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Objective To understand the genetic variations of neuraminidase (NA) genes of avian influenza virus H9N2 in Weining,Guizhou Province,and to provide the scientific evidence for the prevention and control of avian influenza virus.Methods Ribonucleic acids (RNA) were extracted and NA genes were amplified and sequenced from 13 randomly selected H9N2 positive samples from the live poultry market (LPM)environments in north of Weining Yi and Hui and Miao autonomous county (Weining),Guizhou Province during 2015 to 2017.Then the homology,genetic evolution,and sites of stalk deletion areas,potential N-glycosylation,receptor binding regions and drug resistance of H9N2 subtype avian influenza viruses were analyzed by a series of bioinformation software.Results Homology analysis revealed that there were 93.0％-100.0％ and 92.1％-100.0％ similarity among 13 strains H9N2 avian influenza viruses in nucleotide and amino acid of the NA gene,respectively.All strains belonged to DK/HK/Y280/97 sub-lineage,but their genetic sources were complex and diverse.Thirteen strains had a stalk deletion of 3 amino acid residues TEI at positions 63-65 and 3 isolates had mutation QN to QK at positions 39-40.The potential N-glycosylation sites at amino acid residues 86,146,200,and 234 of the NA protein of all strains were highly conserved,while other N-glycosylation sites had quantity and site mutations.There were different mutation types at the three sialic acid binding site areas,especially at 399-404 area.All NA protease activity sites and key sites of the 13 strains had no mutations associated with resistance to the neuraminidase inhibitor drugs.Conclusions All 13 strains H9N2 viruses belongs to DK/HK/Y280/97 sub-lineage in Weining,Guizhou Province during 2015-2017,and their genetic sources are complex and diverse.The mutations on sites of stalk areas,potential N-glycosylation and sialic acid binding site areas are presented at different degrees.Hence,enhancing surveillance and controlling H9N2 avian influenza virus is necessary.
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The number of H7N9 bird flu cases was high and the situation was grim in guizhou province in 2017. To understand the molecular characteristics of the hemagglutinin gene (HA) and the risk of human infection with avian influenza virus A(H7N9) in Guizhou Province, 2017. Homology, genetic evolution and pivotal sites related to receptor binding regions, pathogenicity and potential glycosylation of 14 avian influenza viruses A(H7N9) were analyzed by a series of bioinformation softwares. It was cleared that there was 95.9%-100% similarity among 14 strains in nucleotide of the HA gene, and there were 96.8%-97.8% and 96.8%-97.9% similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO, respectively. Phylogenetic analysis showed that 14 HA genes were directly evolved in the Yangtze River Delta evolution branch, but they could be derived from five diffenrent strains. Then 13 of 14 strains cleavage site sequences of HA protein revealed they were low pathogenic avian influenza viruses, while A/Guizhou-Weining/CSY01/2017 was high pathogenic avian influenza virus. Mutation G186V at the receptor binding sites in the HA was found in all 14 strains, and mutation Q226L in 13 strains besides A/Guizhou-Weining/CSY01/2017. All five potential glycosylation motifs in the HA were conservative.
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Objective To investigate the molecular characteristics of H5 subtype avian influenza viruses (AIV) in Weining, Guizhou Province. Methods Nine representative strains were randomly select-ed from H5 subtype AIV that were identified by real-time PCR in Weining, Guizhou Province from 2015 to 2017. Nucleic acid was extracted from each sample and hemagglutinin (HA) genes were amplified and then sequenced. Homology, genetic evolution and the sites related to pathogenicity, receptor binding regions as well as potential glycosylation of H5 AIV were analyzed by bioinformation software. Results Homology analysis revealed that there was 96. 1%-99. 9% and 95. 7%-100% similarity among the nine strains in nu-cleotide and amino acid of HA gene, respectively. These strains belonged to two branches, H5-1 and H5-2. The cleavage site motifs were PLREKRRKR↓GLF for five strains in H5-1 branch and PQRERRRKR↓GLF for four strains in H5-2 branch, which made them high pathogenic. QSG and QRG at the key receptor bind-ing sites were found in H5-1 and H5-2 branch strains, respectively. They were responsible for receptor bind-ing specificity of AIV. Mutations of 138Q, 139G and 53K were all detected in the nine strains. 129K, 189T, 140K and 282V mutations were discovered in the five strains of H5-1 branch, while 189N, 140M and 282I mutations were found in the four strains of H5-2 branch. Results of the glycosylation motif analysis showed that six sites were conservative, but there was an addition of 124NHT site in two strains of H5-2 branch isolated in 2017. Conclusion Two high pathogenic H5 subtypes of AIV could be epidemic in Wein-ing, Guizhou Province during 2015 to 2017. Although H5 subtype AIV did not possess specific receptor binding regions like human influenza viruses, they were in continuous variation with an increase in glycosyla-tion motifs, which might enhance their virulence and pathogenicity to human beings. Hence, surveillance and study on the molecular properties of H5 subtype AIV should be strengthened.
