Associations of maternal upper respiratory tract infection/influenza during early pregnancy with congenital heart disease in offspring: evidence from a case-control study and meta-analysis.

BACKGROUND: Evidences regarding the associations between maternal upper respiratory tract infection/influenza during pregnancy and the risk of congenital heart disease (CHD) is still controversial. This study was specifically designed to examine the associations by a case-control study and a meta-analysis of the published evidences and our finding. METHODS: A hospital-based case-control study involving 262 children with simple CHD and 262 children with complex CHD, along with 262 control children, was conducted through June, 2016 to December, 2017. All children were aged 0-2 years old. Furthermore, a meta-analysis based on both previously published studies and our case-control study was performed. RESULTS: In the case-control study, after adjusting for possible confounders, maternal upper respiratory tract infection/influenza during early pregnancy was found to be related to an increased risk of CHD (OR = 3.40 and 95% CI: 2.05-5.62 for simple CHD; OR = 2.39 and 95% CI: 1.47-3.88 for complex CHD). After a meta-analysis, the adverse impact was still kept significant (OR = 1.47 and 95% CI: 1.28-1.67 for simple CHD; OR = 1.44 and 95% CI: 1.14-1.75 for complex CHD). The very similar associations were also observed among single type of CHD, herein, ventricular septal defects (VSD) and tetralogy of fallot (TOF) in the case-control study. In the subsequent meta-analysis, however, the significant association only existed in VSD. CONCLUSIONS: Although there is still conflicting in TOF, the results are overall consistent, which provide new enforced evidence that maternal upper respiratory tract infection/influenza during early pregnancy, in general, play an important role in the occurrence of CHD.

Polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy.

Papillomavirus infection is a major antecedent of anogenital malignancy. We have previously established that the L1 and L2 capsid genes of papillomavirus have suboptimal codon usage for expression in mammalian cells. We now show that the lack of immunogenicity of polynucleotide vaccines based on the L1 gene can be overcome with codon modified L1, which induces strong immune responses, including conformational virus neutralising antibody and delayed type hypersensitivity. Conjugation of a ubiquitin gene to a hybrid gene incorporating L1 and the E7 non-structural papillomavirus protein improved E7 specific CTL responses, and induced protection against an E7 expressing tumour, but induced little neutralising antibody. However, a mixture of ubiquitin conjugated and non-ubiquitin conjugated polynucleotides induced virus neutralising antibody and E7 specific CD8 T cells. An optimal combined prophylactic/therapeutic viral vaccine might therefore comprise ubiquitin conjugated and non-ubiquitinated genes, to induce prophylactic neutralising antibody and therapeutic cell mediated immune responses.

Efficiency of delivery of DNA to cells by bovine papillomavirus type-1 L1/L2 pseudovirions.

To investigate the efficiency of encapsidation of plasmid by papillomavirus virus-like particles (PV VLPs), and the infectivity of the resultant PV pseudovirions, Cos-1 cells were transfected with an 8-kb plasmid incorporating a green fluorescent protein (GFP) reporter gene (pGSV), and infected with bovine PV (BPV-1) L1/L2 recombinant vaccinia virus to produce BPV1 pseudovirions. Approximately 1 in 1.5 x 10(4) of dense (1.35 g/ml) PV pseudovirions and 0.3 in 10(4) of less-dense (1.29 g/ml) pseudovirions packaged an intact pGSV plasmid. The majority (>75%) of packaged plasmids contained deletions, and the deletions affected all tested genes. After exposure of Cos-1 cells to BPV-1 pseudovirions at an MOI of 40,000:1, 6% of cells expressed GFP, giving a calculated efficiency of delivery of the pGSV plasmid, by pseudovirions which had packaged an intact plasmid, of approximately 5%. Plasmid delivery was not effected by purified pGSV plasmid, was blocked by antiserum against BPV-1, and was not blocked by DNase treatment of pseudovirions, confirming that delivery was mediated by DNA within the pseudovirion. We conclude that a major limitation to the use of PV pseudovirions as a gene delivery system is that intact plasmid DNA is not efficiently selected for packaging by VLPs in cell-based pseudovirions production systems.

Association of bovine papillomavirus type 1 with microtubules.

Transport of BPV-1 virus from the cell membrane to the nucleus was studied in vitro in CV-1 cells. At reduced temperature (4 degrees C), BPV-1 binding to CV-1 cells was unaffected but there was no transport of virions across the cytosol. Electron microscopy showed BPV-1 virions in association with microtubules in the cytoplasm, a finding confirmed by co-immunoprecipitation of L1 protein and tubulin. Internalization of virus was unimpaired in cells treated with the microtubule-depolymerizing drug nocodazole but virions were retained in cytoplasmic vesicles and not transported to the nucleus. We conclude that a microtubule transport mechanism in CV-1 cells moves intact BPV-1 virions from the cell surface to the nuclear membrane.

