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Objective To understand the gene E sequences of prevalent strains of Dengue fever type Ⅰ virus in Zhaoqing City during 2014.Methods The medical record data and acute stage serum of the patients with Dengue fever in Zhaoqing City during 2014 were collected.Dengue virus was cultured and isolated by using the C6/36 cell culture.Gene E of positive strains was amplified with RT-PCR.The phylogenetic tree was drawn and the bioinformatics analysis was conducted.Results Of 36 samples,20 samples were positive in viral isolation and culture.The gene E sequences of 20 strains of type Ⅰ Dengue virus prevalence in Zhaoqing dur-ing 2014 were obtained;the homology of these sequences was close to that of the 2 prevalent strains found in Zhongshan City,but was distant from that found in Guangzhou City.Conclusion The epidemic situation of Dengue fever in Zhaoqing City is closely re-lated to the prevalence situation of Zhongshan and is characterized by imported prevalence.
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Objective To provide scientific evidences for Zika virus detection by clarifying the means by which Zika virus was discharged and the duration of corresponding processes. Methods Various samples of Zika cases were collected at different times and detected by using real-time RT-PCR. The positive samples were inoculated into cells and suckling mice through intracranial injection. The whole genome se-quences of those isolated Zika virus strain were sequenced and the results were further analyzed by comparing with the sequences of Zika virus from GenBank. Results The positive rates of Zika virus in urine, saliva and serum samples were 82. 4% (14/17), 82. 4% (14/17) and 52. 9% (9/17) respectively. The longest period of detected presence of Zika virus was found in urine samples amongst the three types of samples, fol-lowed by saliva and serum samples. Six Zika virus strains were isolated from 9 positive serum samples. Phy-logenetic analysis showed that the six genomes of Zika virus all belonged to Asia lineage, but located in two branches by Samoa and Venezuela strains. Conclusion This study indicated that urine, saliva and serum all could be used as the samples for routine detection of Zika virus. Urine and saliva samples showed higher detection rates of Zika virus RNA in comparison to serum samples, while Zika virus could be easily isolated from positive serum samples. Suckling mice were better for Zika virus isolation than cell lines.
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Objective To evaluate the clinical efficiency of posterior unilateral open-door laminoplasty and leverage titanium plate internal fixation in the treatment of cervical spondylotic myelopathy ( CSM ) with multi-segmental spinal stenosis.Methods Between Mar 2011 and May 2015, 25 patients with multi-segmental CSM with multi-segmental spinal stenosis were treated by posterior unilateral open-door laminoplasty and leverage fixation .There were 16 males and 9 females, whose mean age was 60.6 ±9.9 years during the surgery.The change of clinical symptoms and signs was recorded during follow-up,and they all received X-ray and MRI.In all the patients, the preoperative and postoperative neurological function, the cervical curvature,cervical vertebra tube volume and axial symptoms were measured , recorded and analyzed. There was statistically significant difference (P<0.05) in the mean Japanese Orthopaedic Association (JOA) score, and Visual Analogue Scale ( VAS) .Results All the 25 patients were followed up for more than 6 months ( 6-24 months ) .No symptoms of C5 nerve root were found in our series .According to the JOA score and VSA score ,the neurological functions of each patient were significantly improved .The preoperative JOA score was 10.16 ±1.35 and the improvement rate 61.24%. There was statistically significant difference between the preoperative VSA score and the postoperative one (6.68 ±1.12 vs 2.32 ±0.84) ( P<0.05).The preoperative and postoperative meansurement of the spinal vertebrai canal diameter was (9.22 ±2.01) and (15.64 ±2.08) mm, respectively,so there was statistically significant difference (P <0.05), indicating that the cavical spinal canal was increased after operation .Conclusion Leverage titanium plate internal fixation can effectively help maintain the expanded vertebral canal after unilateral open -door laminoplasty ,reduce the incidence of postoperative axial symptoms , and maintain the cervical physical curvature .
