Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

African swine fever(ASF)is an infectious disease caused by African swine fever virus(ASFV)with clinical symptoms of high fever,hemorrhages and high mortality rate,posing a threat to the global swine industry and food security.Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection.In this study,a recombinase polymerase amplification(RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF.As a highly sensitive method,RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lw Cas13 a(previously known as C2c2)through quantitative real-time PCR(q PCR)and has the same or even higher sensitivity than the traditional q PCR method.A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of Taq Man q PCR.Likewise,RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity.This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100%for both ASFV positive and negative samples.RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.

Genetic and Molecular Characterization of H9N2 Avian Influenza Viruses Isolated from Live Poultry Markets in Hubei Province, Central China, 2013–2017

H9N2 subtype avian influenza virus(AIV)is an influenza A virus that is widely spread throughout Asia,where it jeopardizes the poultry industry and provides genetic material for emerging human pathogens.To better understand the epidemicity and genetics of H9 subtype AIVs,we conducted active surveillance in live poultry markets(LPMs)in Hubei Province from 2013 to 2017.A total of 4798 samples were collected from apparent healthy poultry and environment.Realtime RT-PCR revealed that the positivity rate of influenza A was 26.6%(1275/4798),of which the H9 subtype accounted for 50.3%(641/1275)of the positive samples.Of the 132 H9N2 viral strains isolated,48 representative strains were subjected to evolutionary analysis and genotyping.Phylogenetic analysis revealed that all H9N2 viral genes had 91.1%–100%nucleotide homology,clustered with genotype 57,and had high homology with human H9N2 viruses isolated from2013 to 2017 in China.Using a nucleotide divergence cutoff of 95%,we identified ten distinct H9N2 genotypes that continued to change over time.Molecular analysis demonstrated that six H9N2 isolates had additional potential glycosylation sites at position 218 in the hemagglutinin protein,and all isolates had I155 T and Q226 L mutations.Moreover,44 strains had A558 V mutations in the PB2 protein and four had E627 V mutations,along with H9N2 human infection strains A/Beijing/1/2016 and A/Beijing/1/2017.These results emphasize that the H9N2 influenza virus in Hubei continues to mutate and undergo mammalian adaptation changes,indicating the necessity of strengthening the surveillance of the AIV H9N2 subtype in LPMs.