The Study Of The Diagnose Human Parvovirus B₁₉ By Nested PCR And The Afection To The Fetus By The Virus Infection

Human Parvovirus B₁9(HPVB₁9) is a single strained liner DNA virus in animal viruses,which belongs to genus parvovirus and has the smallest volume and the simplest structure.The distribution of HPVB19 is worldwide. HPVB19 was found in 1975,is the only kind of virus causing human diseases.Many human diseases,for example,erythema infectiosum, arthritis and granulocyto- pemia,even the severe disease of hematopoietic system,are due to the infection of HPVB19. It can be spread by respiratory tract, blood and Vertical transimssion between maether and baby, leading to local prevalonce in community, nosocomial infection and outbreak.The gene of HPVB19 is about 5.6Kb,which encode the nonstracture protein(NS) and the two capsid proteins (vp1 and vp2). Vpl and vp2 protein from the capsid of parvovirus B19, coating at the surface of the virus and related to the antigenicity of it.the main epitope of neutral antigen is lies in the vplunique area and the vp1-vp2connective area. In 1990,it was confirmed for the first time that there exiets HPVB19 in China. At the same time, stady showed that the protective antibody is low in serum of wamen and children in our country. So that the infection of HPVB19 is easy to happen. Especially in those patients whose immunity are low or absent , HPVB19 can cause persistent infection, severe diseases of hematopoietic system and chronic anemia, even endanger human lives. Followed studies found it is one of the important etiological factors of non-immunity abortion of the pregnent woman, acute aplastic crisis of children and acute idiopathic thrombocytopenic purpura and related to congenital heart diseases. Objective:In order to diagnoses the HPVB19 DNA, a new nested PCR-EB staining assay technique was establishes using two pairs of primers system, meanwhile the specifity and the sensitivity of nested PCR was tested. To make clear the condition of HPVB19 infection in fetus in China,and to testify its effec on relationship between spontaneous abortion, on the relationship between the congenital heart disease and HPVB19 infection, the nested PCR to detect HPVB₁9 DNA, the E1ISA to detect HPVB19 IgM was used. Method:(1)Preparation of samples: preparation of samples for Nested-PCR were based on general procedure, i .e. samples were incubated with proteinase K, then extracted by phenol and precipitated by alcohol.(2)Primers: The sequences of the nested-PCR primers were selected from the conserved region in the HPV B19 gene. The outer primers were at the position 5'-AGGTATAGCCA -TGG-3'(sense) and 5'-ACACTGAGTTTACTAGTGGC-3' (antisense). The inner primers were at the position 5'-CAAAAGCATGTGGAGTGAGG-3' (sense) and 5'-CCTT -ATAATGGTGCTCTGGG-3' (antisense).(3)Nested-PCR: 5μl TE dissolved DNA of the sample was added to 50μ PCR reaction system(first round PCR). 1μ1 of first round PCR product was added to 50μ1 PCR reaction system(second round PCR). The amplification parameters were 94 °C 30 s, 55 °C 30 s and 72 °C 30s, 35 circles for the first round PCR, 30 circles for second round PCR(nested-PCR).The amplified PCR products were visualized by standard gel electrophoresis. A sample containing HPV B19 DNA will produce a distinct 104bp band(positive control). The internal control DNA should be present in every reaction.(4)Nest polymerase chain reaction (PCR) and in situ hybridization (ISH) were used to detect HPVB19-DNA in the tissues of spontaneous abortion and cardiac muscle of CHD,enzyme linked immunosorbent assay (ELISA) to detect HPVB19-IgM.Results:(1) Specificity and sensitivity of Nested-PCR assay: Predicted fragment(104 bp) was amplified from the sample containing the plasmid in which HPV B19 DNA was cloned, however thesamples containing feline parvovirus, Adv3,Adv7, RSV, CMV, HSV, RV were found to be negative results. In the sensitivity test , as few as O.Olfg/ml of target DNA could be detected from a series of dilutions.(2) The assay was carried out in testing 182 specimens of spontaneous abortion tissue, of which 66 specimens were pathological tissue (paraffin embeded during the time of 1991-1994), 116 specimens were fresh tissue of spontaneous abortion. 40 normal tissue were used as negative control group. Results of Nested-PCR showed that 47 specimens from spontaneous abortion tissue were positive(the positive rate was 25.8%). 2 of 40 specimens were also positive(the positive rate was 5.0%). The discrepancy between two groups was significant (P < 0.05). This results suggest that there is a relationship between spontaneous abortion and HPV B19 infection.(3) In another experiment of detecting HPV B19 DNA in heart tissue of congenital heart disease(CHD), 66 specimens of fatal heart tissue of CHD, a pool of 37 peripheral blood from CHD infants were tested respectively by Nested-PCR assay. Meanwhile this 37 specimens of peripheral blood were also detected for anti-HPV B19 IgM antibody level by ELISA. Another group of 30 specimens of non-CHD heart tissue were used as control group. The positive specimens were 12 of 66(18.2%), no positive result was found in the control group. HPV B19 DNA could be found in 7 of 37 CHD infants peripheral blood, while results of ELISA showed thatanti-HPV B19 IgM were negtive in all 37 specimens. These results indicated that HPV B19 infection may be one of the causations of CHD.Conclusion:(1)A high sensitive Nested-PCR assay was developed to detecting HPV B19 DNA domestically. A single-step PCR may not be sufficient for detecting B19 DNA in specimen, a nested PCR is necessary for maximum sensitivity, particularly when investigating fetal tissues in which partially degraded nucleic acids may be recovered. Both Specificity and sensitivity could be much higher after a nested PCR. Serology for HPV B19 has been hampered by limited availability of antigen, because the propagation of B19 in cell cultures is not applicable. Thus the genetic testing of HPV B19 will play a key role in the diagnosis of HPV B19.(2)Diagnosis of fetal parvovirus B19 infection can be accomplished by nested-PCR to detect the virus DNA in abortuses or placental specimens. It is the first report in China that fetal infection with parvovirus B19 associates with spontaneous abortion. Two cases of maternal persistent infection with parvovirus B19 were found.(3)B19 DNA can be detected in cardiac muscle tissue of CHD in autopsy and biopsy is also reported here. DNA of the virus is located in nucleolus of infected heart tissue cell was confirmed. Relation between HPV B19 infection and congenital heart disease was revealed by high positive rate...