Identification of a novel astrovirus from intestinal tissue of a donkey foal with severe diarrhea in China

DEAR EDITOR,Astroviruses are known to cause gastroenteritis and diarrhea symptoms in various hosts.However,no information is currently available on any donkey astrovirus genome.In this study,we collected six intestinal samples from donkey foals that died of severe diarrhea on a donkey farm in Shandong Province,China,in 2021.Based on metagenomic nextgeneration sequencing,a nearly complete donkey astrovirus 1(DAstV-1)sequence was detected as the only possible diarrhea-related mammalian virus.

Virome in healthy pangolins reveals compatibility with multiple potentially zoonotic viruses

Previous studies have identified multiple viruses in dead or severely diseased pangolins,but descriptions of the virome in healthy pangolins are lacking.This poses a greater risk of cross-species transmission due to poor preventive awareness and frequent interactions with breeders.In this study,we investigated the viral composition of 34 pangolins with no signs of disease at the time of sampling and characterized a large number of arthropodassociated viruses belonging to 11 families and vertebrate viruses belonging to eight families,including those with pathogenic potential in humans and animals.Several important vertebrate viruses were identified in the pangolins,including parvovirus,pestivirus,and picobirnavirus.The picobirnavirus was clustered with human and grey teal picobirnaviruses.Viruses with cross-species transmission ability were also identified,including circovirus,rotavirus,and astrovirus.Our study revealed that pangolins are frequently exposed to arthropod-associated viruses in the wild and can carry many vertebrate viruses under natural conditions.This study provides important insights into the virome of pangolins,underscoring the importance of monitoring potential pathogens in healthy pangolins to prevent outbreaks of infectious diseases in domesticated animals and humans.

Repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus-related coronavirus model

Background:Medicines for the treatment of 2019-novel coronavirus(2019-nCoV)infections are urgently needed.However,drug screening using live 2019-nCoV requires high-level biosafety facilities,which imposes an obstacle for those institutions without such facilities or 2019-nCoV.This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019(COVID-19)in a 2019-nCoV-related coronavirus model.Methods:A 2019-nCoV-related pangolin coronavirus GX_P2V/pangolin/2017/Guangxi was described.Whether GX_P2V uses angiotensin-converting enzyme 2(ACE2)as the cell receptor was investigated by using small interfering RNA(siRNA)-mediated silencing of ACE2.The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection.Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2V infection.The anti-viral activities and anti-viral mechanisms of potential drugs were further investigated.Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction(qRT-PCR)and plaque assay,respectively.Results:The spike protein of coronavirus GX_P2V shares 92.2%amino acid identity with that of 2019-nCoV isolate Wuhanhu-1,and uses ACE2 as the receptor for infection just like 2019-nCoV.Three drugs,including cepharanthine(CEP),selamectin,and mefloquine hydrochloride,exhibited complete inhibition of cytopathic effects in cell culture at 10μmol/L.CEP demonstrated the most potent inhibition of GX_P2V infection,with a concentration for 50%of maximal effect[EC50]of 0.98μmol/L.The viral RNA yield in cells treated with 10μmol/L CEP was 15,393-fold lower than in cells without CEP treatment([6.48±0.02]×10-4vs.1.00±0.12,t=150.38,P<0.001)at 72 h post-infection(p.i.).Plaque assays found no production of live viruses in media containing 10μmol/L CEP at 48 h p.i.Furthermore,we found CEP had potent anti-viral activities against both viral entry(0.46±0.12,vs.1.00±0.37,t=2.42,P<0.05)and viral replication([6.18±0.95]×10-4vs.1.00±0.43,t=3.98,P<0.05).Conclusions:Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research.CEP,selamectin,and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection.Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus,and further study of CEP for treatment of 2019-nCoV infection is warranted.

An easy-to-use site-directed mutagenesis method with a designed restriction site for convenient and reliable mutant screening

Site-directed mutagenesis (SDM) has been a very important method to probe the function-structure relationship of proteins. In this study, we introduced an easy-to-use, polymerase chain reaction (PCR)-based SDM method for double-stranded plasmid DNA, with a designed restriction site to ensure simple and efficient mutant screening. The DNA sequence to be mutated was first translated into amino acid sequence and then the amino acid sequence was reversely translated into DNA sequence with degenerate codons, resulting in a large number of sequences with silent mutations, which contained various restriction endonu-clease (RE) sites. Certain mutated sequence with an appropriate RE site was selected as the target DNA sequence for designing a pair of mutation primers to amplify the full-length plasmid via inverse PCR. The amplified product was 5′-phosphorylated, cir-cularized, and transformed into an Escherichia coli host. The transformants were screened by digesting with the designed RE. This protocol uses only one pair of primers and only one PCR is conducted, without the need for hybridization with hazardous isotope for mutant screening or subcloning step.

