【病毒外文文献】2016 Longitudinal surveillance of SARS-like coronaviruses in bats by quantitative real-time PCR

VIROLOGICA SINICA 2016 31 1 78 80 DOI 10 1007 s12250 015 3703 3 LETTER Longitudinal surveillance of SARS like coronaviruses in bats by quantitative real time PCR Dear Editor The 2002 2003 severe acute respiratory syndrome coronavirus SARS CoV Drosten et al 2003 caused human pandemics that began in China and spread glob ally Subsequently diverse SARS like coronaviruses SL CoVs have been detected in horseshoe bats in China Europe and Africa Li et al 2005 Tong et al 2009 Drexler et al 2010 Recently we found SL CoVs with high genetic diversity in a single bat colony pre dominantly roosted by Chinese horseshoe bats Rhinolo phus sinicus in Kunming Yunnan province Ge et al 2013 Two of these SL CoVs are able to use human ACE2 as a receptor for cell entry Ge et al 2013 Men achery et al 2015 highlighting the risk of this group of viruses to humans and the importance of long term sur veillance We conducted a longitudinal surveillance study of bat SL CoVs using quantitative real time PCR qRT PCR targeting the nucleocapsid N and RNA dependent RNA polymerase RdRp genes in one bat population in Yun nan China A total of 431 bat fecal samples were collec ted during 2011 2014 Total RNA extraction was per formed with 200 L of each fecal sample using a High Pure Viral RNA Kit Roche Basel Switzerland accord ing to the manufacturer s instructions Five microliters of RNA was used to screen for all alphacoronaviruses and betacoronaviruses as previously described Ge et al 2013 To construct standard templates fragments of the N and RdRp genes were amplified from WIV1 genomic RNA and cloned into the pGEM T Easy Plasmid Vector Promega Madison USA The primers and probe for the N gene were adopted from our previous report Ge et al 2013 and the primers and probe targeting the con served region of RdRp was newly designed Supplement ary Table S1 Both forward primers contained a 5 T7 RNA polymerase promoter sequence TAATACGACT CACTATAGGG to facilitate in vitro transcription Cor rect clones were transcribed using the MAXIscript Kit Applied Biosystems Waltham USA Following puri fication and quantification ten fold serial dilutions of the RNA transcripts of the N and RdRp genes were used as external standards to calculate viral concentrations which are expressed as genome copies per gram of bat feces copies g The qRT PCRs were performed using an AgPath ID One Step RT PCR Kit Applied Biosystems ac cording to the manufacturer s instructions Each 25 L reaction mixture contained 12 5 L of 2 RT PCR buf fer 1 L of RT PCR Enzyme Mix 400 nmol L each primer 120 nmol L probe primer and 1 L of nucleic acid extract Amplification was carried out in 96 well plates using the StepOne PCR system Applied Biosys tems Thermocycling conditions were as follows 10 min at 45 C for reverse transcription 10 min at 95 C for activation of the Taq DNA polymerase and 40 cycles of 95 C for 15 sec and 60 C for 45 sec Each run in cluded three viral positive control templates and one neg ative control to evaluate assay performance A positive result was defined as a well defined exponential fluores cence curve that crossed the cycle threshold Ct within 38 cycles A specimen with a Ct value 36 was assayed again to exclude operation faults Data were analyzed us ing the analysis of variance ANOVA for continuous variables All comparisons were two tailed and a P value of less than 0 05 was considered significant The analytical detection range and sensitivity of the two real time PCR assays for SL CoVs were investig ated by testing 10 fold serial dilutions of RNA tran scripts and WIV1 genomic RNA The highest dilution of transcripts at which all three replicates were positive was defined as the limit of detection LoD Ct values were plotted against the log10 of gene copy number and lin earity was observed over the entire virus concentration range Supplementary Figure S1 LoD values of N and RdRp transcripts were three and four copies respect ively Linear amplifications for the N assay ranged from 101 to 109 copies reaction efficiency values 132 while for RdRp were from 100 to 109 copies reaction ef ficiency values 109 The LoDs of WIV1 genomic RNA were as low as 2 04 10 2 plaque forming units pfu reaction for both assays Linear amplifications ranged from 2 04 10 2 to 2 04 105 pfu reaction with efficiency values of 85 and 84 respectively Supple mentary Figure S2 The specificity of the two assays was then confirmed using Orthoreovirus isolated from a 78 FEBRUARY 2016 VOLUME 31 ISSUE 1 Wuhan Institute of Virology CAS and Springer Science Business Media Singapore 2016 bat Yang et al 2015 and viral containing bat fecal samples that are positive for paramyxovirus hepatitis A virus hepatitis B virus hepatitis E virus or coronavir uses other than SL CoVs unpublished No false posit ive was observed for any of these samples data not shown Fifty seven of 431 bat fecal samples were positive for SL CoVs by RT PCR screening The detection rate var ied significantly among sampling dates ANOVA F 28 42 P 0 03 from 3 1 to 48 7 The highest de tection rate was observed in September 2012 Standard curves of in vitro transcribed RNA for both assays were obtained with R2 0 99 Supplementary Figure S3 qRT PCR assays for N and RdRp were used to quantify the concentration of SL CoVs in the positive samples Both assays were sensitive and efficient when extracted RNAs were used as templates Additionally the Ct val ues means of triplicates standard deviation for most of