Date Published: May 24, 2018
Publisher: Public Library of Science
Author(s): Ziyaad Valley-Omar, Preetha Iyengar, Claire von Mollendorf, Stefano Tempia, Alexandra Moerdyk, Orienka Hellferscee, Neil Martinson, Meredith McMorrow, Ebrahim Variava, Katlego Masonoke, Adam L. Cohen, Cheryl Cohen, Florette K. Treurnicht, Florian Krammer.
Limited information is available on influenza virus sequence drift between transmission events. In countries with high HIV burdens, like South Africa, the direct and indirect effect of HIV on influenza sequence drift between transmission events may be of public health concern. To this end, we measured hemagglutinin sequence diversity between influenza transmission events using data and specimens from a study investigating household transmission dynamics of seasonal influenza viruses in 2 peri-urban communities in South Africa during the 2013 influenza season. Thirty index cases and 107 of 110 eligible household contacts were enrolled into the study, 47% (14/30) demonstrating intra-household laboratory-confirmed influenza transmission. In this study 35 partial hemagglutinin gene sequences were obtained by Sanger sequencing from 11 index cases (sampled at enrolment only) and 16 secondary cases (8 cases sampled at 1 and 8 cases sampled at 2 time-points). Viral sequence identities confirmed matched influenza transmission pairs within the 11 households with corresponding sequenced index and secondary cases. Phylogenetic analysis revealed 10 different influenza viral lineages in the 14 households. Influenza A(H1N1)pdm09 strains were shown to be genetically distinct between the 2 communities (from distinct geographic regions), which was not observed for the influenza A(H3N2) strains. Intra-host/intra-household influenza A(H3N2) sequence drift was identified in 2 households. The first was a synonymous mutation between the index case and a household contact, and the second a non-synonymous mutation between 2 serial samples taken at days 0 and 4 post enrolment from an HIV-infected secondary case. Limited inter-household sequence diversity was observed as highlighted by sharing of the same influenza strain between different households within each community. The limited intra-household sequence drift is in line with previous studies also using Sanger sequencing, corroborating the presence of strict selective bottlenecks that limit sequence variance. We were not able to directly ascertain the effect of HIV on influenza sequence drift between transmission events.
Within each influenza virus subtype a unique genetic background exists as a result of genetic drift [1–4]. Trends in influenza sequence drift are well documented at national and international levels but are not often explored within individual transmission events as they are rarely observed [1, 5–9]. Household transmission studies (HTS) are an ideal platform to investigate intra-host and intra-household sequence evolution using both Sanger and deep sequencing technologies [1, 7, 9, 10].
Thirty index cases were enrolled, 9 (30%) of 30 tested were HIV infected, 3 of which were taking antiretrovirals, the remaining 21 (70%) were confirmed HIV negative. Within their 30 households, there were 110 eligible household contacts, of whom 107 (97%) were enrolled and 12/107 (11%) were confirmed as HIV-infected, of which 8 were taking antiretrovirals (Fig 1 and Table 1) . The HIV prevalence among individuals aged 2 years and older for Kwazulu-Natal and North-West provinces where our study was conducted was 17.4% (range 15.8–19.2%) and 13.9% (range 12.0–16.1%) respectively in 2012 . Three household contacts were excluded as they were not available for follow-up visits. Of the 107 enrolled household contacts, 24 (22.4%) tested influenza-positive from 19 households during the study period. Among those with available information 3% of index cases (1/29) and 5% of household contacts (5/106) reported receiving one dose of influenza vaccine in the previous 12 months, which included the current influenza season. Of our analysed sample set only the secondary case from household 101 received the influenza seasonal vaccine (Table 1). Five households were excluded from our analysis as influenza types in contacts did not match that of the index case and a further 2 secondary cases could not be subtyped due to low viral load. Therefore only14 households remained in the study of which 6 were infected with influenza A(H1N1)pdm09 and 8 were infected with influenza A(H3N2).
We assessed the intra-host and intra-household variability of influenza A subtypes in peri-urban South African households using Sanger sequencing. The prevalence of influenza virus types/subtypes identified in communities sampled, which showed dominance of A(H1N1)pdm09, was reflective of the influenza virus type/subtype distribution in South Africa during the study period [22, 23]. In the available sample set, while influenza A(H1N1)pdm09 strains did not display any intra-household nucleotide changes in the regions sequenced, a greater level of sequence diversity existed within the communities sequenced. Influenza A(H1N1)pdm09 strains appeared to exhibit a geographic identity segregating according to the communities from which the viral strains were derived, which may be an artefact of limited population sampling. We demonstrated limited nucleotide changes in 2 influenza A(H3N2)-infected households within the hemagglutinin HA1 domain. It is notable that the secondary case that demonstrated a non-synonymous, S91G HA mutation (household 408) between serial samples was the only secondary case known to be HIV infected. Studies have suggested that this specific amino acid (S91) forms part of a conserved set of amino acid residues that can serve as a neutralizing antibody binding domain. S91 specifically serves as one of several hydrogen binding sites for neutralizing antibody binding, where the S91G mutation may act by impairing the antigen-antibody interaction . The HIV viral load for this individual is unfortunately unknown. It should also be noted that the 12 day period between influenza sequences derived from the index and household contact in household 404 may suggest a community-acquired infection in the secondary case and not necessarily be a result of sequence drift between successive transmission events within the household. In contrast to previous HTSs, not all households in this study could be distinguished by unique viral phylogenies as some appeared to share identical influenza strains. This may suggest limited influenza sequence variability within the communities or inter-household transmission, which may be clarified by understanding the social dynamics within these communities. The sequencing depth limitations of Sanger sequencing may have prevented us from attaining necessary sequencing depth to distinguish households with similar strains. Similarly, the lack of sequencing depth has prevented us from determining the statistical significance of intra-household and intra-host sequence identity. The use of full genome deep sequencing technologies in future studies could therefore be instrumental to detect unique ratios of viral quasispecies that exist within households or between transmission pairs with more confidence by providing a sequencing depth greater than the 20% threshold of Sanger sequencing .