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Objective To establish a method for the isolation of Zika virus and to gather experi-ences for viral isolation. Methods Suckling mice at age 1-3 days were inoculated with serum samples posi-tive for Zika virus through intracranial injection. All mice were sacrificed 6 days after the injection. Viral nu-cleic acids were extracted from brain, heart, liver, spleen, lung, kidney, muscle, skin and intestine tissue samples and analyzed by real-time RT-PCR. The supernatants of brain tissues positive for Zika virus were used for subculturing. Nested PCR was performed to amplify the NS5 gene of the isolated virus. The se-quences of NS5 gene were analyzed by using MEGA6. 0 software. Results All of the tissue samples were positive for Zika virus. Higher viral loads were detected in heart and brain tissue samples with cycle thresh-old (Ct) values of 24. 4 and 25. 3, respectively. The second generation of Zika virus was identified in suck-ling mice brain tissues 2 days after infection by using real-time RT-PCR. The amplified product of nested PCR was 972 bp in length. Sequencing analysis showed that the isolated Zika virus ( GDZ16002 strain) be-longed to the Asian lineage. Conclusion A strain of Zika virus was successfully isolated in China by using intracranial injection via a suckling mouse model. The isolated Zika virus belonged to the Asian lineage.
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Objective To study the factors influencing epidemiological characteristics and virulence of Enterovirus 71 (EV71) in Guangdong province from 2008 to 2010. Methods RNA was extracted from collected samples or cultured virus , then reversing transcription into cDNA. We amplified full-length EV71-VP1 using poly-merase chain reaction technology , then conducted sequence alignment and established phylogenetic tree with MEGA software (version 5.0) to confirm the genotype of EV71. The association between severity of clinical symp-toms and sex, age, viral genotype and VP1 variation was also analyzed using Logistic regression. Results The genotype of the predominant epidemical strain was C4a in Guangdong from 2008 to 2010. However , this subtype had already differentiated into 4 subgroups (C4a1- C4a4). There was no correlation between clinical syndrome and sex or viral genotype; the severity of symptoms was negatively correlated with age: before 4 years old, varia-tion A289T can easily lead to severe cases, increasing the risk of infection (P<0.05, OR = 2.360, 95%CI:1.163~ 4.659). Conclusion The main epidemical EV71 strain is C4a1 in Guangdong province. The emerging differen-tiation and simultaneous prevalence should merit attention to strengthen relevant surveillance; and the protection of the susceptible population should be reinforced during EV71 prevalence.
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Objective To research the relation between the time-dependent appearances of myofibroblasts during the repair of contused skeletal muscle in rat and wound age determination. Methods A total of 35 SD male rats were divided into the control and six injured groups according to wound age as fol-lows: 12 h, 1 d, 5 d, 7 d, 10 d and 14 d after injury. The appearances of myofibroblasts were detected by HE staining, immunohistochemistry and confocal laser scanning microscopy. Masson’s trichrome staining was utilized to examine collagen accumulation in the contused areas. Results Immunohistochemical stain-ing showed that α-SMA+ myofibroblasts were initially observed at 5 d post-injury. The average ratio of myofibroblasts was highest at 14 d post-injury, with all samples, ratios more than 50%. In the other five groups, the average of α-SMA positive ratios were less than 50%. The collagen stained areas in the contused zones, concomitant with myofibroblast appearance, were increasingly augmented along with ad-vances of posttraumatic interval. Conclusion The immunohistochemical detection of myofibroblasts can be applied to wound age determination. The myofibroblasts might be involved in collagen deposition during the repair of contused skeletal muscle in rat.
