Abstract
Highly pathogenic avian influenza viruses (HPAIVs) with H5 and H7 hemagglutinin (HA) subtypes are derived from their low pathogenic counterparts following the acquisition of multiple basic amino acids in their HA cleavage site. It has been suggested that consecutive adenine residues and a stem-loop structure in the viral RNA region that encodes the cleavage site are essential for the acquisition of the polybasic cleavage site. By using a reporter assay to detect non-templated nucleotide insertions, we found that insertions more frequently occurred in the RNA region (29 nucleotide-length) encoding the cleavage site of an H5 HA gene that was predicted to have a stem-loop structure containing consecutive adenines than in a mutated corresponding RNA region that had a disrupted loop structure with fewer adenines. In virus particles generated by using reverse genetics, nucleotide insertions that created additional codons for basic amino acids were found in the RNA region encoding the cleavage site of an H5 HA gene but not in the mutated RNA region. We confirmed the presence of virus clones with the ability to replicate without trypsin in a plaque assay and to cause lethal infection in chicks. These results demonstrate that the stem-loop structure containing consecutive adenines in HA genes is a key molecular determinant for the emergence of H5 HPAIVs.
Highlights
Influenza A viruses (IAVs) belong to the genus Alphainfluenzavirus in the family Orthomyxoviridae
We previously showed that nucleotide insertions frequently occur in the stem-loop structure containing the adenine stretch in the RNA sequence encoding the cleavage site of H5 HAs [33]
We further show that increased nucleotide insertions create additional codons for basic amino acids, resulting in the generation of viruses with the ability to replicate in the absence of trypsin
Summary
Influenza A viruses (IAVs) belong to the genus Alphainfluenzavirus in the family Orthomyxoviridae. Artificial introduction of multiple basic amino acids into the HA cleavage site of an H6 LPAIV strain allows the virus to replicate in the absence of trypsin in vitro and to cause systemic infection of chickens [18]. An alternative proposal, reported for the H7 subtype, is recombination of the HA RNA genome with other RNA of host or virus origin [27,28,29,30,31] These studies hypothesized that RNA secondary structure and polymerase errors are involved in the generation of multiple basic amino acids at the cleavage site, empiric evidence was mostly absent. We have shown that the RNA region around the cleavage site of most low-pathogenic H5 and H7 viruses isolated from waterfowl contains characteristic stem-loop structures including more than eight adenine and/or guanine nucleotides.
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