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Background Flesh firmness is a critical factor that influences fruit storability, shelf-life and consumer’s preference as well. However, less is known about the key genetic factors that are associated with flesh firmness in fresh fruits like watermelon. Results In this study, through bulk segregant analysis (BSA-seq), we identified a quantitative trait locus (QTL) that influenced variations in flesh firmness among recombinant inbred lines (RIL) developed from cross between the Citrullus mucosospermus accession ZJU152 with hard-flesh and Citrullus lanatus accession ZJU163 with soft-flesh. Fine mapping and sequence variations analyses revealed that ethylene-responsive factor 1 (ClERF1) was the most likely candidate gene for watermelon flesh firmness. Furthermore, several variations existed in the promoter region between ClERF1 of two parents, and significantly higher expressions of ClERF1 were found in hard-flesh ZJU152 compared with soft-flesh ZJU163 at key developmental stages. DUAL-LUC and GUS assays suggested much stronger promoter activity in ZJU152 over ZJU163. In addition, the kompetitive allele-specific PCR (KASP) genotyping datasets of RIL populations and germplasm accessions further supported ClERF1 as a possible candidate gene for fruit flesh firmness variability and the hard-flesh genotype might only exist in wild species C. mucosospermus. Through yeast one-hybrid (Y1H) and dual luciferase assay, we found that ClERF1 could directly bind to the promoters of auxinresponsive protein (ClAux/IAA) and exostosin family protein (ClEXT) and positively regulated their expressions influencing fruit ripening and cell wall biosynthesis. Conclusions Our results indicate that ClERF1 encoding an ethylene-responsive factor 1 is associated with flesh firmness in watermelon and provide mechanistic insight into the regulation of flesh firmness, and the ClERF1 gene is potentially applicable to the molecular improvement of fruit-flesh firmness by design breeding. [ABSTRACT FROM AUTHOR]

Background: Human parvovirus B19 (B19V) is a frequent contaminant of blood and plasma-derived medicinal products. Three distinct genotypes of B19V have been identified. The distribution of the three B19V genotypes has been investigated in various regions or countries. However, in China, data on the existence of different B19V genotypes are limited. Methods: One hundred and eighteen B19V-DNA positive source plasma pool samples collected from three Chinese blood products manufacturers were analyzed. The subgenomic NS1/VP1u region junction of B19V was amplified by nested PCR. These amplified products were then cloned and subsequently sequenced. For genotyping, their phylogenetic inferences were constructed based on the NS1/VP1-unique region. Then putative recombination events were analyzed and identified. Results: Phylogenetic analysis of 118 B19V sequences attributed 61.86 % to genotype 1a, 10.17 % to genotype 1b, and 17.80 % to genotype 3b. All the genotype 3b sequences obtained in this study grouped as a specific, closely related cluster with B19V strain D91.1. Four 1a/3b recombinants and 5 new atypical B19V variants with no recombination events were identified. Conclusions: There were at least 3 subtypes (1a, 1b and 3b) of B19V circulating in China. Furthermore, putative B19V 1a/3b recombinants and unclassified strains were identified as well. Such recombinant and unclassified strains may contribute to the genetic diversity of B19V and consequently complicate the B19V infection diagnosis and NAT screening. Further studies will be required to elucidate the biological significance of the recombinant and unclassified strains. [ABSTRACT FROM AUTHOR]

Background: Human parvovirus B19 (B19V) is a frequent contaminant of blood and plasma-derived medicinal products. To ensure the quality and safety of plasma-derived products, European regulations, Plasma Protein Therapeutics Association (PPTA) standard and FDA guidelines require testing of manufacturing plasma for parvovirus B19 DNA to limit the load of this virus. In China, however, there have been no related documentation and technical guiding principles for monitoring B19V, moreover, an adequate level of information on the prevalence of B19V in Chinese plasma donations is not available. Findings: By using an in-house quantitative polymerase chain reaction (qPCR) assay adapted for all three genotypes of B19V, 235 source plasma pools from three regional different Chinese manufacturers of blood products were screened and quantified. Results showed that 71.91 % (169/235) of plasma pools were contaminated by B19V, with the concentrations of 5.18 × 102-1.05 × 109 IU/mL. Approximately 31.95 % of the DNA-positive plasma pools were only moderately contaminated (<104 IU/mL), while 68.05 % contained >104 IU/mL. Conclusions: The high level of B19V in plasma pools could present a great risk in plasma derivatives. Therefore, the implementation of B19V NAT (Nucleic Acid Testing) assays capable of detecting all B19V genotypes and discard donations with high titer B19V DNA for Chinese blood products manufacturers seems to be necessary. [ABSTRACT FROM AUTHOR]

Background: Xenotransplantation from animals has been considered to be a preferable approach to alleviate the shortage of human allografts. Pigs are the most suitable candidate because of the anatomical and physiological similarities shared with humans as well as ethical concerns. However, it may be associated with the risk of transmission of infectious porcine pathogens. Porcine endogenous retroviruses (PERVs) are of particular concern because they have been shown to infect human cells in vitro. To date, researches on the molecular characteristics and potential pathogenicity of PERV are still tenuous. In this report, an infectious replication competent clone of PERV from Wuzhishan pigs (WZSPs) in China was generated and characterized. This infectious clone will contribute to studies on PERV virology and control of PERV in xenotransplantation using Chinese miniature pigs. Methods: The proviral DNA of PERV from WZSPs was amplified in two overlapping halves. Then the two fragments were isolated, subcloned and fused to generate pBluescriptαSK+-WZS-PERV recombinant clones. Screened with RT-PCR, a molecular clone of PERV designated as WZS-PERV(2) was selected. Its infectivity and replication competency were characterized in HEK293 cells by PCR, real-time fluorescent quantitative RT-PCR, western blot, indirect immunofluorescence assay as well as sequence analysis. Results: The ability of WZS-PERV(2) to infect human cells and produce infectious virions were shown after transfection of the clone into HEK293 cells and infection of PERV derived from this recombinant clone. The expression of Gag proteins were detected in HEK293 cells infected with the virus derived from the clone by the indirect immunofluorescence assay and western blot. The results of sequences analysis and comparison combined with the PCR based genotyping result demonstrated that the WZS-PERV(2) belonged to PERV-A subgroup. Compared with a previous proviral DNA clone of PERV (PERV-WZSP), G to A hypermutation occurred in the env gene of WZS-PERV(2) was found, whereas APOBEC proteins have the potential to inhibit the replication of a variety of retroviruses through a cDNA cytosine deamination mechanism, so we presumed these G to A hypermutation might be the contribution of porcine APOBEC3F. Conclusions: Altogether, an infectious replication competent clone of PERV from Chinese miniature pigs (WZSPs) termed WZS-PERV(2) was generated, characterized and sequenced. [ABSTRACT FROM AUTHOR]