[A molecular epidemiological study of KI polyomavirus and WU polyomavirus in children with acute respiratory infection in Tianjin, China].

OBJECTIVE: To investigate the relationship of KI polyomavirus (KIPyV) and WU polyomavirus (WUPyV) with acute respiratory infection in children in Tianjin, China. METHODS: A total of 3 730 nasopharyngeal secretions were collected from hospitalized children with acute respiratory infection in Tianjin Children's Hospital from January 2011 to December 2013. Viral nucleic acid was extracted, and virus infection (KIPyV and WUPyV) was determined by PCR. Some KIPyV-positive and WUPyV-positive PCR products were subjected to sequencing. Sequencing results were aligned with the known gene sequences of KIPyV and WUPyV to construct a phylogenetic tree. Amplified VP1 fragments of KIPyV were inserted into the cloning vector (PUCm-T) transformed into E. coli competent cells. Positive clones were identified by PCR and sequencing. The nucleotide sequences were submitted to GenBank. In addition, another seven common respiratory viruses in all samples were detected by direct immunofluorescence assay. RESULTS: In the 3 730 specimens, the KIPyV-positive rate was 12.14% (453/3 730) and the WUPyV-positive rate was 1.69% (63/3 730). The mean infection rate of KIPyV was significantly higher in June and July, while the mean infection rate of WUPyV peaked in February and March. Most of the KIPyV-positive or WUPyV-positive children were <3 years. The co-infections with KIPyV, WUPyV, and other respiratory viruses were observed in the children. The co-infection rate was 2.31% (86/3 730) and there were nine cases of co-infections with WUPyV and KIPyV. Thirty-five KIPyV-positive and twelve WUPyV-positive PCR products were sequenced and the alignment analysis showed that they had high homology with the known sequences (94%-100% vs 95%-100%). The VP1 gene sequences obtained from two KIPyV strains in this study were recorded in GenBank with the accession numbers of KY465925 and KY465926. CONCLUSIONS: For some children with acute respiratory infection in Tianjin, China, the acute respiratory infection may be associated with KIPyV and WUPyV infections. KIPyV infection is common in summer, and WUPyV infection in spring. The epidemic strains in Tianjin have a high homology with those in other regions.

[Detection and analysis of bocavirus in hospitalized children with respiratory infection].

OBJECTIVE: To detect human bocavirus (HBoV) and investigate its genetic and evolutionary characteristics in children with acute respiratory infection in Tianjin, China. METHODS: A total of 1,259 samples of nasopharyngeal aspirates were collected from children with a confirmed diagnosis of acute respiratory infection between January and December, 2012. Viral nucleic acid was extracted, HBoV was detected by real-time quantitative PCR, and the gene segments of nucleocapsid protein of HBoV in positive samples were amplified by PCR. Several products were randomly selected and sequenced.The sequence obtained was compared with the known sequence of HBoV, and a phylogenetic analysis was performed. All the samples were examined to detect for other common respiratory tract viruses. RESULTS: Among the 1,259 samples, the positive rate of HBoV was 4.53% (57/1,259), and among the 57 samples with positive HBoV, 75% (43/57) were positive in children with an age of 6-36 months. The positive rate of HBoV in children peaked in summer (from June to August), and there was a mixed infection with other viruses. Sequence analysis was performed for the PCR products from 36 positive samples, and the presence of HBoV was confirmed, with a higher homology to the known sequence of HBoV. CONCLUSIONS: In Tianjin, acute respiratory infection in some children may be associated with HBoV infection, which is commonly seen in infants with an age of 6-36 months. The peak of HBoV infection occurs in summer. The phylogenetic analysis shows a high homology to the known sequence of HBoV, with few gene sequence variations.

[Characterization of pcr2 gene of Pseudomonas aeruginosa].

Type III secretion system (TTSS) is an important virulence factor encoding by pseudomonas aeruginosa. About 40 genes are involved and they function as structure proteins, chaperons, regulators, and effectors proteins, respectively. Although some genes have been studied previously, functions of many genes remained unknown. Pcr2 gene is the third gene of popN operon that is one of the five operons of the TTSS gene clusters. Its functions were investigated in this study. First, by characterization of the phenotypes of pcr(2-) mutant, we found that the abilities of secreting or translocating effectors proteins were significantly damaged in the absence of Pcr2 protein, suggesting that Pcr2 protein involved in both the secretion and translocation processes of TSS. Second, evidences were provided that no PopN protein was detectable in supernatant of pcr(2-) mutant culture. Combined with the data from the bacterial two-hybrid system, we can conclude that Pcr2 protein might function as part of a chaperone complex for the PopN protein. Third, Pcr2 protein was found secreted in a TTSS-dependent manner, suggesting that secreted Pcr2 may play a role in the TTSS needle biogenesis.

[Type III secretion study of popN- mutant of Pseudomonas aeruginosa and proteases degradation].

Pseudomonas aeruginosa is an important opportunistic human pathogen. It encodes many virulence factors and one of them is type III secretion system (TTSS). Effectors proteins can be delivered into host cells directly by this system, causing necrosis or apoptosis. popN gene is the first gene in the popN operon of TTSS gene cluster. To investigate its function, popN gene deletion mutant was generated in this study, and we found this mutant can secrete effectors proteins constitutively under non-inducting condition in DMEM medium containing serum. The results indicated that PopN is a negative regulator of the TTSS expression. However, no secreted effector proteins were detectable when the popN- mutant was grown in LB medium under non-inducting condition. To investigate the possible reasons, effects of growth status and protease (s) inhibitors on the TTSS were investigated. We present evidences that indicate protease mediated degradation of secreted effector proteins played a key role in the phenotypic inconsistency of popN- mutant.