Since 1997, G1-lineage H9N2 avian influenza viruses have been circulating in Asia and later on in the Middle East, and they have been associated to mild respiratory disease, drops in egg production and moderate mortality in chickens, in particular in the presence of concurrent infections. In this study, we investigated the importance of the G1-lineage H9N2 A/chicken/Israel/1163/2011 virus as a primary pathogen in layers, analyzing its tropism and binding affinity for the oviduct tissues, and investigating the long-term impact on egg production. Besides causing a mild respiratory infection, the virus replicated in the oviduct of 60% of the hens causing different degrees of salpingitis throughout the organ, in particular at the level of the infundibulum, where the detection of the virus was associated with severe heterophilic infiltrate, and necrosis of the epithelium. Binding affinity assays confirmed that the infundibulum was the most receptive region of the oviduct. The drop in egg production was at its peek at 2 weeks post-infection (pi) (60% decrease) and continued up to 80 days pi (35% decrease). On day 80 pi, non-laying birds showed egg yolk peritonitis, and histopathological analyses described profound alteration of the infundibulum architecture, duct ectasia and thinning of the epithelium, while the rest of the oviduct and ovary appeared normal. Our results show that this H9N2 virus is a primary pathogen in layer hens, and that its replication in the infundibulum is responsible for acute and chronic lesions that limits the effective functionality of the oviduct, compromising the commercial life of birds.Electronic supplementary materialThe online version of this article (10.1186/s13567-018-0575-1) contains supplementary material, which is available to authorized users.

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.

Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; hence, an accurate and rapid AIV diagnosis is essential. To date AIV detection relies on molecular methods, mainly RT-qPCR directed against AIV M gene segment. The evolution of AIV represents a relevant issue in diagnostic RT-qPCR due to possible mispriming and/or probe-binding failures resulting in false negative results. Consequently, RT-qPCR for AIV detection should be periodically reassessed both in silico and in vitro. To this end, a specific workflow was developed to evaluate in silico the complementarity of primers and probes of four published RT-qPCR protocols to their target regions. The four assays and one commercially available kit for AIV detection were evaluated both for their analytical sensitivity using eight different viral dilution panels and for their diagnostic performances against clinical specimens of known infectious status. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances. This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions.

Among recurrent sanitary emergencies able to spread rapidly worldwide, avian influenza is one of the main constraints for animal health and food security. In West Africa, Nigeria has been experiencing repeated outbreaks of different strains of avian influenza virus (AIV) since 2006 and is also recognized as a hot spot in the region for the introduction of emerging strains by migratory wild birds. Here, we generated complete genomes of 20 highly pathogenic avian influenza (HPAI) H5N8 viruses collected during active surveillance in Nigerian live bird markets (LBM) and from outbreaks reported in the country between 2016 and 2019. Phylogenetic analysis reveals that the Nigerian viruses cluster into four separate genetic groups within HPAI H5 clade 2.3.4.4b. The first group includes 2016–2017 Nigerian viruses with high genetic similarity to H5N8 viruses detected in Central African countries, while the second includes Nigerian viruses collected both in LBM and poultry farms (2018–2019), as well as in Cameroon, Egypt and Siberia. A natural reassortant strain identified in 2019 represents the third group: H5N8 viruses with the same gene constellation were identified in 2018 in South Africa. Finally, the fourth introduction represents the first detection in the African continent of the H5N6 subtype, which is related to European viruses. Bayesian phylogeographic analyses confirmed that the four introductions originated from different sources and provide evidence of the virus spread within Nigeria, as well as diffusion beyond its borders. The multiple epidemiological links between Nigeria, Central and Southern African countries highlight the need for harmonized and coordinated surveillance system to control AIV impact. Improved surveillance at the Wetlands, LBMs and early warning of outbreaks are crucial for prevention and control of AIV, which can be potentially zoonotic and be a threat to human health.

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV’s major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered.