Phylogenetic and molecular characteristics of Eurasian H9 avian influenza viruses and their detection by two different H9-specific RealTime reverse transcriptase polymerase chain reaction tests

Abstract

Avian influenza viruses (AIVs) of the H9 haemagglutinin subtype are endemic in many Asian and Middle-East countries, causing mortality and morbidity in poultry. Consequently there is a need for accurate and sensitive detection of Eurasian H9 subtype viruses. Two H9 RealTime reverse transcriptase polymerase chain reaction (RRT-PCR) tests, developed by Monne et al. (2008) and Ben Shabat et al. (2010), were originally validated with a limited number of H9 specimens. In the present study, the two tests have been assessed using 66 diverse H9 isolates and 139 clinical specimens from six H9 poultry outbreaks in four geographically disparate Eurasian countries. The Monne et al. (2008) test was modified and successfully detected all H9 viruses from all three Eurasian H9 lineages. Bayesian analysis of the clinical specimens' results revealed this test to be more sensitive (97%) than the Ben Shabat et al. (2010) test (31%). The latter test detected most H9 isolates of the G1 lineage, but no isolates from other H9 lineages. Mismatches in the primer/probe binding sequences accounted for sensitivity differences between the two H9 RRT-PCRs. Genetic analysis of 34 sequenced H9 haemagglutinin genes showed the South Asian and Middle-East H9 isolates to belong to the H9 G1 lineage, and possessed residues that appear to preferably bind alpha 2,6-linked sialic acid receptors which indicate a potential for human infection. European H9s clustered phylogenetically in a broader geographical group that includes recent North American H9 wild bird isolates and contemporary Asian viruses in the Y439 H9 lineage.

Viral interference between low pathogenic avian influenza H9N2 and avian infectious bronchitis viruses in vitro and in ovo

“…In fact, there is a lack in evaluating the growth of individual viruses and the determination of the exact number of progeny viruses produced, during co-infections; the only reported study on AIV and IBV co-infection being performed without any quantitative evaluation of the degree of such interference (Groupé and Pugh, 1952). Owing the importance of such viruses and their impact on the poultry industry, new quantitative techniques such as rRT-PCR were developed, establishing in vitro and in ovo model system in which many different viruses and their interactions could be studied (Slomka et al, 2013).…”

Effect of serial pig passages on the adaptation of an avian H9N2 influenza virus to swine

“…(2009) inoculated pigs intranasally with A/chicken/Belgium/818/1979 (H9N2) and found that this strain was not excreted at all by the inoculated pigs [24]. Though genetic information of this specific isolate is not available, the lack of replication in the inoculated pigs was likely due to the fact that European H9N2 isolates belong to the Y439-like lineage, which likely lack the ability to replicate in pigs [3, 30]. In the second study, Qiao et al .…”

“…Genetic characterization and phylogenetic analysis revealed that most poultry H9N2 viruses belong to one of three different lineages: G1-like, Y280-like and Y439-like, also known as Korean-like [2, 3]. In 1999, H9N2 viruses were for the first time isolated from two patients with mild respiratory symptoms [4].…”

Development and evaluation of a new real‐time RT‐PCR assay for detecting the latest H9N2 influenza viruses capable of causing human infection

“…Molecular diagnostic techniques such as the PCR method can be used as diagnostic tools for virus identification and assessing viral infection. In particular, real‐time RT‐PCR (rRT‐PCR) is one of the most widely used methods for detecting viral genes, and rRT‐PCR assays for detecting H9 viruses have been reported . However, the sequences of probes and primers used in rRT‐PCR in previous studies were designed for detecting viruses of the North American lineage or past circulated G1‐lineage H9 AIV.…”