Human blood is conventionally considered sterile but recent studies suggest the presence of a blood microbiome in healthy individuals. Here we characterized the DNA signatures of microbes in the blood of 9,770 healthy individuals using sequencing data from multiple cohorts. After filtering for contaminants, we identified 117 microbial species in blood, some of which had DNA signatures of microbial replication. They were primarily commensals associated with the gut (n = 40), mouth (n = 32) and genitourinary tract (n = 18), and were distinct from pathogens detected in hospital blood cultures. No species were detected in 84% of individuals, while the remainder only had a median of one species. Less than 5% of individuals shared the same species, no co-occurrence patterns between different species were observed and no associations between host phenotypes and microbes were found. Overall, these results do not support the hypothesis of a consistent core microbiome endogenous to human blood. Rather, our findings support the transient and sporadic translocation of commensal microbes from other body sites into the bloodstream.

The poxviruses are large, linear, double-stranded DNA viruses about 130 to 230 kbp, that have an animal origin and evolved to infect a wide host range. Variola virus (VARV), the causative agent of smallpox, is a poxvirus that infects only humans, but other poxviruses such as monkey poxvirus and cowpox virus (CPXV) have crossed over from animals to infect humans. Therefore understanding the biology of poxviruses can devise antiviral strategies to prevent these human infections. In this study we used a system-based approach to examine the host responses to three orthopoxviruses, CPXV, vaccinia virus (VACV), and ectromelia virus (ECTV) in the murine macrophage RAW 264.7 cell line. Overall, we observed a significant down-regulation of gene expressions for pro-inflammatory cytokines, chemokines, and related receptors. There were also common and virus-specific changes in the immune-regulated gene expressions for each poxvirus-infected RAW cells. Collectively our results showed that the murine macrophage RAW 264.7 cell line is a suitable cell-based model system to study poxvirus host response.

Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy.

Influenza A virus (IAV) is a major public health concern, being responsible for the death of approximately half a million people each year. Zoonotic transmissions of the virus from swine and avian origin have occurred in the past, and can potentially lead to the emgergence of new IAV stains in future pandemics. Pulmonary macrophages have been implicated in disease severity in the lower airway, and understanding the host response of macrophages infected with avian influenza viruses should provide new therapeutic strategies. We used a systems-based approach to investigate the transcriptome response of primary murine lung macrophages (PMФ) infected with the mouse-adapted H1N1/WSN virus and low pathogenic avian influenza (LPAI) viruses H5N2 and H5N3. The results showed that the LPAI viruses H5N2 and H5N3 can infect PMФ with similar efficiency to the H1N1/WSN virus. While all viruses induced antiviral responses, the H5N3 virus infection resulted in higher expression levels of cytokines and chemokines associated with inflammatory responses. The LPAI H5N2 and H5N3 viruses are able to infect murine lung macrophages. However, the H5N3 virus was associated with increased expression of pro-inflammatory mediators. Although the H5N3 virus it is capable of inducing high levels of cytokines that are associated with inflammation, this property is distinct from its inability to efficiently replicate in a mammalian host.

Although the influenza A virus H7N9 subtype circulates within several avian species, it can also infect humans with a severe disease outcome. To better understand the biology of the H7N9 virus we examined the host response to infection in avian and human cells. In this study we used the A/Anhui/1/2013 strain, which was isolated during the first wave of the H7N9 epidemic. The H7N9 virus-infected both human (Airway Epithelial cells) and avian (Chick Embryo Fibroblast) cells, and each infected host transcriptome was examined with bioinformatic tools and compared with other representative avian and human influenza A virus subtypes. The H7N9 virus induced higher expression changes (differentially regulated genes) in both cell lines, with more prominent changes observed in avian cells. Ortholog mapping of differentially expression genes identified significant enriched common and cell-type pathways during H7N9 infections. This data confirmed our previous findings that different influenza A virus subtypes have virus-specific replication characteristics and anti-virus signaling in human and avian cells. In addition, we reported for the first time, the new HIPPO signaling pathway in avian cells, which we hypothesized to play a vital role to maintain the antiviral state of H7N9 virus-infected avian cells. This could explain the absence of disease symptoms in avian species that tested positive for the presence of H7N9 virus.

Seasonal H3N2 influenza evolves rapidly, leading to an extremely poor vaccine efficacy. Substitutions employed during vaccine production using embryonated eggs (i.e., egg passage adaptation) contribute to the poor vaccine efficacy (VE), but the evolutionary mechanism remains elusive. Using an unprecedented number of hemagglutinin sequences (n = 89,853), we found that the fitness landscape of passage adaptation is dominated by pervasive epistasis between two leading residues (186 and 194) and multiple other positions. Convergent evolutionary paths driven by strong epistasis explain most of the variation in VE, which has resulted in extremely poor vaccines for the past decade. Leveraging the unique fitness landscape, we developed a novel machine learning model that can predict egg passage substitutions for any candidate vaccine strain before the passage experiment, providing a unique opportunity for the selection of optimal vaccine viruses. Our study presents one of the most comprehensive characterizations of the fitness landscape of a virus and demonstrates that evolutionary trajectories can be harnessed for improved influenza vaccines.