Study On The Pregnant Women Infected With Parvovirus B19 Cloning And Sequencing Of B19 Virus Genome From Zhengzhou Isolate Of China

Background and Objective:Being the smallest and simplest DNA viruses able to infect mammalian cells, humanParvovirus B19 (B19) is the only member of the family Parvoviridae known to bepathogenic in humans. Its infection is generally spread in the community via respiratoryroute and contaminated blood products. Since 1990, the Parvovirus B19 infection was firstconfirmed in China, many subsequent reporting have proved that parvovirus B19 is theprimary cause of non-immune abortion, children acute transient aplastic crisis, acutethrombocytopenic purpura and so on. B19 can be transmitted transplacentally from aninfected mother to her fetus, which may lead to non-immune fetal hydrops (NIHF),spontaneous abortion or intrauterine fetal death (IUFD). In our country, however, mostwomen are non-immune and do not possess neutralizing antibodies to B19 and, therefore,are susceptible to infection by this virus. Confirmation of the diagnosis is primarily basedon detection of specific antibodies by enzyme-linked immunosorbent assay and detection ofviral DNA by PCR. In spite of that, B19 infection in pregnant women have not beenreported in Zhengzhou, and besides, there are few nucleotide sequence analysis and noresearch about the complete sequencing of B19 in China, and moreover, data aboutdifference between the epidemic strain of B19 virus in China and overseas strains is notadequate. Therefore, we detected B19 IgM, B19 IgG and B19-DNA in the serum usingELISA and PCR respectively in order to investigate human parvovirus B19 infection inpregnant women in Zhengzhou. At the same time, we extracted B19-DNA from their sera to serve as PCR reaction templates and afterwards get whole genome cloning of B19 andsequenced, then the sequence date was compared with the known sequence in Genbank. Asa result, we offered the data about Zhengzhou for B19 sequence analysis in China.Methods:There were 29 serum samples collected from unidentified reason abortion or stillbornfoetus pregnant women and 237 control serum samples from normal pregnant women.B19-DNA, B19 IgM and B19 IgG in these specimens were detected using PCR and ELISArespectively, and moreover, we analyzed this datum using statistical software.According to the full-length B19 sequence published by Genbank NC₀00883, wedesigned 7 pairs of overlapping primers. Using PCR and molecular cloning technique, we get7 gene segments which overlapped each other. Each gene segment was inserted into pGEM-Tplasmid, which was transformed into JM109. Furthermore, Ampicillin fastness and blue/whitescreening was also used to select the positive clone and PCR was used to identify thesequence of DNA for the purpose of the correctness of objective fragment cloned, thereby, weconstructed and amplified T vector containing the complete genome sequence of B19 virusfor sequence.Results:1. The positive rate of B19 IgG and B19 IgM in the serum were counted as 51.7ï¼…(15/29) and 6.9ï¼…(2/29) in 29 unidentified reason abortion or stillborn foetus pregnantwomen. By contrast, the positive rate of B19 IgG and B19 IgM were counted as 30.4ï¼…(72/237) and 6.9ï¼…(2/29) in 237 normal pregnant women, respectively.2. The positive rate of PCR in the serum were counted as 13.8ï¼…(4/29) in 29 unidentifiedreason abortion or stillborn foetus pregnant women. By contrast, the positive rate of PCRwere counted as 2.5ï¼…(6/237) in 237 normal pregnant women.3. Seven recombinant plasmids T vectors respectively contained seven gene segmentswhich overlapped each other were constructed and cloned successfully.4. Compared with the known sequence in GenBank NC₀0088, The complete genome ofB19 virus has 10 bases differences between them. Furthermore, only two caused amino aciddifference.Conclusions: The datum showed that the positive rate of B19 IgG,B19 IgM and B19-DNA in 237normal pregnant women matched with the report all over the world and the infection ofhuman parvovirus B19 exists in the pregnant women in Zhengzhou.Compared with the nucleotide sequence from GenBank, The complete genome sequenceof B19 virus from Zhengzhou are all more homologous to the NC00088. there are 10 baseschanged, no base insertion or deletion, and only two mutations caused amino acid difference.This result is identical with the overseas reports that the sequence of B19 virus is relativelysteadily.