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Objective To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province.Methods RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017.Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package.Results Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8％-99.2％ and 99.2％ similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO,respectively.Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2％-99.3％ and 97.6％-98.8％ similarities with vaccine strain A/Hunan/02650/2016,respectively.Other 6 stains detected in 2017 shared 99.1％-99.4％ and 98.9％-99.3％ similarities with strain A/Guangdong/17SF003/2016,respectively.Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch,but they were derived from different small branch.PEVPKRKRTAR ↓ GLF was found in 6 of 24 strains cleavage site sequences of HA protein,indicating the characteristic of highly pathogenic avian influenza virus.Mutations A134V,G186V and Q226L at the receptor binding sites were found in the HA.All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein,and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/ 18980/2017.In addition,potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains.Condusions HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017.The mutations of key sites might enhance the virulence of the virus,human beings are more susceptible to it.Hence,the risk of infection is increasing.
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Objective To understand the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) as well as the disease risk of influenza virus A H7N9 in Guizhou province.Methods RNAs were extracted and sequenced from HA and NA genes of H7N9 virus strains obtained from 18 cases of human infection with H7N9 virus and 6 environmental swabs in Guizhou province during 2014-2017.Then the variation and the genetic evolution of the virus were analyzed by using a series of bioinformatics software package.Results Homology analysis of HA and NA genes revealed that 2 strains detected during 2014-2015 shared 98.8％-99.2％ and 99.2％ similarities with vaccine strains A/Shanghai/2/2013 and A/Anhui/1/2013 recommended by WHO,respectively.Two strains detected in 2016 and 14 strains detected in 2017 shared 98.2％-99.3％ and 97.6％-98.8％ similarities with vaccine strain A/Hunan/02650/2016,respectively.Other 6 stains detected in 2017 shared 99.1％-99.4％ and 98.9％-99.3％ similarities with strain A/Guangdong/17SF003/2016,respectively.Phylogenetic analysis showed that all the strains were directly evolved in the Yangtze River Delta evolution branch,but they were derived from different small branch.PEVPKRKRTAR ↓ GLF was found in 6 of 24 strains cleavage site sequences of HA protein,indicating the characteristic of highly pathogenic avian influenza virus.Mutations A134V,G186V and Q226L at the receptor binding sites were found in the HA.All the strains had a stalk deletion of 5 amino acid residue "QISNT" in NA protein,and drug resistance mutation R294K occurred in strain A/Guizhou-Danzhai/ 18980/2017.In addition,potential glycosylation motifs mutations NCS42NCT were found in the NA of 9 of 24 strains.Condusions HA and NA genes of avian influenza A (H7N9) virus showed genetic divergence in Guizhou province during 2014-2017.The mutations of key sites might enhance the virulence of the virus,human beings are more susceptible to it.Hence,the risk of infection is increasing.
