Establishment Of Nucleic Acid Testing System For Detecting Blood-borne Human Parvoviruses And Molecular Characterizationof Human Parvovirus B19

Viral safety of plasma-derived medicinal products(PDMPs)has always been particularly concerned.With the development and application of the blood-borne pathogen detection as well as viral inactivation/removal technologies,the risk of human immunodeficiency virus(HIV),hepatitis B virus(HBV)and hepatitis C virus(HCV)transmission through PDMPs has been dramatically reduced.Human parvovirus B19(B19V)and human parvovirus 4(PARV4)become the emerging pathogen threats to safety of PDMPs and are attracting more concerns.In order to reduce the possible risk of B19 V transmission by PDMPs,international organizations and developed countries have taken monitoring measures to control the contamination status of B19 V in source plasma and PDMPs.They proposed nucleic acid amplification technology(NAT)for B19 V as an in-process test and recommended or stipulated a limit of 104 IU/m L for plasma pools destined for manufacturing the PDMPs.PARV4 causes wide attention because of the similar biological,physical and chemical properties with B19 V.But there are not any related guidelines about PARV4 in source plasma.In China,there are not any related documentations and technical guiding principles for monitoring the human parvoviruses.Abundant data on the prevalence and level of B19 V in Chinese blood donors and in source plasma pools as well as plasma derivatives are not available.The characteristics of the human parvoviruses circulating in China have not been systemically studied.On the basis of the background and according to our national conditions,some researches have been done in our study as follows.(1)Optimization of the competitive internal control in the human parvovirus B19 nucleic acid testing system and evaluationOn the basis of our previous studies of the B19 V nucleic acid testing system,the production method of competitive internal control(IC)was optimized.The internal control DNA sequence was cloned into the M13mp18 vector and transfected into XL1-Blue cells to produce the competitive phage IC,designated as M13mp18-IC,which consisted of single-strand DNA packaged in a protein coat.Following optimization,the IC was added to each plasma sample to give a final concentration of 10 copies/?L.Finally,the sensitivity,specificity,reproducibility and accuracy of the assay were evaluated.Results indicated that,the limit of quantification(LOQ)of the assay was determined to be 10 copies/?L,meaning that we can quantified the B19 V DNA as low as 492 IU/m L in plasma samples.This assay detected three genotypes of B19 V DNA rather than other blood-borne viruses.The intra-and interassay coefficients of variation(CV)for Cq(quantification cycle)values were calculated to be lower than 2%,while the mean CV value for copy number evaluation were calculated to be 8.60% and 9.17% in the range 5×107~5×101 copies/?L.The accuracy of the assay is high(slope=1.005)in the range 1×108~1×104 copies/?L.The B19 V universal nucleic acid testing system established in this study has good sensitivity,specificity,reproducibility and accuracy,and can prevent false negative results.This system can be used to screen plasma pools destined for manufacturing PDMPs for the presence of B19 V DNA,which will be significant for ensuring the safety of PDMPs in China.Besides,this system can also be used in the early diagnosis of B19 V infection,which will be beneficial for the further development of B19 V diagnostic kit.(2)Establishment and evaluation of a duplex nucleic acid testing system for human parvovirus B19 and human parvovirus 4Firstly,the oligonucleotide primers and probes for the detection of B19 V and PARV4 were synthesized respectively while the primers and probe for noncompetitive IC were designed and synthesized according to the sequence of helper phage M13K07.Subsequently the reference plasmids of B19 V,PARV4 and noncompetitive IC were prepared or constructed,respectively.Then the q PCR reactions were optimized with respect to annealing temperature,probe and primer concentrations,and final Mg2+ concentrations.Following optimization,the IC was added to each plasma sample to give a final concentration of 2.5 copies/?L.Finally,the sensitivity,specificity and reproducibility of the duplex assay were evaluated.Results indicated that,the limit of detection(LOD)was determined to be 5 copies/?L both for B19 V DNA and PARV4 DNA.This assay detected three genotypes of B19 V and PARV4 rather than other blood-borne viruses.The intra-and interassay CV for Cq values were calculated to be lower than 2% for B19 V and PARV4.The average of intraassay CV for copy number evaluation were calculated to be 4.74% for B19 and 5.97% for PARV4 respectively in the range 5×106~5×101 copies/?L,while the average of interassay CV for copy number evaluation were respectively 8.22% for B19 and 12.29% for PARV4.The duplex nucleic acid testing system for B19 V and PARV4 established in this study has good sensitivity,specificity and reproducibility,and can prevent false negative results.This system will be beneficial for the detection of B19 V and PARV4 in Chinese blood/plasma donors.(3)Detection of blood-borne parvoviruses in Chinese blood/plasma donorsBy using the duplex nucleic acid testing system for B19 V and PARV4 developed in our study,a total of 1151 single donations collected from Beijing and Xi'an were screened and quantified.Results showed that,in total,five blood donors(0.43%)were infected with B19 V,with the concentrations of 6.80×102~1.38×105 IU/mL.Three blood donors in Xi'an region(0.3%)were infected with B19 V,while two in Beijing(1.32%).All of the donations were PARV4 DNA negative.By using the B19 V universal nucleic acid testing system developed in our study,235 source plasma pools from three different regional Chinese PDMPs manufacturers were screened and quantified.Results showed that 71.91%(169/235)of plasma pools were contaminated by B19 V,with the concentrations of 5.18×102~1.05×109 IU/m L.Approximately 68.05%(115/169)of the DNA-positive plasma pools were higher than 104 IU/mL.These results indicated that the prevalence and titre of B19 V and PARV4 in blood donors of this study were low.But the high level of B19 V in plasma pools could present a great risk in PDMPs.Therefore,the implementation of B19 V NAT assays for Chinese PDMPs manufacturers seems to be necessary and is significant for ensuring the safety of PDMPs in China.(4)Molecular characterization of human parvovirus B19 in Chinese blood/plasma donorsThe subgenomic NS1/VP1 u region junction of B19 V derived from a total of 123 B19V-DNA positive plasma samples was cloned and subsequently sequenced.Multiple sequence alignment was carried out on B19 V sequences derived from samples together with reference sequences of B19 V downloaded from GenBank.Then phylogenetic analysis and recombination identification were performed.Phylogenetic analysis indicated that there were at least three subtypes(1a,1b and 3b)of B19 V circulating in China and no genotype 2 was detected.Genotype 1a(78/123,63.41%)dominated in China,followed by genotype 3b(21/123,17.07%)and 1b(12/123,9.76%).Four B19 V 1a/3b recombinants and five new atypical B19 V variants with no recombination events were identified.Besides,near-full-length genome of B19 V was cloned and sequenced,followed by multiple sequence alignment and phylogenetic analysis.A total of six near-full-length sequences of B19 V were obtained and phylogenetic analysis indicated that these sequences belonged to B19 V genotype 1a.The present data provides,for the first time,evidence that B19 V subtypes 1a,1b and 3b are currently circulating in Chinese plasma donors.Furthermore,this is the first report of the putative B19 V 1a/3b recombinant and unclassified strains in China as well.This study will be helpful for insight into the prevalence and genotypic characterization of B19 in China as well as the mutation trend of B19 V.This study will be significant for B19 V infection prevention and control and also important for ensuring the safety of both blood and PDMPs in China.