Surveillance of Highly Pathogenic Avian Influenza Viruses (H5Nx Subtypes) in Wild Birds in Iran, 2014-2019.

After the emergence of the highly pathogenic avian influenza viruses (HPAIV) subtypes H5N6 in 2013 and H5N8 in 2014, a surveillance study using molecular epidemiology approaches was carried out during 2014 - 2019 in Iran to discover any potential introduction or outbreak of HPAIV in wild bird populations. All sick and dead wild birds found in nature, or in cases of an outbreak, a collection of representative samples was tested using the specific molecular methods for HPAIV H5 subtypes. Additionally, wild bird species in wetlands, several zoos, zoological gardens, or rehabilitation centers were tested for HPAIV. During the active surveillance plan, several individual and outbreak cases of HPAIV and orthoavulaviruses were identified. In general, more than 900 fecal materials, cloacal and oropharyngeal swabs, and/or tissue samples were collected from apparently healthy live birds representing several different species and families. In addition, tissue and swab samples were collected and investigated from any reported wild birds' mortality cases in different parts of Iran in the framework of this study. No positive bird was found among apparently healthy live birds; however, the highly pathogenic influenza viruses of H5N1, H5N2, H5N6, and H5N8 were found in individual dead birds or mass die-off cases.

Monitoring of influenza A viruses in wild bird populations in Kazakhstan in 2002-2009.

A comprehensive influenza virus monitoring study of wild birds was carried out at important flyway resting places and wintering sites in Kazakhstan over eight years. More than 3200 birds belonging to 155 species were sampled. Nearly three-fourths of the birds belonged to the orders Anseriformes and Charadriiformes. In total, 118 hemagglutinating agents were isolated, and 95 of them were identified as influenza A viruses. The influenza viruses comprised eight different subtypes with a high prevalence of H13 and H3 viruses and also included low-pathogenic H5 viruses. The vast majority of the H13 viruses were isolated from members of the family Laridae, whereas the H3 viruses mostly originated from members of the family Anatidae, both in concordance with other monitoring studies. All virus isolates were recovered from cloacal swabs or fecal samples only. The influenza viruses were identified mainly in wetlands north of the Caspian Sea. These findings should be integrated in the design of further wild-bird-monitoring activities.

No virological evidence for an influenza A - like virus in European bats.

New members of the influenza A virus genus have been detected recently in bats from South America. By molecular investigations, using a generic real-time RT-PCR (RT-qPCR) that detects all previously known influenza A virus subtypes (H1-H16) and a newly developed RT-qPCR specific for the South American bat influenza-like virus of subtype H17, a total of 1571 samples obtained from 1369 individual bats of 26 species from Central Europe were examined. No evidence for the occurrence of such influenza viruses was found. Further attempts towards a more comprehensive evaluation of the role of bats in the ecology and epidemiology of influenza viruses should be based on more intense monitoring efforts. However, given the protected status of bats, not only in Europe, such activities need to be embedded into existing pathogen-monitoring programs.

Continuing evolution of equine influenza virus in Central Asia, 2007-2012.

Equine influenza (EI) continues to be an important respiratory pathogen of horses worldwide. Since 2007 several outbreaks of EI have occurred in Central Asian countries, including Kazakhstan, western Mongolia, India and western China. Phylogenetic analysis showed that two H3N8 equine influenza virus (EIV) isolates from Kazakhstan, A/equine/Almaty/26/2007 and A/equine/South Kazakhstan/236/12, were related to Florida sublineage 2, with high similarity to EIVs circulating in the same period in neighbouring countries. New outbreaks of EI during 2011 and 2012 in Kazakhstan and other Central Asian countries were caused by viruses of the same lineage. Genetic characterization of the viruses showed formation of a small EIV cluster with specific genetic signatures and continued evolution of this lineage in Central Asia between 2007 and 2012. The main genetic changes were observed in hemagglutinin gene without any antigenic drift. Although no vaccination policy was carried out in Kazakhstan, application of Florida clade 2-based vaccines is recommended.

Consecutive natural influenza a virus infections in sentinel mallards in the evident absence of subtype-specific hemagglutination inhibiting antibodies.

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand-raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT-qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full-length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian-type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re-introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein-specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds.

Understanding the ecological drivers of avian influenza virus infection in wildfowl: a continental-scale study across Africa.

Despite considerable effort for surveillance of wild birds for avian influenza viruses (AIVs), empirical investigations of ecological drivers of AIV prevalence in wild birds are still scarce. Here we used a continental-scale dataset, collected in tropical wetlands of 15 African countries, to test the relative roles of a range of ecological factors on patterns of AIV prevalence in wildfowl. Seasonal and geographical variations in prevalence were positively related to the local density of the wildfowl community and to the wintering period of Eurasian migratory birds in Africa. The predominant influence of wildfowl density with no influence of climatic conditions suggests, in contrast to temperate regions, a predominant role for inter-individual transmission rather than transmission via long-lived virus persisting in the environment. Higher prevalences were found in Anas species than in non-Anas species even when we account for differences in their foraging behaviour (primarily dabbling or not) or their geographical origin (Eurasian or Afro-tropical), suggesting the existence of intrinsic differences between wildfowl taxonomic groups in receptivity to infection. Birds were found infected as often in oropharyngeal as in cloacal samples, but rarely for both types of sample concurrently, indicating that both respiratory and digestive tracts may be important for AIV replication.

Differentiation of two distinct clusters among currently circulating influenza A(H1N1)v viruses, March-September 2009.

Analysis of all complete genome sequences of the pandemic influenza A(H1N1)v virus available as of 10 September 2009 revealed that two closely related but distinct clusters were circulating in most of the affected countries at the same time. The characteristic differences are located in genes encoding the two surface proteins - haemagglutinin and neuraminidase - and four internal proteins - the polymerase PB2 subunit, nucleoprotein, matrix protein M1 and the non-structural protein NS1. Phylogenetic inference was demonstrated by neighbour joining, maximum likelihood and Bayesian trees analyses of the involved genes and by tree construction of concatenated sequences.

Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses.

Accurate identification of hemagglutinin (HA) and neuraminidase (NA) subtypes of influenza A viruses is an integral part of monitoring programs targeting avian influenza viruses (AIV). Use of highly sensitive molecular screening methods such as pan influenza-specific real-time RT-PCR (rRT-PCR) yields an increasing number of samples which are positive for AIV RNA but negative by virus isolation and, therefore, require molecular, instead of serological, subtyping. We developed specific RT-PCR assays for all known nine AIV NA subtypes. Validation using 43 reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity. On basis of serial tenfold dilution series of reference isolates a benchmark value of C(t) 32 in an M gene-specific rRT-PCR became evident below which all nine NA subtypes were readily detectable by the subtype-specific RT-PCRs. For subtypes N1, N2, N4 and N6 detection was extended to dilutions with C(t) values of up to 35. Diagnostic applicability of the whole set of conventional NA-specific RT-PCRs was evaluated by analysis of 119 different diagnostic samples from wild birds which proved to be positive for AIV by M gene-specific rRT-PCR. Diagnostic sensitivity and specificity was confirmed by sequencing NA amplicons from 41 field isolates generated from this set and by NA inhibition assays. A universal molecular HA/NA subtyping algorithm for rRT-PCR positive avian influenza virus monitoring samples is proposed which may complement classical serological subtyping of influenza A virus isolates.