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Objective@#To investigate the molecular characteristics and tracing of the hemagglutinin (HA) gene, and to analyze the risk of human infection with influenza virus A (H7N9) in Guizhou Province, so that to provide evidence for the prevention and control of highly pathogenic avian influenza A (H7N9).@*Methods@#Nucleic acids of 5 strains of H7N9 including 1 sample of the patient′s nasopharyngeal swab and 4 samples of the live poultry market (LPM) environment were extracted and HA genes were amplified and sequenced. Then the homology, genetic evolution and the pivotal sites related to receptor binding regions, pathogenicity and potential glycosylation of the avian influenza A (H7N9) viruses were analyzed by a series of bioinformatics softwares.@*Results@#Homology analysis revealed that the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains from the patient and LPM in Weining County, Guizhou Province were 99.8% and 99.6%, respectively, while those of 4 strains from LPM were both 100%. The homologies of nucleotide and amino-acid of the HA gene of H7N9 strains were the highest with the strain of A/Guangxi/5/2017 isolated from a Guangxi infected patient (99.7%-99.9% and 99.4%-99.8%, respectively), while those with the strain isolated from LPMs environment at the end of 2016 (A/Environment/Guangdong/C16283222/2016) were 99.0%-99.2% and 98.9%-99.2%, respectively. However, the homologies of nucleotide and amino-acid of the HA gene of H7N9 strains with A/Shanghai/2/2013 recommended by world health organization and the candidate vaccine strain A/Anhui/1/2013 were 96.8%-97.0% and 95.8%-96.2%, respectively. Phylogenetic analysis showed that the 5 strains had the nearest genetic distance to the strain A/Guangxi/5/2017. All the 5 strains cleavage site sequences of HA protein showed mutation of PEVPKRKRTAR↓GLF, and they were highly pathogenic avian influenza viruses mutant strains, which all had mutation of G186V at the receptor binding sites of HA gene, while no Q226L mutation was found. All 5 strains had new mutation of A363S, and new mutations of R56K and I297V were only found in the strain isolated from the patient. Among the five potential glycosylation motifs in the HA, only 421NWT and 493NNT had variation of the position post shift.@*Conclusions@#All the 5 H7N9 strains isolated in Weining County, Guizhou Province are highly pathogenic avian influenza mutative viruses. The current candidate vaccine may not provide a very good protection. The mutations of cleavage site of HA protein, G186V as well as other new mutation sites of HA may enhance the susceptibility and pathogenicity to human beings.
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Objective To understand the epidemic characteristics and regularity of influenza B virus in Guizhou Province, and provide scientific evidence for the control and prevention of influenza.Methods Results of reverse transcription polymerase chain reaction(PT-PCR) of influenza B virus in Guizhou Province from April 1, 2013 to March 31, 2016 were statistically analyzed.Results A total of 1 904 samples were detected influenza B virus by RT-PCR, B/Yamagata (By) lineage and B/Victoria (Bv) lineage were 1 215 and 642 respectively.In April 2013-March 2014 and April 2014-March 2015, the predominant strains of influenza B were both By lineage, in April 2015-March 2016, the predominant strains of influenza B were Bv and By lineages, the epidemic peaks were in winter and spring;there's a higher positive percentage of influenza B in male, accounting for 56.83%;the highest detection rate of influenza B virus was found in population aged <15 years(70.80%),Bv and By lineages were the highest in the 0~ (42.37%) and 5~ age groups (35.56%) respectively;the main pathogen causing mixed infection was By+Bv (67.65%),mixed infection with influenza B virus accounted for 95.59%.Conclusion There are two lineages By and Bv epidemic in Guizhou Province, the epidemic peaks of influenza B are in winter and spring, male cases are higher than female, people under 15 years old are the high-risk group for influenza B, it is of great significance to strengthen the vaccination and surveillance of influenza in low age population.
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Objective@#To conduct an epidemiological investigation of two leptospirosis death cases reported in Guizhou Province in 2014.@*Methods@#The information of the patients were investigated and analyzed. The serological detection, samples of the two patients was detected using ELISA and microscopic agglutination test (MAT). Leptospira carrier status of murine host animal in the living environment of the two patients was investigated in October and November of 2014. Leptospires in the kidney were cultured and isolated, the isolates were identified using Leptospira specific PCR and further identified with serogroup specific PCR and the conventional MAT. The relativity between the carrier status of murine and the death cases of human leptospirosis was analyzed.@*Results@#The two death cases of human leptospirosis came from Liping County and the clinical symptoms were consistent with the diagnosis criteria for Leptospirosis. The results of ELISA detection showed that the anti-Leptospira antibody was positive for one of the death cases, MAT identified the serum reacted with sera-group icterohaemorrhagiae Leptospira, while the serum sample of the other case was failed to perform antibody detection due to hemolysis. 1 600 traps were placed in the living environment of the two death cases and 183 murine rodents were trapped. The murine density was 11.44% (183/1 600); 40 leptospirea suspected strains were isolated and all of them were isolated from Apodemus agrarius. The positive rate was 21.86% (40/183); 95 Apodemus agrarius were trapped and the murine density was 5.93% (95/1 600). Species specific PCR identified all the 40 strains as Leptospire. Serogroup specific PCR further identification showed that they were iterohaemorrahgiae serogroup Leptospria. interrogans.@*Conclusion@#Anti-iterohaemorrahgiae Leptospira antibody was detected from one of the two patients. 40 strains of iterohaemorrahgiae serogroup Leptospira interrogans were isolated and all of them were isolated from Apodemus agrarius in the living environment and the serogroup of the Leptospira matched with the serological detection results from patients, which indicated that the two death cases were caused by the infection of iterohaemorrahgiae serogroup Leptospira interrogans, and Apodemus agrarius were the potential source of infection.
