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Since 2003, H5N1-subtype avian influenza viruses (AIVs) with both a deletion of 20 amino acids in the stalk of the neuraminidase (NA) glycoprotein (A−) and a deletion of five amino acids at positions 80 to 84 in the non-structural protein NS1 (S−) have become predominant. To understand the influence of these double deletions in the NA and NS1 proteins on the pathogenicity of H5N1-subtype AIVs, we selected A/mallard/Huadong/S/2005 as a parental strain to generate rescued wild-type A−S− and three variants (A−S+ with a five-amino-acid insertion in the NS1 protein, A+S− with a 20-amino-acid insertion in the NA stalk, and A+S+ with insertions in both NA and NS1 proteins) and evaluated their biological characteristics and virulence. The titers of the AIVs with A− and/or S− replicated in DEF cells were higher than that of A+S+, and the A−S− virus exhibited a replication predominance when co-infected with the other variants in DEF cells. In addition, A−S− induced a more significant increase in the expression of immune-related genes in peripheral blood mononuclear cells of mallard ducks in vitro compared with the other variants. Furthermore, an insertion in the NA and/or NS1 proteins of AIVs resulted in a notable decrease in virulence in ducks, as determined by intravenous pathogenicity index, and the two insertions exerted a synergistic effect on the attenuation of pathogenicity in ducks. In addition, compared with A+S+ and A+S−, the A−S+ and A−S− viruses that were introduced via the intranasal inoculation route exhibited a faster replication ability in the lungs of ducks. These data indicate that both the deletions in the NA stalk and the NS1 protein contribute to the high pathogenicity of H5N1 AIVs in ducks.

H9N2 avian influenza virus is a zoonotic agent with a broad host range that can contribute genetic information to H5 or H7N9 subtype viruses, which are significant threats to both humans and birds. Thus, there is a great need for a vaccine to control H9N2 avian influenza. Three mutant viruses of an H9N2 virus A/chicken/Taixing/10/2010 (rTX-NS1-73, rTX-NS1-100, and rTX-NS1-128) were constructed with different NS1 gene truncations and confirmed by western blot analysis. The genetic stability, pathogenicity, transmissibility, and host immune responses toward these mutants were evaluated. The mutant virus rTX-NS1-128 exhibited the most attenuated phenotype and lost transmissibility. The expression levels of interleukin 12 in the nasal and tracheal tissues from chickens immunized with rTX-NS1-128 were significantly upregulated on day 3 post-immunization and the IgA and IgG antibody levels were significantly increased on days 7, 14, and 21 post-immunization when compared to chickens that received an inactivated vaccine. rTX-NS1-128 also protected chickens from challenge by homologous and heterologous H9N2 avian influenza viruses. The results indicate that rTX-NS1-128 can be used as a potential live-attenuated vaccine against H9N2 avian influenza.

The pathogenicity and transmissibility of H9N2 influenza viruses has been widely investigated; however, few studies comparing the biological characteristics of H9N2 viruses isolated from different hosts have been performed. In this study, eight H9N2 viruses, isolated from chickens (Ck/F98, Ck/AH and Ck/TX), pigeons (Pg/XZ), quail/(Ql/A39), ducks (Dk/Y33) and swine (Sw/YZ and Sw/TZ) were selected, and their biological characteristics were determined. The results showed that all H9N2 viruses maintained a preference for both the avian- and human-type receptors, except for Sw/TZ, which had exclusive preference for the human-type receptor. The viruses replicated well in DF-1 and MDCK cells, whereas only three isolates, Ck/F98, Ck/TX and Sw/TZ, could replicate in A549 cells and also replicated in mouse lungs, resulting in body weight loss in mice. All H9N2 viruses were nonpathogenic to chickens and were detected in the trachea and lung tissues. The viruses were shed primarily by the oropharynx and were transmitted efficiently to naïve contact chickens. Our findings suggest that all H9N2 viruses from different hosts exhibit efficient replication and contact-transmission among chickens, and chickens serve as a good reservoir for the persistence and interspecies transmission of H9N2 influenza viruses.

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Naturally occurring pentacyclic triterpenes, such as betulinic acid (BA) and its derivatives, exhibit various pharmaceutical activities and have been the subject of great interest, in particular for their antiviral properties. Here, we found a new anti-influenza virus conjugate, hexakis 6-deoxy-6-[4-N-(3β-hydroxy-lup-20­(29)-en-28-oate)­aminomethyl-1H-1,2,3-triazol-1-yl]-2,3-di-O-acetyl-α-cyclodextrin (CYY1-11, 1), in a mini library of pentacyclic triterpene–cyclodextrin conjugates by performing a cell-based screening assay and then exploring the underlying mechanisms. Our results showed that conjugate 1 possessed a high-level activity against the influenza virus A/WSN/33 with an IC50 value of 5.20 μM (SI > 38.4). The study of the mechanism of action indicated that conjugate 1 inhibited viral replication by directly targeting the influenza hemagglutinin protein (K D = 1.50 μM), thus efficiently preventing the attachment of the virion to its receptors on host cells and subsequent infection. This study suggests that multivalent BA derivatives have possible use as a new class of influenza virus entry inhibitors.

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