Identification of tick-borne pathogen diversity by metagenomic analysis in Haemaphysalis longicornis from Xinyang,China

Background:A wide variety of pathogens could be maintained and transmitted by Haemaphysalis longicornis.The aim of this study is to systematically examine the variety of pathogens carried by Haemaphysalis longicornis,an importnatn vector,in tick-borne diseases epidemic area,and to estimate the risk of human infection imposed by tick bites.Methods:Adult questing ticks were collected in Xinyang,central China.Genomic DNA and RNA were extracted from 144 H.longicornis ticks individually,and sequenced respectively as the templates for high-throughput sequencing.Clean reads were compared against the database of NCBI nucleotide collection and specific PCR was performed to confirm the presence of pathogen.Phylogenetic analysis was performed to explore the evolutionary status of pathogens.Results:The assignment of reads to taxa based on BLASTN results revealed the existence of several potential pathogens,including Anaplasma spp.,Rickettsia spp.,Babesia sp.,as well as severe fever with thrombocytopenia syndrome bunyavirus(SFTSV).Comfirmantory PCR assays revealed the existence of Anaplasma bovis(13/144,9.03%),Anaplasma centrale(2/144,1.39%),Rickettsia heilongjiangensis(3/144,2.08%),Rickettsia sp.LON-13(1/144,0.69%),Rickettsia raoultii(5/144,3.47%),Babesia sp.(1/144,0.69%).SFTSV accounted for the highest detected pathogen with a positive rate of 18.75%(27/144).Three of the ticks(2.08%)were co-infected with SFTSV and A.bovis.Conclusion:Our study provided a broadened list of microorganism that harbored by H.longicornis.In previously unrecognized endemic regions,prokaryotic and eukaryotic infection including Anaplasma spp.,Rickettsiae spp.,and Babesia spp.should be considered,along with the well-known SFTSV for patients with tick bites history.A novel Babesia species was identified in local natural foci,which needs further investigation in the future.

A large-scale fatal outbreak of Pasteurella multocida among wild rodents in southwestern China

Background:Pasteurella multocida is an important and old zoonotic pathogen worldwide which has an impressive host spectrum including numerous domestic and wild animals as well as birds,causing specific diseases or outbreak with great economic impact.It has never been reported that P.multocida can cause an epidemic in wild rodents.In June 5–17,2016,more than 1000 rodent deaths of an unknown cause quickly spread in the PuEr City,Yunnan province,southwestern China.Methods:The rodents in affected areas and outside of the epidemic areas were collected and screened for possible known pathogens including Yersinia pestis,rabies virus and hantavirus as well as other bacteria.The possible bacterial pathogens were isolated both by culture medium and by mouse inoculation in parallel.The isolates were identified by the Vitek GNI card and PCR assays for 16S rRNA genes.The pathogen strains were selected for whole genome sequencing analysis.Results:A total of 123 rodents were collected from 25 sample sites at affected area,among of which,all 119 dead rodents were negative for the pathogen under consideration except P.multocida,and all four live rodents were negative for P.multocida.In addition,480 rodents collected from other 23 counties outside of the epidemic area in Yunnan were negative for with P.multocida.A total of 14 strains of P.multocida(six directly isolated from the field rodents and eight from the experimental mice that were injected with the organ substrates from the dead rodents)belonged to serogroup A and serogroup F represented by 9 N and 20 N were identified in these epidemic areas.Whole genome sequencing revealed that the serogroup F strain shared 99%similarity to P.multocida Pm70 from chicken,but contained a 50 k bp insertion sequence.The serogroup A strain shared 95%similarity to P.multocida FDAARGOS_385 from a human patient,but contained four large structural differences.Histological abnormalities were identified in the livers,lungs,hearts and brains of the inoculated mice.Conclusions:The simultaneous occurrence of both serotypes of P.multocida may have caused this sudden onset of mortality across the local rodent population in Yunnan Province,China.Further attention should be paid to this old bacterium in the world.