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Objective To understand the genetic variations of influenza B virus outbreaks in Guizhou province in 2016,and to compare the matching situation of outbreak epidemic strains with the vaccine strains recommended by WHO and representative strains in China.Methods The haemagglutinin HA1 gene of 8 strains isolated from two episodes of influenza B virus outbreaks in Tongren area was amplified and sequenced.The sequencing products were analyzed by bioinformatics software DNAStar. Results The two episodes of influenza outbreaks were both caused by influenza B Victoria lineage virus (BV).The homologies of the isolated strains were 99.8%—100.0% in nucleotide and 99.5 %—100.0%in amino acid.Mutation was only detected at 274 site in some strains.Compared with reference strain B/Victoria/2/87,the homologies were 91 .8%—92.0% and 91 .5 %—92.0%,respectively.Mutations developed at 17 amino acid sites,among which,I143V,V163I and V201I site were associated with the main antigenic determinant area B,C and D.Compared with previous vaccine strain B/Brisbane/60/2008, the homologies were 98.2%—98.3% and 98.5 %—99.0%,respectively,and mutations were detected at 3 sites.Mutations at I143V and N155D were detected in all 8 strains and at T247I in some strains.The mutation of I143V was associated with antigenic determinant area B.Compared with the representative strain B/Chongqing-Yuzhong/1384/2010,the homologies were 96.7%—96.8% and 97.0%—97.5 %, respectively.A total of 6 sites developed mutations,among which,5 sites were P84L,I143V,N155D, V172I and T223N mutations.The mutation of T247I was detected in some strains,and I143V was associated with area B.Compared with the epidemic strain in Guizhou in 2016,the homologies were 99.8%—100.0% and 99.5 %—100.0%,respectively.Mutation was only detected at site 247 in some strains and was not associated with the main antigenic determinant area.Conclusions The two episodes of influenza outbreaks in Guizhou are caused by the same BV lineage epidemic virus strain.Haemagglutinin gene of BV lineage virus is constantly changing.However,there is no new mutation emerged at important site.Compared with previous influenza vaccine strain B/Brisbane/60/2008 recommended by WHO,BV lineage virus is well matched and could provide a positive protection effect.
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Objective To analyze the effects of Leptospira interrogans ( L. interrogans) infection on the activation of NLRP3 in THP-1 and J774A. 1 cells and to further understand the mechanism of inflam-mation caused by L. interrogans in different hosts. Methods Human mononuclear macrophage cell line (THP-1) and murine mononuclear macrophage cell line (J774A. 1) were infected with L. interrogans strain 56601. The expression of NLRP3 at mRNA and protein levels were measured by using real-time RT-PCR and flow cytometry analysis, respectively. The NLRP3-mediated secretion of IL-1β, IL-18 and IL-33 was detec-ted by ELISA combined with the NLRP3 inhibitory test. Results Compared with the normal cells, the ex-pression of NLRP3 at mRNA level in L. interrogans-infected THP-1 cells was respectively increased by 4. 05, 0. 34, 0. 33, 0. 06 and 1. 66 times at the time points of 1 h, 2 h, 4 h, 12 h and 24 h after infection ( P0. 05). Results of the inhibitory test showed that the up-regulation of IL-1β , IL-18 and IL-33 in THP-1 and J774A. 1 cells were effectively inhibited by the specific inhibitor of NLRP3. Conclusion NL-RP3 inflammasome was activated and involved in the production of specific inflammatory cytokines IL-1βand IL-18 in both THP-1 and J774A. 1 cells after L. interrogans infection, but the inflammatory cytokines induced by L. interrogans infection varied in different cells. L. interrogans induced earlier and higher level of IL-1βand IL-18 production in human macrophages than in murine macrophages.