the bat fecal samples were within 36 The virus con centrations of these specimens are listed in supplement ary Table S2 Of note the concentration of individual samples varied significantly ANOVA F 4 03 P 0 001 from 105 to 1011 copies g bat feces in the RdRp assay or 106 to 1011 copies g in the N assay Figure 1A The sample with the highest virus concentration was col lected in September 2012 1 71 1011 copies g for the RdRp assay and 4 58 1011 copies g for the N assay Ten samples 17 had virus concentrations of greater than 109 copies g four of which were collected in September 2012 and four in July 2013 Additionally the average of bat SL CoVs for the seven sampling times was evalu ated As shown in Figure 1B three significant peaks were observed ANOVA F 1 5 P 0 05 The first peak corresponding to the highest virus detection rate was observed in September 2012 The second peak was in July 2013 and the third peak was in October 2014 Both assays sensitively and efficiently detected SL CoV RNA in environmental samples However the vir Figure 1 qRT PCR assays of SL CoVs in bat fecal samples A SL CoV concentration in individual bat fecal samples Circles represent the N assay diamonds represent the RdRp assay B SL CoV concentrations for seven sampling dates Mei Niang Wang et al www virosin org FEBRUARY 2016 VOLUME 31 ISSUE 1 79 us concentrations for the RdRp assay were slightly lower than those of the N assay normally by one order of mag nitude To assess whether the discrepancy was caused by a systematic error repeatability was assessed by replicat ing the standard controls at least three times The relat ive abundance of the subgenomic mRNA for N was higher than that of RdRp during virus replication con sistent with previous observations Lu et al 2014 in dicating that sensitivity was higher for the N assay than the RdRp assay Although higher virus concentrations were generally observed for the N assay compared to the RdRp assay three samples contained less virus in the N assay with Ct values abnormally exceeding 36 These results indicate that the N assay is more sensitive and the RdRp assay is more stable and accurate The combina tion of these two assays could greatly reduce the false positive rate in future surveillance studies of SL CoVs Bats in this unique cave excreted 105 1011 copies g SL CoVs from 2011 to 2014 The N and RdRp assays re vealed dynamic changes in SL CoV concentrations in the longitudinal surveillance Fecal pellets with high vir us concentrations were typically collected in either September 2012 or July 2013 Additionally the average virus concentrations in July September and October were over 5 fold higher than those observed in April and May These results indicated that SL CoV amplification was more efficient from the late summer to autumn Sim ilar qRT PCR results for other bat viruses such as al phacoronaviruses Drexler et al 2011 henipaviruses Chua et al 2002 and filoviruses Pourrut et al 2007 have been reported Bat borne RNA viruses appear to have increased amplification and transmission efficien cies from the late summer to autumn unlike DNA vir uses Drexler et al 2011 Hypothetically the higher concentrations of RNA viruses in bats at specific times may be related to the life habit of bats Virus amplifica tion after July may be associated with the establishment of a susceptible subpopulation of newborn bats who had not yet mounted their own adaptive immunity during the parturition period Drexler et al 2011 Our longitudin al survey of SL CoVs in one bat population over 4 years provides valuable data for surveillance efforts to monit or the potential transmission of these viruses to humans FOOTNOOTES This work was jointly funded by the National Natural Science Foundation of China 81290341 China Mega Project for Infec tious Disease 2014ZX10004001 003 from the Minister of Sci ence and Technology of the People s Republic of China and US NIAID R01AI110964 The authors declare that they have no conflict of interest This article does not contain any studies with human or animal subjects performed by any of the authors Supplementary figures tables are available on the website of Virologica Sinica www virosin org 12250 Mei Niang Wang1 Wei Zhang1 Yu Tao Gao1 Ben Hu1 Xing Yi Ge1 Xing Lou Yang1 Yun Zhi Zhang2 Zheng Li Shi1 1 Key Laboratory of Special Pathogens and Biosafety Wuhan Institute of Virology Chinese Academy of Sciences Wuhan 430071 China 2 Yunnan Provincial Key Laboratory for Zoonosis Control and Prevention Yunnan Institute of Endemic Diseases Control and Prevention Dali 671000 China Correspondence Phone 86 27 87197240 Email zlshi ORCID 0000 0001 8089 163X Published online 18 February 2016 REFERENCES Chua KB Koh CL Hooi PS et al 2002 Microbes Infect 4 145 151 Drexler JF Corman VM Wegner T et al 2011 Emerg Infect Dis 17 449 456 Drexler JF Gloza Rausch F Glende J et al 2010 J Virol 84 11336 11349 Drosten C Gunther S Preiser W et al 2003 N Engl J Med 348 1967 1976 Ge XY Li JL Yang XL et al 2013 Nature 503 535 538 Li W Shi Z Yu M et al 2005 Science 310 676 679 Lu XY Whitaker B Sakthivel SKK et al 2014 J Clin Microbiol 52 67 75 Menachery VD Yount BL Jr Debbink K et al 2015 Nat Med 21 1508 1513 Pourrut X Delicat A Rollin PE et al 2007 J Infect Dis 196 S176 S183 Tang XC Zhang JX Zhang SY et al 2006 J Virol 80 7481 7490 Tong S Conrardy C Ruone S et al 2009 Emerg Infect Dis 15 482 485 Vijaykrishna D Smith GJD Zhang JX et al 2007 J Virol 81 4012 4020 Wang LF Shi ZL Zhang SY et al 2006 Emerg Infect Dis 12 1834 1840 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