Role of budding morphology in pathogenesis and transmission of avian influenza A virus
Item statusRestricted Access
Embargo end date27/11/2020
Conceição, Carina Liliete Brito Tomé da
Pleomorphism is a characteristic of influenza A virus (IAV), which can produce spherical particles of ~100 nm in diameter and filaments up to 30 μm in length. Laboratory-adapted strains mainly produce spherical virions, whereas clinical isolates of human and equine strains of IAV are mostly filamentous, thus suggesting that this latter virion morphology may be involved in viral fitness in vivo. Furthermore, M1 is known to be the main morphology determinant of mammalian strains of IAV, and a “filamentous signature” within its amino acid sequence has been shown to switch virus budding between spherical and filamentous phenotypes. Even though filamentous virions have been consistently detected in clinical isolates of human and equine IAV, their biological significance still remains unclear, although recent studies have implied a role in transmission. In contrast, budding morphology of avian strains of IAV remains less well characterised with several unanswered questions, such as: (i) what is the predominant budding phenotype of avian IAV; (ii) is segment 7 also a major morphology determinant and, if so, is the mammalian “filamentous signature” present; and (iii) if avian IAVs do produce filamentous virions, what is their biological significance in this host? Therefore, the aim of this study was to answer these questions, starting with characterising the budding phenotype of 8 avian isolates by immunofluorescence assays (IF), which showed that avian isolates of IAV are as pleomorphic as mammalian isolates. Furthermore, using reverse genetics we identified avian segment 7 as the main determinant of budding morphology. Thus, in order to test a higher number of avian strains, complete nucleotide sequences of avian segment 7s available online were collected and organised into a phylogenetic tree, which was divided into clades (1 to 11) and fitted with segment 7s from avian strains available in the laboratory. For the clades with no representation, segment 7s were randomly selected, synthesised and cloned in a reverse genetics plasmid vector (pDUAL). Using this method, a total of 17 avian segment 7s were characterised as 7:1 reassortant viruses in the background of the laboratory strain A/PR/8/34 (PR8). Similar to mammalian segment 7s, avian segment 7s showed a pleomorphic budding phenotype in the PR8 backbone and some were able to produce filaments up to 10 μm in length. However, when sequence alignment was performed with the avian M1 amino acid sequences, grouped according to budding phenotype, this showed that avian strains do not contain the same mammalian virus “filamentous signature”. and, in fact, do not show a consistent pattern of amino acid alterations that would explain the different morphologies. However, by performing site-directed mutagenesis on closely related avian strains with opposite budding phenotypes, we identified M1 positions 59, 169 and 234 as morphology determinants. Furthermore, it was observed that chicken viruses were mainly spherical while duck viruses were predominantly filamentous. Bioinformatics coupled with statistical analysis led to the hypothesised identification of position 234 of M1 as a novel avian host species-dependent morphology determinant of IAV budding, as we observed that spherical chicken viruses mainly possessed isoleucine at this position while filamentous duck viruses had leucine. To test this, a spherical chicken virus (A/chicken/Pakistan/UDL-01/2008 H9N2 [Ck/Pakistan]) with isoleucine at position 234 of M1 was mutated to encode a leucine at this position. This resulted in conversion of a spherical chicken virus to a filamentous virus. Next, all the M1-mutant viruses and their WT counterparts were characterised in vitro and in ovo, showing that in vitro, spherical or filamentous morphologies were equally advantageous, whereas in ovo the spherical viruses showed a fitness advantage in some of the viruses characterised. Additionally, the ability to alternate the budding phenotype of Ck/Pakistan by a single amino acid change at position 234 of M1 provided the opportunity to determine the biological significance of filaments in vivo. Contact-infected birds with the filamentous mutant virus showed prolonged but similar higher-titre buccal shedding than the equivalent spherical virus. This observation correlated with the longer-lasting persistence of the filamentous virus in the environment and in vivo. Additionally, within the chicken host, spherical and filamentous morphologies replicated to comparable titre in the nasal tissues, but the filamentous virus showed a replication advantage in the tracheal tissues. Overall, this study showed that avian IAVs are as pleomorphic as human and equine IAVs. It also showed that avian IAVs budding morphology is determined by segment 7 but that these do not follow the genetic determinants identified in mammalian strains as defining budding morphology. Instead, this study identified a novel position within M1 that was able to determine budding phenotype in an avian host species-dependent manner. Lastly, the in vivo data corroborated the hypothesis that filaments are associated with viral transmission, perhaps due to their ability to readily cross the mucous barrier of the trachea when compared with the spherical-equivalent virus.