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Objective To investigate the effects of Leptospira interrogans ( L. interrogans) infec-tion on the activities of NADPH oxidase ( nicotinamide adenine dinucleotide phosphate-oxidase) and the lev-els of reactive oxygen species (ROS) in THP-1 and J774A. 1 cells and to understand the bactericidal mecha-nisms of macrophages in different hosts against L. interrogans. Methods Human mononuclear macrophage cell line (THP-1 cells) and murine mononuclear macrophage cell line (J774A. 1 cells) were infected with L. interrogans strain 56601. The activities of NADPH oxidase and the levels of superoxide ion ( O-2 ) were measured with spectrophotography. Changes in the levels of ROS were detected with immunofluorescence as-say. Results Compared with the normal cells, the activities of NADPH oxidase in L. interrogans-infected J774A. 1 cells changed from 0. 619 0 μmol · min-1 · mg-1 to 0. 305 5 μmol · min-1 · mg-1 , 6. 141 5μmol·min-1 ·mg-1 , 1. 487 1μmol·min-1 ·mg-1 and 0. 964 6μmol·min-1 ·mg-1 after 2, 4, 12 and 24 hours of infection, respectively (P<0. 05), while the activities of NADPH oxidase in L. interrogans-infected THP-1 cells were up-regulated from 0. 723 5μmol·min-1 ·mg-1 to 0. 884 2μmol·min-1 ·mg-1 , 1. 897 1μmol·min-1 ·mg-1 , 1. 125 4 μmol·min-1 ·mg-1 and 0. 562 7 μmol·min-1 ·mg-1 , respectively ( P<0. 05). The levels of O-2 in L. interrogans-infected J774A. 1 cells at the time points of 2 h, 4 h, 12 h and 24 h after infection increased from 0. 189 0μmol/L to 0. 236 3μmol/L, 0. 297 7μmol/L, 0. 324 0μmol/L and 0. 305 7 μmol/L, respectively (P<0. 05), while the levels of O-2 in L. interrogans-infected THP-1 cells rose from 0. 123 7 μmol/L to 0. 149 3 μmol/ L, 0. 249 0 μmol/ L, 0. 270 0 μmol/ L and 0. 272 7μmol/L, respectively (P<0. 05). The fluorescence intensity of ROS in THP-1 and J774A. 1 cells increased gradually after infection with L. interrogans for 2 h and decreased after reaching the peak at 24 h. Conclu-sion Both the activities of NADPH oxidase and the levels of O-2 in J774A. 1 and THP-1 cells were signifi-cantly upregulated after infected with L. interrogans, especially in J774A. 1 cells. The results of this study provided references for further elucidating the bactericidal mechanisms of macrophages in different hosts against L. interrogans.
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Objective To investigate the expression of inducible nitric oxide synthase ( iNOS) and the levels of nitric oxide (NO) in THP-1 and J774A. 1 cells during Leptospira interrogans (L. interrogans) infection for a better understanding of the mechanism of macrophages involved defense against L. interrogans strains in different hosts. Methods The human mononuclear macrophages (THP-1) and the murine mono-nuclear macrophages (J774A. 1) were infected with L. interrogans strain 56601. The expression of iNOS at mRNA and protein levels were determined by using real-time RT-PCR and flow cytometry analysis. The lev-els of NO were detected with Griess test. Results The expression of iNOS at mRNA level in J774A. 1 and THP-1 cells infected with L. interrogans strains for 2, 4, 12 and 24 hours were respectively 1. 37, 2. 82, 25. 76, 27. 47 times and 1. 59, 3. 98, 3. 89, 8. 81 times than that of cells without infection (P<0. 05). The expression rates of iNOS protein in J774A. 1 cells were increased from 34. 16% to 85. 85%, 93. 82%, 91. 77% and 93. 65% along with the increased time of infection time (P<0. 05). The expression rates of iNOS protein in THP-1 cells were up-regulated from 22. 08% to 72. 64%, 81. 33%, 80. 03% and 65. 72%after 2, 4, 12 and 24 hours of infection (P<0. 05), respectively. Results of the Griess test indicated that the levels of NO in J774A. 1 and THP-1 cells were respectively increased from 0. 1588 μmol/L to 0. 2208μmol/L, 0. 2668μmol/L, 0. 3808μmol/L, 0. 3828μmol/L and from 0. 0988μmol/L to 0. 2848μmol/L,0. 3228 μmol/L, 0. 2608μmol/L and 0. 3308μmol/L after infection with L. interrogans strains for 2, 4, 12 and 24 hours (P<0. 05). Conclusion The expression of iNOS and the levels of NO in J774A. 1 and THP-1 cells were significantly increased during L. interrogans infection. This study might help to explain the bactericidal mechanism of macrophages derived from different hosts against L. interrogans infection.
