Influenza A Virus Transmission Bottlenecks Are Defined by Infection Route and Recipient Host

Transcription

1 Cell Host & Microbe, Volume 16 Supplemental Information Influenza A Virus Transmission Bottlenecks Are Defined by Infection Route and Recipient Host Andrew Varble, Randy A. Albrecht, Simone Backes, Marshall Crumiller, Nicole M. Bouvier, David Sachs, Adolfo García-Sastre, and Benjamin R. tenoever

2 Supplemental Information Figure S1, as related to Figure 1. Replication and deep sequencing of barcoded influenza A viruses. (A) Multicycle growth curve of wt or barcode-containing A/California/04/2009 ((MOI = 0.01) 10,000 plaque forming units) in MDCK cells, harvested at indicated time post-infection. Viruses were divided into three subgroups: (1) those that replicated in eggs following library infection, (2) those that were efficiently transmitted following library infection, or (3) those that neither grew in eggs nor transmitted. Virus numbers correspond to barcode identifiers in Supplementary Tables 1, 2, and 3. Data represented as mean SEM, LOD=limit of detection. (B) Percentage of individual barcodes in relation to the overall virus population in an infected guinea pig. Sample was sequenced in duplicate in two independent sequencing runs. Corresponding values for individual barcodes were plotted for (B), replicate samples on flow cell one, (C), replicate samples on flow cell two, (D), replicate one on flow cells one and two, (E), replicate two on flow cells one and two. Line details best-fit linear regression based on depicted data.

3 Figure S2, as related to Figure 2. Avian adaptation of barcoded influenza A viruses library. (A) Growth of barcode-containing A/California/04/2009 following inoculation of embryonated-chicken eggs with indicated viruses from the original viral library. 10,000 plaque-forming units were injected into eggs and harvested at 48 hours post infection. Viruses were divided into two subgroups: those that replicated in eggs from library infection or those that were not amplified in eggs during library infection. Virus numbers correspond to barcode identifiers in Supplementary Tables 1, 2, and 3. Data represented as mean SEM. (B) Sequencing of HA segment from viruses propagated in eggs and MDCKs. Viruses were plaque purified following inoculation of the virus library (10000 plaque forming units) into embryonated-chicken eggs or MDCKs and collected at 48 hours post infection. Ten viruses were purified from two independent infections of MDCK cells or eggs. The HA segment was sequenced and compared to the consensus sequence of the original virus library. Solid lines represent matches to the consensus sequence, while changes in amino acids are depicted in yellow highlight along with the surrounding amino acid context.

4 Amino acid positions are stated at the beginning and end of the sequence. The number of viral clones corresponding to that sequence is denoted on the left. The globular head region in the HA schematic is defined by the cysteines that form the disulfide bond at the base of the globular head. (C) Table denoting the percentage of the original virus library detected following in ovo propagation. Embryonated-chicken eggs were inoculated with viruses from the original viral library or viruses from an egg-adapted barcode library. 10,000 plaque-forming units were injected into eggs and harvested at 48 hours post infection. Virus present in supernants was deep-sequenced and the number of viral clones was quantified. (D) Graphical representation of (C). Two-tailed Students t-test was used to calculate P value, **** P<

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6 Figure S3, as related to Figure 5. Dynamics of direct and airborne infection. (A) Viruses were plaque purified at the time point of peak virus titers in nasal washes during the ferret transmission experiment. Animals were inoculated with plaque-forming units (pfu) of the virus barcode library, and naïve animals were housed into direct and airborne contact one day post infection. Ten viruses were purified from two experiments for each condition. The HA segment was sequenced and compared to the consensus sequence from the original virus library. Solid lines represent matches to the consensus sequence, while changes in amino acids are depicted in yellow highlight along with the surrounding amino acid context. Amino acid positions are stated at the beginning and end of the sequence. The number of viral clones corresponding to that sequence is denoted on the left. The globular head region in the HA schematic is defined by the cysteines that form the disulfide bond at the base of the globular head. (B) Comparison of clones detected during direct and airborne transmission events in all ferret and guinea pig experiments. Data depicted as percentage of the original viral library detected in the recipient animal where blue shapes indicate samples collected from guinea pigs, and red shapes data from ferrets. (C) Plot representing viral populations. Each color depicts a unique barcode whose relative proportion corresponds to its abundance in the virus population in the indicated sample. Library denotes starting virus material whereas IN mouse represents mice infected intranasasally. Mice were infected with 10,000 pfu and total lungs were harvested three days post-infection. (D) As in (C) where AI represents mice infected with nebulized virus. (E) Graph depicting percentage of the original viral library detected in the recipient animal from (C). Two-tailed Students t-test was used to calculate P value, * P<0.05, **** P<

7 Figure S4, as related to Figure 7. Predictor Model Results for Bronchus and Nasal Turbinate. Monte- Carlo simulations as described in Figure 6A as applied to bronchus and nasal turbinate subsets of the data. (A) The model served as a poor predictor for the transmission rates in the bronchus, with observed results being highly statistically unlikely (p=.003). (B) Nasal turbinate data showed greater conformity to model's predictions, with results likely to have been produced from the model (p=.73). Table S1, as related to Figure 1. Barcode Distribution Data from in vitro and in ovo Infections. Illumina miseq-based data detailing the overall percentage of each viral barcode sequenced during in vitro (MDCKs and A549s) or in ovo (embryonated chicken eggs)infections. Numbers are derived from individual/total barcode reads. Table S2, as related to Figure 3. Barcode Distribution in Guinea Pigs following Direct and Contact Infection. Percentage of viral barcodes as determined by deep sequencing following direct infection of guinea pigs. IAV Library v2 denotes the starting library (purified virus). Additional table headings depict the day (d2, d6, or d8) post infection, the cage location (A,B,C, or D), and whether the infection was initiated by direct infection (DI) or contact infection (CI). Numbers are derived from individual/total barcode reads. Table S3, as related to Figure 4. Barcode Distribution in Guinea Pigs following Group Contact Infections. Illumina miseq-based data detailing the overall percentage of each viral barcode sequenced during direct infection of a single guinea pig co-caged with three naïve animals. Table headings are as described in Table S2. Numbers are derived from individual/total barcode reads. Table S4, as related to Figure 5. Barcode Distribution in Ferrets Following Virus Transmission. Illumina miseq-based data detailing the overall percentage of each viral barcode in the starting library (IAV Library V2) as well as from Ferrets that were directly infected (DI), contact infected (CI), or aerosol infected (AI). Figure legends depict days post infection and cage location. NW denotes nasal wash. BR denotes bronchus-derived sample. Numbers are derived from individual/total barcode reads.