Ferret upper respiratory tract microbiome in influenza infection

Barb Methe & Elodie Ghedin, JCVIAdolfo Garcia-Sastre, Mt Sinai, NYC

Questions study would address

• Following Influenza viral infection of the host, how is the bacterial community structure affected?

• Can we see a change in the bacterial community during the course of the infcetion?

• Once the host recovers from viral infection, how resilient is the bacterial community structure?

Influenza Genome Project

Pilot to determine best conditions

• 2 uninfected (ferrets “94” and “96”)– Nasal swab versus nasal wash– Day 0 and Day 2– Total specimens: 8

• 2 infected (ferrets “01” and “08”) + 1 uninfected control (“06”)– Nasal washes only– Day 0, Day 2, Day 3– Total specimens: 9

Replicates and controls

• Total of 17 specimens– 3 technical replicates of 16S amplicons– 2 regions for each: V1-V3 and V3-V5– For each round of PCR, 1 positive and 1 negative

control

• Total amplicons (test + controls): 114

• Sequencing: average of 4175 classifiable reads per sample

STUDY 1Infected versus uninfected ferrets

Infected V1-V3

Infected V3-V5

Fuso

bacteriu

m

Porphyromonas

Control uninfected ferret: Day 0

Ureaplasma

Cloacibacte

rium

Control uninfected ferret: Day 2

Ureaplasma

Exiguobacte

rium

Control uninfected ferret: Day 3

V1-V3

V3-V5

Bacteria rank differently dependingon region studied

Ferret “01”

Ferret “08”

Infected ferrets: Day 0

Ferret “01”

Ferret “08”

Infected ferrets: Day 2

Ferret “01”

Ferret “08”

Infected ferrets: Day 3

V1-V3

V3-V5

Summary

• Important differences depending on region analyzed (V1-V3 vs V3-V5)

• Ferret effect

STUDY 2Nasal Swab versus Wash

Wash captures more species than swab

V1-V3 vs V3-V5:Some species overlap and many unique

captured by each region

Conclusions• Based on the results we have obtained thus far,

the nasal washes are recovering the greatest diversity using either 16S variable region and are likely to be the most useful samples going forward.

• Not surprisingly, each 16S variable region returns a slightly different result and one can argue for either region. The V1V3 in general performs better in the lab and it is the region of the 16S gene that is evolving faster than the remainder of the gene (so in theory more discrimination).

Teams

JCVIBarb Methe

Elodie GhedinKelvin Li

Monika BihanSana Scherbakova

Mt. SinaiAdolfo Garcia-

SastreRafael Medina

Funding:NIAID

Infected

V1-V3 V3-V5

Uninfected