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<p><b>OBJECTIVE</b>To identify and characterize the Brucella strains from Guizhou province in 2010-2013.</p><p><b>METHODS</b>A total of 12 strains of Brucella suspicious bacteria were isolated in Guizhou province from 2010 to 2013. Four strains (GZLL3, GZLL4, GZLL11 and SH2) were isolated from goat blood samples and eight strains (SH4, GZZY, GZSQ, GZZA, BR13001, BR13004, BR13005 and BR13006) were isolated from blood samples of patient 12 Brucella suspicious strains were identified and characterized using conventional methods. Brucella genus specific gene BCSP31-based PCR (BCSP31-PCR) was used to identify the genus of Brucella and IS711 insert sequence-based PCR (AMOS-PCR) was applied to identify the species of Brucella strains. Goats and patients originated Brucella strains were comparatively analysed using Pulse-field Gel Electrophoresis (PFGE).</p><p><b>RESULTS</b>Both of conventional methods and PCR identified the 12 Brucella suspicious strains as B. melitensis biotype 3. BCSP31-PCR identification results showed that a specific DNA bands (223 bp) were detected in all the 12 strains and positive control samples with no DNA band in negative samples. AMOS-PCR amplified a 731 bp-DNA bands in all the 12 strains, with 731 bp, 498 bp and 275 bp in M5, S2 and A19 strains, respectively, and no DNA band was detected in the negative control samples. PFGE analysis showed that 12 Brucella isolates from patients and goats showed consistent PFGE patterns with the digestion of restriction enzyme Xba I.</p><p><b>CONCLUSION</b>The epidemic species/type of Brucella in both human and animal in Guizhou province was B. melitensis biotype 3 and goat was the main animal source of infection of brucellosis in Guizhou province.</p>
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Animales , Humanos , Técnicas de Tipificación Bacteriana , Brucella , Clasificación , Brucelosis , Epidemiología , China , Epidemiología , ADN Bacteriano , Cabras , Tipificación Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Objective To explore the effect of cryopreserved canine kidney cells (MDCK)single -layer on the isolation and proliferation of influenza virus B.Methods Revived P17 MDCK cells were passage for 2 -4 gener-ations,and subsequently preserved in 4℃ refrigerator for 3,6 and 9 days,respectively.Under same experimental con-ditions,the 4℃ refigerater preserved cells were co -incubated with influenza -like illness(ILI)throat swab speci-mens.Cytopathic effect (CPE)was observed,and the proliferation of virus was determined using real -time PCR and the hemagglutinin titers were determined by serological test.Results (1)CPE:The CPE of the MDCK cells pre-served in 4℃ refrigerator for 3 or 6 days had no significant differences compared with that in the control group,while the cell preserved in 4℃ refrigerator for 9 days showed CPE fastly and maintained for a short time.(2)Real -time PCR:the proliferation of influenza virus B in the MDCK cells preserved with 4℃refrigerator for 3 or 6 days (25.86 × 105 -30.25 ×106 ,26.31 ×105 -30.54 ×106 )had on difference compared with that of the control group (24.82 × 105 -29.86 ×106 ),with the proliferation rate of 105 to 106 times,while the proliferation cell with 4℃ cryopreserved for 9 days(19.72 ×104 -28.34 ×105 )the proliferation in cells preserved for 9 days was sharply decreased,with pro-liferation rate of 104 to 105 times.(3)The HA titer:The virus strains with hemagglutination titer above or equal to 116 (P >0.05)isolated with MDCK cells preserved in 4℃ refrigerator 3 or 6 days were not significantly different from that of the control group (10.92 ±0.79).And the cells with 4℃ cryopreserved for 9 days were significantly dicreased (P <0.01).Conclusion No significant effects on the isolation and proliferation of influenza virus B using MDCK cell preserved in 4℃ frigerator for near one week were observed in the present study.
