(980) Adult reinoculation of microbes is insufficient for restoring microbiome and normal resistance against congenital viruses

Zika virus (ZIKV) causes congenital disease in association with placental replication and fetal infection. We have previously demonstrated in a germ-free (GF) mouse model that the absence of microbes in pregnant mice increases ZIKV vertical transmission, leading to greater fetal demise. Here, we hypothesized that reinoculation of GF mice with its natural microbiome would restore endogenous resistance against ZIKV. Our aim was to measure differences in the ZIKV loads and the microbiome in GF, adult recolonized, and control mice.

Study Design:

Timed pregnant specific-pathogen-free (SPF) or GF Swiss-Webster mice were mock injected or received 4 doses of 1x10⁴ PFU first passage ZIKV on embryonic days 4 through 7. Adult GF mice that were microbiome recolonized by first co-housing in an SPF cage were also infected with ZIKV. Mouse placentas and fetal pups were extracted via sterile Cesarean on embryonic day E18.5, homogenized, and then analyzed via RT-qPCR ZIKV load. Stool was collected on E18.5 to assess differences in maternal microbiome via V4 16S sequencing.

Results:

GF mouse placentas did not have significantly higher ZIKV loads or incidence of infection than placentas of SPF mice. However, adult recolonized dams had significantly greater ZIKV loads by several orders of magnitude and incidence of infection (100%) compared to SPF or GF (p < 0.0001, Fig 1). The microbiomes were not significantly different between SPF mock- or ZIKV-infected mice. In contrast, adult recolonized ZIKV+ mice had microbiomes significantly different from both SPF groups (p=0.025, Fig 2).

Conclusion:

We tested the novel hypothesis that reintroduction of microbes to GF mice would restore normal resistance to ZIKV by restoring the normal microbiome. Interestingly, the microbiome of adult recolonized mice was not restored, and concurrently the endogenous resistance to ZIKV was significantly less with worse rates of ZIKV infection. These observations indicate that adult recolonized mice should not be considered “normal” mice, and the normal microbiome prior to onset of adult life is needed for endogenous congenital viral resistance.