A risk marker of tribasic hemagglutinin cleavage site in influenza A (H9N2) virus

Abstract

Low pathogenic avian influenza A(H9N2) virus is endemic worldwide and continually recruit internal genes to generate human-infecting H5N1, H5N6, H7N9, and H10N8 influenza variants. Here we show that hemagglutinin cleavage sites (HACS) of H9N2 viruses tended to mutate towards hydrophilic via evolutionary transition, and the tribasic HACS were found at high prevalence in Asia and the Middle East. Our finding suggested that the tribasic H9N2 viruses increased the viral replication, stability, pathogenicity and transmission in chickens and the virulence of mice compared to the monobasic H9N2 viruses. Notably, the enlarged stem-loop structures of HACS in the RNA region were found in the increasing tribasic H9N2 viruses. The enlarged HACS RNA secondary structures of H9N2 viruses did not influence the viral replication but accelerated the frequency of nucleotide insertion in HACS. With the prevailing tendency of the tribasic H9N2 viruses, the tribasic HACS in H9N2 viruses should be paid more attention.

In Silico Analyses of the Role of Codon Usage at the Hemagglutinin Cleavage Site in Highly Pathogenic Avian Influenza Genesis

“…Therefore, all subsequent analyses were performed on this stretch of 12 nucleotides/4 amino acids. As mentioned above, it has been shown that the number of As in the region immediately preceding the HA cleavage site is positively associated with acquisition of insertions [ 24 , 28 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. We therefore set out to compare the total number of As present in the P4 to P1 region of all sequences and their disposition into stretches.…”

“…However, HAs of LPAIVs from other subtypes that have so far never evolved to HPAIVs present similar conserved structures (e.g., H6) [ 26 ], suggesting that the presence of a stem-loop is not sufficient for insertion generation at the HA cleavage site. It has also become clear that the nucleotide sequence of the region coding for the HA cleavage site has a strong impact on the generation of insertions, with sequences rich in adenines (A; cRNA orientation) and particularly with long stretches of As being insertion-prone [ 24 , 28 , 30 , 31 , 32 , 33 , 34 , 35 , 36 ]. Serial passaging experiments have revealed that insertions are more easily acquired in H5 and H7 viruses containing substitutions, leading to more A-rich R or K codons at the HA cleavage site than viruses having a consensus LPAIV motif [ 28 , 30 , 31 , 32 , 33 , 34 , 35 ].…”

“…Serial passaging experiments have revealed that insertions are more easily acquired in H5 and H7 viruses containing substitutions, leading to more A-rich R or K codons at the HA cleavage site than viruses having a consensus LPAIV motif [ 28 , 30 , 31 , 32 , 33 , 34 , 35 ]. It has been shown using in vitro minigenome assays that increasing the number of As in the loop region of H5 and H9 HAs via substitutions (and thereby the number of basic amino acids) significantly increases the likelihood of insertion introduction by the RdRp [ 24 , 28 , 36 ].…”

Real-Time Visualization of the Infection and Replication of a Mouse-Lethal Recombinant H9N2 Avian Influenza Virus

“…In Asia, the H9N2 AIVs have become endemic in poultry in different countries, posing a huge economic loss to the poultry farming industry (8)(9)(10)(11)(12). Furthermore, H9N2 AIVs have conduced to some zoonotic events by providing the internal segments to reassortment viruses such as H7N9, H5N6, and H10N8 (13,14). Strikingly, it has been reported that H9N2 AIVs continuously cross the species barriers to infect mammalians, including dogs, pigs, and humans (15)(16)(17).…”

Airborne Transmission of Avian Origin H9N2 Influenza A Viruses in Mammals

“…However, LPAIVs have been shown to replicate in the oviducts and kidneys of some infected birds, suggesting the presence of proteases in other tissues that may be able to process monobasic cleavage sites [13][14][15]. Recent H9N2 isolates from Asia and Middle East have increasingly shown the presence of a tri-basic HA cleavage site, which was associated with increased pathogenicity and transmission in chickens in comparison with viruses with mono-basic cleavage sites [16]. However, these viruses with the tri-basic cleavage site did not demonstrate a HPAI phenotype but rather an intermediate step towards a gain in pathogenicity, and were still considered LPAIV.…”