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<p><b>OBJECTIVE</b>To analyze causes of growing hepatitis E (HE) cases reported in Guizhou province, and probe into existing problems faced by medical institutions in diagnosis of clinical and laboratory-confirmed cases, for the purpose of improving the quality of HE surveillance system.</p><p><b>METHODS</b>Six hospitals reporting greater HE cases from 2007 to 2011 were pinpointed, whose reported cases rose suddenly in 2011. Such cases were investigated by means of impatient medical record review, results of laboratory test and clinician interview.</p><p><b>RESULTS</b>136 of the 354 reported HE cases investigated were found compliant with the diagnostic criteria of HE with an accordance rate of 38.42%. Difference of the HE diagnostic accordance rate among individual years, hospitals and reporting departments was statistically significant. Such rate of hospital reports was found to be the lowest in 2011, ranging from 0 to 18.18% respectively; HE cases reported by non-infectious departments accounted for 61.30% of total cases reported, with its accordance rate considerably below the infectious departments (8.29%). HE positive cases and HE positive rate in 2011 were significantly higher than that of preceding years.</p><p><b>CONCLUSION</b>Such increase of reported HE cases in 2011 in the province was mostly attributable to more HE laboratory tests made by the hospitals, yet the accordance rates were lower than satisfactory. In this regard, the medical institutions in question were advised to enhance their competency training for HE diagnosis and case report quality.</p>
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Humanos , China , Epidemiología , Hepatitis E , Diagnóstico , Epidemiología , Hospitales , Laboratorios , Proyectos de InvestigaciónRESUMEN
<p><b>OBJECTIVE</b>This study was to evaluate the effects of prevention and control regarding programs on typhoid fever and paratyphoid fever, in Guizhou province, from 2007 to 2012, to provide evidence for the improvement of related programs.</p><p><b>METHODS</b>Data on typhoid fever and paratyphoid including information on epidemics, individual, cases, measures for prevention and control programs taken and relative government documents were collected and analyzed in Guizhou province, from 2007 to 2012. Information related to the average annual incidence, nature of outbreaks, time span before confirmed diagnosis was made, unit which carried the case report, proportion of laboratory confirmed diagnosed cases and case-management were compared between 2007-2009 and 2010-2012 descriptively while chi-square test with Excel and EpiInfo software were used for data analysis.</p><p><b>RESULTS</b>In the period of 2007-2009, a total of 5 978 typhoid fever and paratyphoid fever cases were reported in Guizhou province with the average yearly incidence as 5.29/100 000. In the period of 2010-2012, 2 765 cases were reported with the average yearly incidence as 2.57/100 000. When compared to the former, data from the latter period showed that the average yearly incidence had declined 51.31% in all the prefectures. There were still some outbreaks appeared but the total number of cases involved reduced 87.50%. The time span before the confirmation of diagnosis became shorter but the difference was not statistically significant (χ² = 0.08, P = 0.99). Number of cases reported by hospitals at county or above had 11.51% of increase while those cases reported at the township hospitals or below decreased for 61.47% . The proportion of laboratory diagnosed cases increased 23.63%. Rates of timeliness on cards being filled in, input and audited showed increase of 8.44%, 6.76% and 2.40% respectively.</p><p><b>CONCLUSION</b>Successful measures for prevention and control on typhoid fever and paratyphoid fever had been remarkably taken in Guizhou province, but the potential risk of outbreaks still existed in some areas, suggesting that health education and surveillance programs including laboratory diagnosis, should be strengthened.</p>
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Humanos , China , Epidemiología , Control de Enfermedades Transmisibles , Métodos , Fiebre Paratifoidea , Epidemiología , Fiebre Tifoidea , EpidemiologíaRESUMEN
To identify the isolated suspicious strain of Campylobacter jejuni from the blood of bacteremia patient in Guizhou Province ,China ,conventional and molecular techniques (specific mPCR and NAP-mPCR) were used to identify suspi-cious bacteria strains .Results showed that Campylobacter jejuni suspicious colonies were cultured in bacteremia patient blood samples .The strain was identified as Campylobacter jejuni ssp . jejuni by conventional tests and was identified as Campy-lobacter jejuni by genus specific mPCR .Then the strain was classified as Campylobacter jejuni ssp . jejuni by subspecies NAP-mPCR .The strain was identified as Campylobacter jejuni ssp .jejuni isolated from the blood of bacteremia patient and Campylobacter jejuni can be identified subspecies by NAP-mPCR .
