SUMMARY: H9N2 is the most widespread avian influenza virus subtype in poultry worldwide. It infects a broad spectrum of host species including birds and mammals. Infections in poultry and humans vary from silent to fatal. Importantly, all AIV, which are fatal in humans (e.g. H5N1, H7N9) acquired their ‘internal’ gene segments from H9N2 viruses. Although H9N2 is endemic in the Middle East (ME) and North Africa since the late 1990s, little is known about its epidemiology and genetics on a regional level. In this review, we summarised the epidemiological situation of H9N2 in poultry and mammals in Iran, Iraq, Kuwait, Qatar, United Arab Emirates, Oman, Bahrain, Yemen, Saudi Arabia, Jordan, Palestine, Israel, Syria, Lebanon, Turkey, Egypt, Sudan, Libya, Tunisia, Algeria and Morocco. The virus has been isolated from humans in Egypt and serosurveys indicated widespread infection particularly among poultry workers and pigs in some countries. Some isolates replicated well in experimentally inoculated dogs, mice, hamsters and ferrets. Insufficient protection of immunised poultry was frequently reported most likely due to concurrent viral or bacterial infections and antigenic drift of the field viruses from outdated vaccine strains. Genetic analysis indicated several distinct phylogroups including a panzootic genotype in the Asian and African parts of the ME, which may be useful for the development of vaccines. The extensive circulation of H9N2 for about 20 years in this region where the H5N1 virus is also endemic in some countries, poses a serious public health threat. Regional surveillance and control strategy are highly recommended.

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses. The identification of influenza virus-like sequences in two different bat species has generated great interest in understanding their biology, ability to mix with other influenza viruses, and their public health threat. Unfortunately, bat-influenza viruses couldn't be cultured from the samples containing the influenza-like nucleic acids. We used synthetic genomics strategies to create wild type bat-influenza, or bat-influenza modified by substituting the surface glycoproteins with those of model influenza A viruses. Although influenza virus-like particles were produced from both synthetic genomes, only the modified bat-influenza viruses could be cultured. The modified bat-influenza viruses replicated efficiently in vitro and an H1N1 modified version caused severe disease in mice. Collectively our data show: (1) the two bat-flu genomes identified in other studies are replication competent, suggesting that host cell specificity is the major limitation for propagation of bat-influenza, (2) bat-influenza NS1 antagonizes host interferon response more efficiently than that of a model influenza A virus, (3) bat-influenza has both genetic and protein incompatibility with influenza A or B viruses, and (4) that these bat-influenza lineages pose little pandemic threat.

Extensive vaccination against Newcastle disease virus (NDV) induced more selective immune pressure from hosts that enhanced the evolutionary process of NDV. Herein, we characterized 13 recently isolated NDV isolates from vaccinated chicken flocks during 2014-2017. Sequence analysis of F gene showed the presence of (112)RRQKRF(117) velogenic cleavage motif in 11 isolates, whereas the other two isolates (Ck/ME3/Eg/16 and Ck/ME5/Eg/16) showed the monobasic motif (112)GGRQGRL(117). Interestingly, isolate Ck/ME5/Eg/16 showed 100% and 99.82% nucleotide identity with the LaSota F gene hypervariable region and full-length gene, respectively. On the other hand, isolate Ck/ME3/Eg/16 revealed natural recombination with strains NDV/Ck/Egypt/3/2006 and NDV/Teal/VRLCU/Egypt/2015 that indicates re-emergence of that old strain. Interestingly, all 13 isolates showed high intracerebral pathogenicity (ICPI) and mean death time (MDT) despite the presence of lentogenic motif in both Ck/ME5/Eg/16 and Ck/ME3/Eg/16 isolates. Comparative analysis of F antigenic epitopes in our isolates with other published sequences from Egypt revealed high sequence conservation; recent isolates had one fixed amino acid substitution (K78R) and a novel V168I substitution, whereas a D170N substitution was detected in older strains (NDV-EG-35-2014 and NDV-KFR-B7-2012). Taken together, our results support the first isolation of virulent NDV isolates with a lentogenic motif; isolate Ck/ME5/Eg/16 might be generated in nature from LaSota live vaccine, whereas isolate Ck/ME3/Eg/16 is emergent from NDV/Ck/Egypt/3/2006. We conclude that the current diagnostic evaluation of the virulence of NDV isolates by characteristic amino acid residues at the F protein cleavage site is insufficient. There is a need to link virologic and epidemiologic data together, and routine and emergency LaSota vaccination protocols should be carefully and optimally applied, with regards to the timing and presence of co-infecting agents in the field.