Molecular Epidemiological Analysis Of Blood-borne Human Parvoviruses And Study On The Function Of Human Parvovirus B19 Nonstructural Proteins

Human parvovirus B19(B19V)and Human parvovirus 4(PARV4)can infect human and can be transmitted through blood and blood products.Chinese plasma-derived medicinal products manufactures do not screen them when they produce blood products.It is difficult to remove / inactivate human parvovirus effectively by conventional virus removal / inactivation technique.Therefore,the potential threat of B19 V and PARV4 to clinical blood and blood products has attracted more and more attention.In order to protect the viral safety of clinical blood and blood products in China,it is necessary for us to carry out molecular epidemiological analysis of B19 V and PARV4 in blood / plasma donation.Therefore,in the first part of this study,We used B19V-PARV4 duplex nucleic acid testing system to detect the nucleic acid of B19 V and PARV4 in 3200 single blood donation plasma samples from Xi'an area and 54 mixed source plasma samples from a Chinese plasma-derived medicinal products manufacture.In order to understand the distribution of different genotypes of B19 V,the nucleotide sequence of NS1-VP1 u region or the near-full-length sequence of the genome of B19 V were cloned and sequenced in this study.After multiple sequence alignment,phylogenetic analysis was carried out based on the nucleotide sequence or near-full-length sequence of the NS1-VP1 u region.Genotyping of B19 V nucleic acid positive plasma samples was carried out.The results show that there are 12 positive samples of B19 V nucleic acid in plasma of 3200 single donations in Xi'an of China,with a total positive rate of 0.38%,while 4 samples were positive for PARV4 nucleic acid with a total positive rate of 0.13%.Four samples were identified as 1a subtype of B19 V and 2 samples were identified as 1b subtype of B19 V.There are 5 positive samples of B19 V nucleic acid in the Chinese plasma-derived medicinal products manufacture,with a total positive rate of 9.26%,while 11 samples are positive for PARV4 nucleic acid with a total positive rate of 20.37%.B19 V nucleic acid positive sample had one near-full-length genome clone.Its genotype is identified as subtype 1b of B19 V.B19V infects the host cell and causes cell damage or death.When the host cell autophagy level increases,the host cell infected with B19 V is more likely to survive.We think that it is necessary to study the function of B19 V protein in autophagy.Most viruses synthesize a large number of viral proteins in the endoplasmic reticulum of host cells after infecting the cells.Endoplasmic reticulum stress may be associated with autophagy.Therefore,in the second part of this study,we explored the role of B19 V protein in inducing autophagy and endoplasmic reticulum stress.We constructed the eukaryotic expression plasmids which can express B19 V non-structural proteins X and 7.5 kDa in the transfected UT7/Epo-S1 cells,respectively.We analyzed the transformation of LC3B-? by Western blot and observed the phenomenon of membrane type LC3B-? aggregation on the membrane of autophagosome.The results show that protein 7.5kDa can increase the level of autophagy in UT7/Epo-S1 cells significantly.But protein X do not have the effect.The result suggests that non-structural protein 7.5 kDa may play an important role in the molecular mechanism of autophagy in the cells infected with B19 V.In order to study the effects of these two nonstructural proteins on endoplasmic reticulum stress,We analyzed the expression of endoplasmic reticulum stress-related proteins(Bip and CHOP)in transfected cells by Western blot.The results show that the presence of non-structural proteins X and 7.5 kDa could not up-regulate the expression of protein Bip and CHOP in UT7/Epo-S1 cells.It implies that non-structural protein X and 7.5 kDa could not induce endoplasmic reticulum stress in UT7/Epo-S1 cells.In conclusion,this study is divided into two parts.The first part is the molecular epidemiological analysis of B19 V and PARV4 in Chinese blood / plasma donors.The genotypes of B19 V were identified,which was helpful for us to know about the prevalence of B19 V and PARV4 and the distribution of B19 V genotypes in China.The second part is about the function of human parvovirus B19 nonstructural protein 7.5 kDa and X in inducing autophagy and endoplasmic reticulum stress.It is useful for elucidating the molecular mechanism of B19 V infected cells.