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Eight patients with suspected cases of C .jejuni were etiologically diagnosed and analyzed in this study to pro-vide scientific basis for the confirmation of the cases of human campylobacteriosis in Guizhou Province ,China .Blood or feces of 8 suspected patients were employed to isolate bacteria strains .Conventional and multi-PCR techniques were applied to identify suspicious bacteria strains .The C .jejuni strains were analyzed by using Pulsed Field Gel Electrophoresis (PFGE) .Suspicious strains of C .jejuni were isolated from all the 8 suspected patients of campylobacteriosis and anticipated genes fragment were detected with multi-PCR .With the digestion of restriction enzyme SmaI ,the 8 C .jejuni strains were divided into 7 PFGE pat-terns with 7-10 DNA bands .Cluster analysis showed that the gross similarity of 8 strains of C . jejuni was more than 50% . The similarity of PFGE patterns between strain GZ201004 and GZ201005 from diarrhea patients was as high as 100% ,while the similarity of strain GZ201201 and GZ201007 was 66 .7% .Moreover ,C . jejuni were detected from all the suspected pa-tients of campylobacteriosis .PFGE results indicated that strains GZ201004 and GZ201005 were from the same source ,while all the 8 isolates showed PFGE polymorphism .
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Objective To master the prevalence of plague and its trend in Guizhou Province,and to analyze the plague monitoring results from 2000 to 2012.Methods The report of infectious disease,the information of plague natural focus and the epizootic monitoring data of Xingyi City,Anlong County and Dingxiao Distract of Guizhou Province from 2000 to 2012 were collected and the status of the plague natural focus was analyzed.Results There were 137 cases of gland plague in Xingyi City and Arlong county from 2000-2003,1 death,and mt plague occurred in 66 villages.Fifty-four strains of Yersiniapestis were detected and 49 rats were plague antigen F1 positive(49/160).No human plague occurred between 2004-2012.A total of 4 plague antigen F1 positive rats were detected in Dingxiao District and Xingyi City in 2005 and 2006.There was no Yersinia pestis and F1 antibody in 2007-2012.The epidemic stage of plague was from 2000-2003; the active stage was from 2004-2006; and the quiescent stage was from 2007-2012.The dominant species of the plague natural focus was Rattus flavipestus (42.83％,7 966/18 597),but was replaced by Rattus norvegicus at the epidemic stage (47.22％,1 480/3 134) and the active stage(35.35％,2 071/5 196).The density of rodents was 5.34％ at the epidemic stage,which was higher than that of the active stage (3.27％) and the quiescent stage (1.71％,x2 =2 286.15,P ＜ 0.01).Xenopsylla cheopis(56.34％,10 034/17 811) was the dominant species,and the index was 1.537 9,which was greater than those of the active stage(0.959 6) and the quiescent stage(0.540 4,x2 =492.68,P ＜ 0.01).Conclusions The plague of Guizhou Province is at the quiescent stage.Both the density of rodents and the Xenopsylla cheopis index are lower than the national standard of controlling.
RESUMEN
Objective To evaluate the inhibitory effects of shRNAs targeting genes encoding viral capsomeres VP1-VP4 on enterovirus 71 (EV71) replication when used alone or in combination.Methods Short hairpin RNAs (shRNAs) targeting genes encoding VP1-VP4 protein of EV71 were designed and then respectively inserted into lentiviral vector pLKD-CMV-GFP-U6 to construct the recombinant plasmids.The expression plasmids together with psPAX2 and pMD2.G were transfected into 293T cells to induce the expression of recombinant lentiviruses,which were collected on the third day after transfection.The titers of recombined lentiviruses were determined by real-time PCR.The effects of shRNAs used alone or in combination on the expression of EV71 at mRNA and protein levels were respectively detected by real-time PCR and Western blot.Results The inhibitory effects of shRNAs on EV71 replication showed no significant differences among various strains (isolated from fatal cases,severe cases,mild cases and FY0805) (P＞0.05).Their inhibition rates were 51.6％ (sh-VP1-1),85.1％ (sh-VP1-2),76.4％ (sh-VP1-3),57.5％ (sh-VP2-1),81.4％ (sh-VP2-2),79.5％ (sh-VP2-3),68.9％ (sh-VP3) and 56.7％ (sh-VP4) respectively,and they were in a dosage dependent manner.sh-VP1-2 in combination with sh-VP2-2 showed the highest inhibition rate reaching up to 96.6％.Moreover,shRNAs used in combination showed better effects than any one used alone even at double dosage.Conclusion All shRNAs targeting viral capsid VP1-VP4 genes showed inhibitory effects on EV71 replication with inhibition rates over 50％ and the effects could be strengthened when using shRNAs in combination.