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Objective To study the percentages of polymorphonuclear leukocytes (PMN), mononuclear cells (MNC) and fibroblastic cells (FBC) in different post-traumatic intervals after skeletal muscle me-chanical injury in rats. Methods The rat model of skeletal muscle mechanical injury was established. The rats were divided into injured groups (6 h, 12 h, 1 d, 3 d, 7 d, 10 d and 14 d after injury ) and con-trol group. The percentages of PMN, MNC and FBC in different post-traumatic intervals after skeletal muscle mechanical injury were assessed with HE staining and image analysis. Results At post-injury 6-12 h, the percentages of PMN and MNC infiltration appeared in injured sites and that of PMN reached peak. At 1 d, the percentage of MNC infiltration appeared and reached peak, while that of PMN de-creased. At 3-7 d, the percentage of FBC gradually increased, while that of PMN and MNC decreased. At 10-14 d, the percentage of FBC reached peak. Conclusion The percentages of PMN, MNC and FBC in injured zones showed time-dependent changes, which might be used as reference index for determination of age of skeletal muscle injury.
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Objective To investigate the susceptibility to oseltamivir of influenza pandemic A (H1N1) 2009 viruses in Guangdong province during 2009,provide valuable information of prescription for clinics,and elucidate the variant trend of the epidemic strain based on phylogenetic analysis.Methods During April to December 2009,clinical specimens were collected from sentinel hospitals covering the whole Guangdong province.Virus isolation was performed by in MDCK cells or embryonated chicken eggs.A fluorescence-based neuraminidase (NA) enzyme inhibition assay was conducted to measure influenza susceptibility.The NAI susceptibility of influenza virus was expressed as the concentration of NAI needed to inhibit the NA enzyme activity by 50％.A subset of 68 viruses were performed NA sequencing for detecting resistant mutations and studying variant trends.Results During surveillance,221 influenza pandemic A ( H1N1 ) viruses were isolated.All strains were sensitive to oseltamivir inhibition assay,with a median IC50 of 0.24 nmol/L (range 0.02 -1.66 nmol/L).No mutation related to resistance was found.Phylogenic analysis illustrated that these NA genes were homology high to 99.5％ - 100.0％ with those from other countries.Conclusions influenza pandemic A (H1N1) 2009 viruses were sensitive to oseltamivir in Guangdong,and useful for prophylaxis and treatment of influenza infection.Little selective pressure was found by phylogenic analysis.Our laboratory will continue to observe antiviral-resistance among circulating influenza viruses.
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Objective To discuss the prevalence of non-EV71,non-CA16 virus strains of hand,foot and mouth disease(HFMD) in Guangdong province between 2008 and 2009,and analyze the genetic evolution of these non-EV71,non-CA16 virus strains.Methods Isolated viruses from stool samples collected from outpatient and in-patient cases of HFMD between 2008 and 2009 by human rhabdomyosarcoma(RD) cell and HEp-2 cell,cultures that exhibited a characteristic enterovirus cytopathic effect were evaluated by RT-PCR.Those strains which identified non-EV71,non-CA16 were analyzed by VP1 sequencing and then were identified by BLAST program.A phylogenetic tree was constructed using the Neighor-Joinning method in the MEGA 4.0 software.Results Twenty-two virus strains of non-EV71,non-CA16 were obtained,and nine of the twenty-two virus strains in 2008 were classified into CA2,CA4,and CB3 by BLAST; thirteen of the twenty-two virus strains in 2009 were classified into EV80,Echo13,Echo30,CBS,Echo24,CA10,CA6,and poliovirus 1 by BLAST.The honology of all strains was low,and all the strains belonged to CA,CB,Echoviruses,Enterovirus and poliovirus subgroup.Conclusion Except for EV71 and CA16 was a major causative agent in prevail of HFMD in Guangdong province between 2008 and 2009,there also existed other subgroup Enterovirus.The other twenty-two strains respectively belonged to CA,CB,Echoviruses,Enterovirus and poliovirus subgroup,and none of those strains was predominant.Muti-species Enterovirus occurred concomitantly.
