Identification critical host factors for Japanese encephalitis virus replication via CRISPR screening of human sgRNA library.

Japanese encephalitis virus (JEV) is a pathogen with a substantial impact on both livestock and human health. However, the critical host factors in the virus life cycle remain poorly understood. Using a library comprising 123411 small guide RNAs (sgRNAs) targeting 19050 human genes, we conducted a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based screen to identify essential genes for JEV replication. By employing knockout or knockdown techniques on genes, we identified eleven human genes crucial for JEV replication, such as prolactin releasing hormone receptor (PRLHR), activating signal cointegrator 1 complex subunit 3 (ASCC3), acyl-CoA synthetase long chain family member 3 (ACSL3), and others. Notably, we found that PRLHR knockdown blocked the autophagic flux, thereby inhibiting JEV infection. Taken together, these findings provide effective data for studying important host factors of JEV replication and scientific data for selecting antiviral drug targets.

HVT-vectored H7 vaccine protects chickens from lethal infection with the highly pathogenic H7N9 Avian influenza virus.

Since mid-2016, the highly pathogenic H7N9 subtype avian influenza virus (AIV) has threatened both public health and the poultry industry. Although a vaccination strategy has been deemed imperative to manage the virus, the most commonly used inactivated vaccines today are susceptible to interference from maternal antibodies and associated with an over-reliance on humoral immunity. In response, we developed a recombination vaccine with the herpesvirus of turkeys (HVT) as the vector to squeeze HPAI H7N9 and assessed its protective efficiency in immunized chickens. By inserting an enhanced green fluorescent protein (EGFP) expression cassette (i.e., MCMV+EGFP+SV40 polyA) into the HVT065 and HVT066 positions, we obtained the recombinant HVT expressing EGFP (i.e., rHVT-EGFP). Electroporation and EGFP tags improved the efficiency of transfection compared with transfection using expression plasmids without any fluorescent labeling and traditional liposomes. Using limiting dilution analysis and ultrasonic cell disruption techniques, we screened and purified a cell-bound herpes virus based on rHVT-EGFP and consequently constructed a recombinant HVT expressing the hemagglutinin (HA) of H7N9 (i.e., rHVT-H7HA), which was able to proliferate similarly to the parental strain, stably pass for at least 15 generations in vitro, and replicate stably in multiple organs in vivo. After chickens were immunized with rHVT-H7HA, the average antibody titers reached up to 3 log2 at 35 d post-vaccination and remained stable. Those results suggest that rHVT-H7HA can protect chickens against H7N9 with a dose-independent immune protection rate of 90% and significantly reduce the lung virus titer 4 d post-challenge.