Papillomavirus virus-like particles for the delivery of multiple cytotoxic T cell epitopes.

Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial "polytope" minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class I alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems.

BPV1 E2 protein enhances packaging of full-length plasmid DNA in BPV1 pseudovirions.

We studied determinants of efficient encapsidation of circular DNA, incorporating a PV early region DNA sequence (nt 584-1978) previously shown to enhance packaging of DNA within papillomavirus (PV)-like particles (VLPs). Insect coelomic cells (Sf-9) and cultured monkey kidney cells (Cos-1) were transfected with an 8-kb reporter plasmid incorporating the putative BPV packaging sequence and infected with BPV1 L1 and L2 recombinant baculovirus or vaccinia virus. Heavy (1.34 g/ml) and light (1.30 g/ml) VLPs were produced, and each packaged some of the input plasmid. In light VLPs, truncated plasmids, which nevertheless incorporated the PV-derived DNA packaging sequence, were more common than full-length plasmids. Packaging efficiency of the plasmid was estimated at 1 plasmid per 10(4) VLPs in both Cos-1 and Sf-9 cells. In each cell type, expression of the BPV1 early region protein E2 in trans doubled the quantity of heavy but not light VLPs and also increased the packaging efficiency of full-length circular plasmids by threefold in heavy VLPs. The resultant pseudovirions incorporated significant amounts of E2 protein. Pseudovirions, comprising plasmids packaged within heavy VLPs, mediated the delivery of packaged plasmid into Cos-1 cells, whereby "infectivity" was blocked by antisera to BPV1 L1, but not antisera to BPV1 E4. We conclude that (a) packaging of DNA within PV L1+L2 pseudovirions is enhanced by BPV1 E2 acting in trans, (b) E2 may be packaged with the pseudovirion, and (c) E2-mediated enhancement of packaging favors 8-kb plasmid incorporation over incorporation of shorter DNA sequences.

Nucleotides 1506-1625 of bovine papillomavirus type 1 genome can enhance DNA packaging by L1/L2 capsids.

We have previously described a DNA-packaging assay using bovine papillomavirus type 1 (BPV-1) virus-like particles (VLPs) and have identified a region of the BPV genome that assists in packaging. In this study, we identify a specific BPV sequence involved in DNA packaging by BPV-1 VLPs. In the initial screening of BPV-1 genomic sequences essential for DNA packaging, we observed that a plasmid with deletions between nucleotides (nt) 948 and 2113 failed to be packaged into BPV-1 VLPs. However, plasmids containing nt 948 to 2113 were efficiently packaged, suggesting that this 1.2-kb fragment contains a packaging enhancement sequence (PES). Further mapping of the BPV-1 genome showed that this packaging sequence lies between nt 1506 and 1625. Furthermore, this packaging sequence is also recognized by HPV6b VLPs, suggesting that a common packaging mechanism may be used by the two papillomavirus types. Given the phylogenetic difference between these two viral types, it is likely that other papillomavirus types may also use the same packaging mechanism. Identification of the PES has allowed a minimal viral genome sequence to be used in the packaging assay, improving the usefulness of the assay in studying the process of papillomavirus DNA encapsidation.

DNA packaging by L1 and L2 capsid proteins of bovine papillomavirus type 1.

Encapsidation of circular DNA by papillomavirus capsid protein was investigated in Cos-1 cells. Plasmids carrying both an SV40 origin of replication (ori) and an E. coli ori were introduced into Cos-1 cells by DNA transfection PV capsid proteins were supplied in trans by recombinant vaccinia viruses. Pseudovirions were purified from infected cells and their packaged DNA was extracted and used to transform E. coli as an indication of packaging efficacy. VLPs assembled from BPV-1 L1 alone packaged little plasmid DNA, whereas VLPs assembled from BPV-1 L1 + L2 packaged plasmid DNA at least 50 times more effectively. BPV-1 L1 + L2 VLPs packaged a plasmid containing BPV-1 sequence 8.2 +/- 3.1 times more effectively than a plasmid without BPV sequences. Using a series of plasmid constructs comprising a core BPV-1 sequence and spacer DNA it was demonstrated that BPV VLPs could accommodate a maximum of about 10.2 kb of plasmid DNA, and that longer closed circular DNA was truncated to produce less dense virions with shorter plasmid sequences. The present study suggests that packaging of genome within PV virions involves interaction of L2 protein with specific DNA sequences, and demonstrates that PV pseudovirions have the potential to be used as DNA delivery vectors for plasmids of up to 10.2 kb.

Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris.

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7-14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2-3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l(-1) IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.