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Objective To understand the evolutionary characterization of hemagglutinin (HA)gene of pandemic H1N1 influenza virus in Guangdong during 2009-2011. MethodsWe selected 83 pandemic H1N1 strains isolated in Guangdong during 2009-2011. The HA1 genes were sequenced and analyzed comparatively by Bioedit 7.0 and MEGA 4.0. ResultsThe evolutionary rate of Hal gene of pandemic H1N1 and seasonal H1N1 viruses was 5.2×10-3 substitutions/site/year, higher than that of seasonal H1N1 viruses. Most amino acid changes in HA1 molecules accumulated on the surface of the molecule and were partly located in antigenic sites. Two fatal infections were detected with a mutation at HA residue 222, in one virus with a change D222G, and in one virus D222N. ConclusionThe phylogenetic analysis demonstrates that the influenza epidemic in Guangdong at the beginning of 2011 are due to occurrence of genetic changes of pandemic H1N1 virus. The amino acid change at residue 222 of the HA1 are likely to be associated with severe or even fatal illness.
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Objective To analyze the genetic characterization(evolution, antigenicity, enzyme activity sites and glycosylation sites)of the neuraminidase(NA)gene of the novel influenza virus A/H1N1 in 2009 pandemic in Guangdong Province. Methods The viral RNA was extracted from 69 isolates of influenza virus A/H1N1 from patients in 2009 pandemic in Guangdong Province. NA gene fragments were amplified by reverse transcription polymerase chain reaction (RT-PCR) and sequenced. The other 52 NA gene sequences of influenza virus A in different years and different regions were retrieved from GenBank. The analysis of evolution and amino acid sequences were analyzed by MEGA 4.0 software. Results The homology of 2009 novel H1N1 influenza viruses in Guangdong and avian H5N1 influenza virus strains was high(＞85 % ). The amino acid distributions of potential antigenic sites were identical. The enzyme activity sites of NA genes of all virus strains were strictly conserved, which had eight glycosylation sites. But there were amino acid substitutions in 5 glycosylation sites, while it was identical with the 2001 avian H5N1 influenza virus. Conclusion The NA genes of 2009 novel H1N1 influenza viruses in Guangdong are high homologous with avian H5N1 influenza virus and the viral specific binding sites of neuraminidase inhibitor are not changed.
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Objective To understand the genetic characteristics of Enterovirus 71 ( EVT1 ) circu-lating strains of Guangdong province in 2008. Methods We isolated an EV71 strain from the fatal case of the hand, foot and mouth disease during an epidemic of Guangdong in 2008. Its complete genome was se-quenced and analyzed comparatively. Results The results showed that the full length of EV71 GDFS-3 ge-nome( not including poly A tail ) is 7405 bp. No insertion or deletion is detected in the coding region. There are several insertions and deletions in 5'and 3'UTR. Phylogenetic analysis of GDFS-3 and reference strains showed GDFS-3 strain shares the highest nueleotide homology with TW984 strain(96.0% ) but low homology with SIN5865, MS and BrCr( about 81.0% ). GDFS-3 strain also shares the highest amino acid homology with TW984 strain(99.0% ). It clustered with reference strains of CA subgenotype in the phylogenetie tree. The nucleotide identity with CA reference strains is 91.0% -95.0%. Conclusion The phylogenetic analysis based on the entire genome demonstrates that GDFS-3 strain has the nearest genetic relationship with TW984 strains ( isolated in 2004). GDFS-3 may belong to the same subgenogroup ( CA ) with Taiwan predominant strains. Otherwise,Some mutations in 5'UTR of EV71 may play the very important role in heightened viru-lence.
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Oncogene c-jun is a member of jun family,the immediately early genes(IEGs),and belongs to one of the nuclear transcription factors of basic leucine zipper(bZIP)family.Combined with many gene promotors,c-jun is involved in the regulation of gene transcription.Its products play important roles in regulating gene expression,cell proliferation,differentiation and apoptosis.The structure,biological funetion,regulation of c-jun and its roles contributing to tissue damage are reviewed in this article,which may provide understanding for severity of tissue injury and wound age estimation in the field of forensic pathology.
