Novel real-time PCR-based patho- and phylotyping of potentially zoonotic avian influenza A subtype H5 viruses at risk of incursion into Europe in 2017

Abstract

Since November 2016, Europe witnesses another wave of incursion of highly pathogenic avian influenza (HPAI) A(H5) viruses of the Asian origin goose/Guangdong (gs/GD) lineage. Infections with H5 viruses of clade 2.3.4.4b affect wild bird and poultry populations. H5 viruses of clades 2.2, 2.3.1.2c and 2.3.4.4a were detected previously in Europe in 2006, 2010 and 2014. Clades 2.2.1.2 and 2.3.2.1.c are endemic in Egypt and Western Africa, respectively and have caused human fatalities. Evidence exists of their co-circulation in the Middle East. Subtype H5 viruses of low pathogenicity (LPAI) are endemic in migratory wild bird populations. They potentially mutate into highly pathogenic phenotypes following transmission into poultry holdings. However, to date only the gs/GD H5 lineage had an impact on human health. Rapid and specific diagnosis marks the cornerstone for control and eradication of HPAI virus incursions. We present the development and validation of five real-time RT-PCR assays (RT-qPCR) that allow sequencing-independent pathotype and clade-specific distinction of major gs/GD HPAI H5 virus clades and of Eurasian LPAI viruses currently circulating. Together with an influenza A virus-generic RT-qPCR, the assays significantly speed up time-to-diagnosis and reduce reaction times in a OneHealth approach of curbing the spread of gs/GD HPAI viruses.

Novel HPAIV H5N8 Reassortant (Clade 2.3.4.4b) Detected in Germany

“…Pathological examination determined trauma as a cause of death; nevertheless, initial testing for avian influenza virus RNA revealed very high virus loads in mixed tissue homogenates (lung and gut tissue) with RT-qPCR Cq-values of RT-qPCR H5 Cq = 16.1 and RT-qPCR N8.2 Cq = 14.1. Primary sub-and pathotyping results achieved via qPCR [7] revealed a HPAIV H5N8 of clade 2.3.4.4b (RT-qPCR H5 HP 2344b DE Cq = 14.1). Subsequently, a severe necrotizing polioencephalitis, typical of H5N8 infection in waterfowl [8], was detected by histopathology, most likely causing disorientation and predisposing the goose to the traumatic event.…”

“…Shortly after, on 6 February 2020, chickens (Gallus gallus domesticus) from a small holding in the federal state of Baden-Wuerttemberg ( Figure 1) also tested positive for HPAIV H5N8 of clade 2.3.4.4b (RT-qPCR H5 HP 2344b DE Cq = 22.9-25.9; RT-qPCR N8.2 Cq = 22.2-25.4) following the same testing protocol as described [7]. In this case, RNA was extracted from swab samples.…”

“…Subsequently, after library preparation with the Rapid Barcoding Kit (RBK-004, Oxford Nanopore Technology, Oxford, UK; ONT) according to the manufacturer's instructions, full genome sequencing was performed on the MinION platform in combination with a R9.4 flow cell (ONT), the MinIT (v19.12.1; ONT) and basecaller Guppy (v3.2.9; ONT), facilitating realtime basecalling to produce quality checked, demultiplexed, and trimmed raw data. Shortly after, on 6 February 2020, chickens (Gallus gallus domesticus) from a small holding in the federal state of Baden-Wuerttemberg ( Figure 1) also tested positive for HPAIV H5N8 of clade 2.3.4.4b (RT-qPCR H5 HP 2344b DE Cq = 22.9-25.9; RT-qPCR N8.2 Cq = 22.2-25.4) following the same testing protocol as described [7]. In this case, RNA was extracted from swab samples.…”

Investigation of suspected Newcastle disease (ND) outbreaks in Egypt uncovers a high virus velogenic ND virus burden in small-scale holdings and the presence of multiple pathogens

“…Viral RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen; Hilden, Germany), and RNA extracts were screened by real-time RT-PCR (RT-qPCR) for different respiratory pathogens: speciesspecific RT-qPCRs recognizing AAvV-1 independent of the pathotype included M- (Wise et al, 2004), NP- (Ramp et al, 2012) and L-gene (Fuller et al, 2010) specific tests. In addition, a pathotype-specific assay detecting virulent NDV (F(vir), (Wise et al, 2004)) was applied as well as tests for avian influenza virus (AIV) H5N1 (Naguib et al, 2017b), H9N2 (Monne et al, 2008), and infectious bronchits virus (IBV) (Naguib et al, 2017a). For detecting lentogenic vaccine type strains a specific probe F(vac) was generated using the F-specific primers of the RT-PCR described by Wise et al (2004).…”

Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry

“…However, due to the continuous circulation of H5Nx HPAI and other virus clades in avian species, it is very difficult to control viral transmission in poultry [3]. To effectively control and rapidly diagnose suspected infection on poultry farms, it is important to develop a rapid diagnostic assay for the simultaneous detection of different H5 HPAI viruses [20].…”