Influenza A Virus Reassortment Is Limited by Anatomical Compartmentalization following Coinfection via Distinct Routes

Abstract

Exchange of gene segments through reassortment is a major feature of influenza A virus evolution and frequently contributes to the emergence of novel epidemic, pandemic and zoonotic strains. It has long been evident that viral diversification through reassortment is constrained by genetic incompatibility between divergent parental viruses. In contrast, the role of virus-extrinsic factors in determining the likelihood of reassortment has remained unclear. To evaluate the impact of such factors in the absence of confounding effects of segment mismatch, we previously reported an approach in which reassortment between wild-type (wt) and genetically tagged variant (var) viruses of the same strain is measured. Here, using wt/var systems in the A/Netherlands/602/2009 (pH1N1) and A/Panama/2007/99 (H3N2) strain backgrounds, we have tested whether inoculation of parental viruses into distinct sites within the respiratory tract limits their reassortment. Using a ferret model, matched parental viruses were either co-inoculated intranasally, or one virus was instilled intranasally while the second was instilled intratracheally. Dual intranasal inoculation resulted in robust reassortment for wt/var viruses of both strain backgrounds. In contrast, when infections were initiated simultaneously at distinct sites, strong compartmentalization of viral replication was observed and minimal reassortment was detected. The observed lack of viral spread between upper and lower respiratory tissues may be attributable to localized exclusion of super-infection within the host, mediated by innate immune responses. Our findings indicate that dual infections in nature are more likely to result in reassortment if viruses are seeded into similar anatomical locations and have matched tissue tropisms.Genetic exchange between influenza A viruses (IAVs) through reassortment can facilitate the emergence of antigenically drifted seasonal strains and plays a prominent role in the development of pandemics. Typical human influenza is concentrated in the upper respiratory tract; however, lower respiratory tract (LRT) infection is an important feature of severe cases, which are more common in the very young, the elderly, and individuals with underlying conditions. In addition to host factors, viral characteristics and mode of transmission can also increase the likelihood of LRT infection: certain zoonotic IAVs are thought to favor the LRT and transmission via small droplets allows direct seeding into lower respiratory tissues. Toward gauging the likelihood of reassortment in co-infected hosts, we assessed the extent to which initiation of infection at distinct respiratory sites impacts reassortment frequency. Our results reveal that spatially distinct inoculation results in anatomical compartmentalization of infection, which in turn strongly limits reassortment.

Influenza A viruses are transmitted via the air from the nasal respiratory epithelium of ferrets

“…The site of generation of airborne IAVs is the URT. In order to understand from which anatomical site of the respiratory tract airborne transmissible IAVs are expelled, untagged and genetically tagged variant (var) versions of the A/Netherlands/ 602/2009 virus (A/H1N1 and A/H1N1 var ) 30 were used to inoculate donor animals in the ferret transmission model 18,32 . The A/H1N1 var virus carries a single synonymous substitution per gene segment, so that it can be differentiated from the A/ H1N1 virus.…”

“…We have previously shown that double inoculations at different anatomical sites of the respiratory tract of ferrets led to compartmentalization of the viruses, which greatly restricted reassortment 30 . Therefore, next-generation sequencing was performed on only one gene, the PB2 gene, to determine whether the throat and nose swabs collected from the donor and recipient ferrets contained the A/H1N1, A/H1N1 var viruses or a mix of both viruses.…”

“…(A/H1N1) viruses were were initially described in ref. 30 . The tagged A/H1N1 var virus carries a single synonymous nucleotide substitution per segment relative to the untagged A/H1N1 virus, as follows (nucleotide numbering is from the 5′ end of the cRNA): PB2 C273T, PB1 T288C, PA C360T, HA C305T, NP A351G, NA G336A, M G295A, NS C341T.…”

“…However, it still remains unknown from which exact anatomical site of the respiratory tract airborne transmissible IAVs are expelled and whether α−2,6-SA preference is critical at the donor or recipient level, or both. To determine whether airborne transmissible IAVs originate from tissues of the URT or LRT, we here investigate the transmission of untagged and genetically tagged variant IAVs 30,31 upon simultaneous inoculation at a different anatomical site: the URT or LRT of ferrets. Our results demonstrate that airborne transmissible IAVs are transmitted from the URT, more specifically from the nasal turbinates of ferrets, which correlates with high infectivity in the ferret and human nasal respiratory epithelium in vivo and in vitro, respectively.…”

Collective interactions augment influenza A virus replication in a host-dependent manner

“…Reassortment data was used to calculate viral genotypic diversity; that is, the diversity of the 256 possible WT/VAR genotypes present in a given sample. Diversity was quantified as reported previously 80 , by calculating Simpson’s Index, given by D = sum( p i 2 ), where p i represents the proportional abundance of each genotype 81 . Simpson’s Index accounts for both the raw number of species and variation in abundance of each, and is sensitive to the abundance of dominant species.…”