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Bacillus megaterium strains are commonly used in microbial fertilizer(MF) . MF products are often contaminated by other B. cereus group members,which have similar phenotype such as Bacillus cereus,B. thuringiensis,B. mycoide. For quality control and safety of MF,a rapid and accurate method is needed to distinguish the strains of Bacillus megaterium from B. cereus group. Based on specific nucleotide sequences of the spoOA genes,2 pairs of species-specific primers were designed and a multiplex-PCR(mPCR) was developed for this purpose. When the optimized mPCR was used to detect the DNAs of 24 reference strains from three genera of Bacillus,Paenibacillus,and Brevibacillus,all B. megaterium strains showed singlefragment of 443 bp and Bacillus cereus group showed a fragment of 411 bp. However,no any amplified product was from the other bacteria. The sensitivity of mPCR was 105 CFU/mL. The mPCR results of 10 isolates of B. megaterium/B. cereus group and 8 products of MF coincided with the biochemical assay. Taken together,our newly developed mPCR assay was species-specific and effective in application. It can be used to detect and identify the strains of B. megaterium and B. cereus group from microbial fertilizer products.
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Objective To study the molecular characters of porA and porB genes which encode outer membrane proteins (OMP), and predominated clonal complex of Neisseria meningitidis isolates from Guangdong province. Methods Eighteen Neisseria meningitidis isolates from Guangdong province during year 1967 to 2007 were recovered and reconfirmed by API NH biochemical system, and serogrouped by antiserum. The characters of porA and porB gene were analyzed by DNA sequencing. The allele profiles and the sequence types (ST) were determined by multilocus sequence typing (MLST). Based on their allelic profiles, the evolution relationship was analyzed by PHYLIP software. The predominant clonal complex was determined through comparing with the information of reference strains from the PubMLST database. Results For porA gene, type 20 was more frequently in the variable region (VR) 1 and type 9 in VR2 before year 2004. However, for porB gene, type 4 was more frequently in VR Ⅰ, type7 in VR Ⅳ, type 11 in VR Ⅴ,and type 10 in VR Ⅵ, respectively. The multi-types character was presented in VR Ⅴ and VR Ⅵ after2004. VR Ⅶ and VR Ⅷ can not be found among all the isolates except for one W135 isolate in 2007. Among the seven housekeeping genes, the polymorphism of abcZ was the lowest one with 4 allele numbers, while pgm was the highest one with 13 allele numbers. The predominant clonal complex was ST-5 before 2004. The ST-4821 clone complex appeared in 2004 and caused cases every year since then. More important, highly invasive ST-11 clonal complex firstly appeared in Guangdong in 2007. Conclusion The molecular characteristic of OMP genes presents polymorphism for the Neisseria meningitidis isolates from patients in Guangdong province during 1967 to 2007. ST-5 is the predominant clonal complex before 2004 and the highly invasive clonal complex is circulating in recent 3 years. It suggests that the surveillance based on laboratory should be further enhanced.
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Objective To investigate the expressions of caspase-6,-7 during skin wound healing and the applicability of the time-dependent expressions of caspase-6,-7 to determination of the wound age.Method The expressions of caspase-6,-7 were studied in skin incised wound in mice by immunohistochemical SABC method,and the non-incised mouse skin was used as control.Results Expressions of caspase-6,-7 were detectable in polymorphonuclear cells (PMNs) in the wound specimens aged 6h. In the wound specimens aged from 12h to 24h after injury, caspase-6,-7 were identified in a large number of infiltrating PMNs and part of mononuclear cells (MNCs). Afterwards, the MNCs and fibroblastic cells (FBCs) accounted for the most part of the caspase-6,-7-positive cells. Morphometrically, the ratios of the number of the caspase-6,-7-stained PMNs, MNCs and FBCs to total number of the cells in the wounds were evaluated and calculated. The ratios of the caspase-6,-7-positive cells were low in the wound specimens aged from 0h to 3h, and maximized in the wound specimens aged 3 day. Thereafter, the ratios decreased and minimized in the specimens aged 14 day.Conclusion The results suggest that caspase-6,-7 were expressed in neutrophils, macrophages and fibroblasts during healing process of skin incised wound in mice. Caspase-6,-7 may be used as a marker for the wound age determination.
