MicroRNA-based strategy to mitigate the risk of gain-of-function influenza studies

Abstract

Recent gain-of-function studies in influenza A virus H5N1 strains revealed that as few as three-amino-acid changes in the hemagglutinin protein confer the capacity for viral transmission between ferrets. As transmission between ferrets is considered a surrogate indicator of transmissibility between humans, these studies raised concerns about the risks of gain-of-function influenza A virus research. Here we present an approach to strengthen the biosafety of gain-of-function influenza experiments. We exploit species-specific endogenous small RNAs to restrict influenza A virus tropism. In particular, we found that the microRNA miR-192 was expressed in primary human respiratory tract epithelial cells as well as in mouse lungs but absent from the ferret respiratory tract. Incorporation of miR-192 target sites into influenza A virus did not prevent influenza replication and transmissibility in ferrets, but did attenuate influenza pathogenicity in mice. This molecular biocontainment approach should be applicable beyond influenza A virus to minimize the risk of experiments involving other pathogenic viruses.

Figure 1. Species-specific miRNA expression.

Small RNAs were isolated from the human lung epithelial cell line A549, ferret lung and MDCK cells. Small RNAs were cloned and deep sequenced. Graph depicts percent of each miRNA among total miRNA, and displays the ten miRNAs most highly expressed in A549 cells.

Figure 2. Species-specific miRNAs can be exploited to control influenza A virus tropism.

(a) Schematic illustrating WT (unmanipulated) and engineered (control and 192t) hemagglutinin viral segments. For the engineered segments the complete packaging signal (black) was duplicated, allowing for insertion of the control or miR-192 target sequences. Gray area represents hemagglutinin open reading frame. (b) MDCK and MDCK192 cells (left panel) or A549 cells (right panel) were infected with WT, control or 192t H5 HAlo virus. After a single cycle of infection, abundance of viral hemagglutinin (HA) and nucleoprotein (NP) was determined by western blot analysis. Actin, loading control. (c) Replication kinetics of viruses depicted in a in MDCK and MDCK192 cells. Data points indicate plaque quantifications performed in triplicate at indicated hours post-infection. Error bars, mean ± s.e.m. Data are representative of two independent experiments.

Figure 3. Species-specific miR-192 targeting attenuates influenza A virus infection in mice.

(a,b) Mice were infected with 100 plaque forming units (p.f.u.) of WT, control or 192t H5 HAlo virus (or with 1,000 p.f.u. of 192t virus, indicated as 10×) intranasally and weight loss a, and mortality b, were assessed daily (n = 9 WT, 9 control, 10 192t and 5 10×192t mice per group). (c) Mice were infected as in a and virus titered from whole lung at 3 and 5 days post-infection (n = 3 mice per group; each dot represents an individual mouse). (d) Mice were infected as in a and viral hemagglutinin and nucleoprotein protein expression from whole mouse lung were determined by western blot analysis 3 d after infection. Actin, loading control. Data are representative of two independent experiments. Morbidity and mortality data were pooled from two experiments with 4 or 5 mice per group in each experiment. Error bars, mean ± s.e.m.

Figure 4. Species-specific miRNA targeting of influenza A virus does not affect virus capacity to infect, replicate or transmit between ferrets.

(a–c) Ferrets were directly infected with WT (a), control (b) or 192t (c) H3N2 viruses and monitored by nasal wash on indicated days post-infection (d.p.i.). Infections induced by direct contact (left panels) or respiratory contact (right panels) were determined by nasal wash of naive animals co-housed with directly infected animals. Open and closed bars represent individual ferrets.