The emergence of drug resistance to virus(i.e.,acyclovir(ACV)to herpesviruses)has been termed one of the common clinical issues,emphasizing the discovery of new antiviral agents.To address it,a genome-wide clustered regularly interspaced short palindromic repeats(CRISPR)screening was performed in mouse haploid embryonic stem cells infected with pseudorabies virus(PRV),anα-herpesvirus causing human and pig diseases.The results demonstrated that type 2 voltage-gated chloride channels(CLC-2)encoded by one of the identified genes,CLCN2,is a potential drug target for anti-herpesvirus therapy.CLC-2 inhibitors,omeprazole(OME)and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid(DIDS),can efficiently inhibit infection of multiple herpesviruses in cellulo(i.e.,PRV,HSV and EBV),and effectively treat murine herpes simplex encephalitis(HSE).Additionally,DIDS was found to inhibit HSV-1 replication by blocking the PI3K/Akt pathway.Most importantly,both DIDS and OME were able to inhibit ACV-resistant HSV-1 strain infection.The study's findings suggest that targeting host-cell factors such as CLC-2 may be a promising approach to tackling herpesvirus drug resistance.The discovery of CLC-2 as a potential drug target for anti-herpesvirus therapy provides a new direction for the development of novel antiviral agents.
Carassius auratus herpesvirus(CaHV)is a pathogen isolated from crucian carp(Carassius auratus)associated with high mortality.A diagnosis method that can detect the virus at an early stage,specifically and accurately,is an urgent requirement for the prevention of CaHV transmission.In the present study,a droplet digital PCR(ddPCR)method based on the tumor necrosis factor receptor(TNFR)gene was established to detect and quantify CaHV DNA with high specificity and no cross-reactions with other aquatic viruses.Skin mucus samples were collected from infected crucian carp from Day 1–8 after infection,and positive amplification was detected on the first day by ddPCR(0.54 copies/μL),whereas the presence of CaHV was not detected by routine PCR until Day 6.Tissue DNA was then collected from the head kidney of 20 fishes which were injected with CaHV and died during the experiment.The five negative samples checked by routine PCR were detected by ddPCR and real-time PCR(qPCR),respectively.The results showed that the positive detection rate of ddPCR(100%)was higher than that of qPCR(40%).The detection limit of the ddPCR was found to be 0.52 copies/μL,which was much lower than the 50.12 copies/μL determined by qPCR.Overall,ddPCR offers a highly promising diagnosis method for the absolute quantification of CaHV in carrier fish and samples from the skin mucus and head kidney with low viral concentrations.
Frequent outbreaks of emerging infectious diseases in fish,such as Carassius auratus herpesvirus(CaHV)infection has caused great economic losses in China.However,the lack of a sensitive cell culture system has limited studies of CaHV.In the present study,a new cell line(gibel carp skin cell,GiCS)derived from gibel carp(Carassius gibelio)skin tissue was established to create a valuable tool for research of the virus.The GiCS cells consisted mainly of epithelial-like cells,which grew well at 25℃in L-15 medium supplemented with 10–20%fetal bovine serum.Chromosomal analysis revealed that the skin cell line remained amphitriploid,with most chromosome counts being 156(54%).The GiCS cells can be efficiently transfected and expressed exogenous genes.In particular,the GiCS cells showed high susceptibility to CaHV infection,which was confirmed by virus infection tests,detection of viral gene expression,and ultrastructural observation.To our knowledge,it is the first cell line that is highly permissive to CaHV infection.In addition,the cells also showed susceptibility to several aquatic animal viruses from different families including Iridoviridae,Rhabdoviridae,and Reoviridae.In conclusion,these results indicated that the establishment of the GiCS cell line is a significant advance that will be beneficial to future studies of CaHV and other aquatic animal viruses.