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Focal adhesion kinase(FAK)and extracellular signal-regulated protein kinase(ERK),which are thought to play major roles in transducing surface receptor-derived signals into nucleus,are pivotal signal transduction molecules in mammalian cells.FAK is activated through phosphorylation by extracellular stimuli.p-FAK activates the downstream of ERK which changes cell action.FAK-ERK signal conduction factors are involved in a variety of biological responses,such as cell proliferation,differentiation,stress,apoptosis and malignant change.The biologic characters of FAK-ERK and advances in the study on its roles in tissue damage are reviewed in this article,which may provide a more effective way for estimation of wound age in forensic medicine.
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Objective To evaluate the influence of different antigen retrieval methods and pH values of fixatives on immunostaining of incised wound skin.Methods 3d-post-incision mouse skin were sampled and fixed in 4% formalin buffered with PBS at pH 5.0,6.0,7.0,8.0 and 9.0.The deparaffinized sections were treated 0.01mmol citrate buffuer(pH 6.0)in high pressure cooker or by microwave heating,or with pepsin,trypsin or microwave+pepsin.Thereafter,immunostainings of caspase-3,caspase-6,caspase-7,IL-8,and IL-10 were evaluated by immunohistochemical SABC method with ratio of immunostaining-positive cells,respectively.Results The immunostaining-positive cell ratios in sections treated by heating retrieval was higher than by proteinase treatment.The positive cell ratio was slightly higher by high pressure than by microwave heating.Sections were more easier peeled off during high pressure treatment than during micrwave heating.Sections treated with pepsin were fallen off easier than with trypsin digestion.The highest positive cell ratio was detected in sections by microwave+trypsin treatment without section peeling off among all antigen treatment methods.The best result was obtained in sections fixed in buffered formalin at pH 7.0 and 8.0.Conclusion The best immunostaining result may be obtained in mouse incised wound sections fixed in buffered formalin at pH 7.0 and 8.0 with microwave+trypsin treatment.
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Hitherto, it is reported that caspase family includes 14 members at least, which play important roles in executing cell apoptosis. Experimental studies have proved that caspases are expressed and activated in the injured skin and during skin wound healing, which suggests that cell apoptosis induced by caspases may occur. Further investigation on the roles that caspases play in the injured skin and during skin wound healing may offer a new way for the clinical treatment of the skin wound and the medico-legal wound age determination. The article reviews the biologic characters of caspases and the advances in the studies on the caspases in the skin wound and wound healing. It is suggested that caspases exert significant parts during skin wound healing, which are worthy of further investigation.
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To explore the applicability of interleukin 8(IL 8)to determination of wound age in the incised and stabbed human skin samples,an immunohistochemical study on the temporal expression of IL 8 was performed on 52 human skin wounds at different posttraumatic intervals.Expression of IL 8 was detectable in polymorphonuclear cells(PMNs)in the wound specimens aged 4h.In the wound specimens aged from 12～24h after injury,IL 8 was identified in a large number of infiltrating PMNs and part of mononuclear cells(MNCs).Afterwards,the MNCs and fibroblastic cells(FBCs)accounted in the most part of the IL 8 positive cells.Morphometrically,the ratio of the number of the IL 8 stained PMNs,MNCs and FBCs to total number of the cells in the wounds was evaluated and calculated.The ratio of the IL 8 positive cells was low in the wound specimens aged 4 and 6h(16 0?10 1%),and maximized in the wound specimens aged between 1 and 4 days(59 6%?8 7%).Thereafter,the ratio decreased and minimized in the specimens aged from 19 to 21 days(27 5?5 9%).The results suggest that IL 8 can be used as a marker for the wound age determination in both incised and stabbed human skin.