-time RT-PCR methods were developed and evaluated for AIV HA subtyping (H1–H4, H6–H8, H10–H16), H5 and H9 being excluded on the basis of the current validity of the European Union (EU) recommended specific assays. Specific primers and probes sets for each HA-subtype were designed to hybridise the largest isolates range within each single subtype, considering the Eurasian lineage as a major target. The robustness and general application of the 14 HA-subtype methods were verified by the analysis of 110 AIV isolates belonging to all 16 HA-subtypes, performed in different laboratories. The developed real-time RT-PCR assays proved to be highly specific and revealed suitable sensitivity, allowing direct HA-subtyping of clinical material. In summary, this study provides for the first time a panel of molecular tests using specific hydrolysis probes for rapid and complete AIV HA-subtype identification.

Complete Genome Sequence of an H12N8 Avian Influenza Virus Isolated from Wild Bird Feces in Hunan East Dongting Lake National Nature Reserve

An H12N8 subtype avian influenza virus (AIV) was isolated from a wild bird in China in 2011. It is the first report of isolation of the H12N8 subtype AIV in Asia. Phylogenetic analysis results suggested it is a reassortant, and all eight gene segments belong to the Eurasian gene pool.

Phylogenetic studies of H3 low pathogenic avian influenza viruses isolated from wild mallards in Poland

In order to study the variation of low pathogenic avian influenza viruses (AIV) of H3 subtype in the natural reservoir, partial genetic characterisation of four AIV isolates of H3 subtype, recovered from wild mallards in Poland in 2006-2010, was performed. Phylogenetic analysis clearly confirms that there is a constant flow of AIV H3 between wild birds in Eurasia and Africa, and, to a limited degree, to North America (Alaska), with an occasional spill-over to poultry. The analysis of the PA gene of one isolate from 2010 suggests that it is closely related to several HPAI H5N1 viruses belonging to clade 2.3.2 and that, therefore, a reassortment event has occurred recently between low pathogenic and H5N1 highly pathogenic AIV.

Suitability of specimen types for isolation of avian influenza viruses from poultry

In view of the outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus in poultry in India, its impact on global public health and growing concerns of avian influenza (AI) viruses, surveys in wet poultry markets were conducted in the states of Maharashtra, West Bengal and Jharkhand in India during the period 2009-2012. During these surveys various types of samples from poultry were collected. During outbreaks and surveys in poultry, tracheal swabs (TS), cloacal swabs (CS), poultry drinking water (PDW) samples and fecal samples (FS) are preferred samples for AI diagnosis. The suitability of various types of poultry samples for AI virus isolation was analyzed. The parameters such as availability of specimen, ease of collection, quality of the specimen for the presence of contaminants such as organic debris or solid matter were considered for the analysis. A total of 2,405 samples were collected, which included 1,297 TS, 1,012 CS, 79 PDW, and 17 FS. Out of 2,309 TS and CS samples 1,752 samples were paired samples, collected from 876 birds. All samples were processed for virus isolation and identification. Of the 2,405 samples AI H9N2 was isolated from 199 samples (8.27%). The virus isolation rate was significantly higher in PDW samples (21.5%) (P<0.05) and TS samples (12.1%), in comparison with CS (2.3%) (P<0.001). Other viruses isolated were AI H4N6 and HPAI H5N1viruses; however the number of isolates of AI H4N6 and H5N1 were not sufficient for comparison. In conclusion, the PDW and TS samples were suitable for AI H9N2 virus isolation from poultry.