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.

Objective To study the composition of variant and point mutations of Norovirus GⅡ.4 in Guiyang regions.Methods From June to November 2010,cases information and fecal specimens were collected from guard-hospitals in Guiyang regions,who had caught the acute-gastroenteritis.Noroviruses in specimens were detected by a real-time reverse transcription polymerase chain reaction(real-time RT-PCR),and then partial genotyped norovirus-positive clinical samples (in random) were cloned and sequenced in VP1 gene code.Furthermore,the gene sequences were compared with the published variants at home and abroad of norovirus(GⅡ.4),including the phylogenetic analyses of genomes and variation of amino acids within individual sites.Results Those 267 specimens were GⅡ-norovirus-positive(62.68％) in 426 clinical samples.There were nine GⅡ.4-norovirus-positive VP1 gene-sequences available,and two subtype-norovirus variants (GⅡ.4 2008a and G Ⅱ.4 2008b variant) were epidemic in 2010,Guizhou province.The homology between and in subgroups were 95.90％-96.72％ and 99.45％-100％.Two amino acids within individual sites were apt to mutate.Conclusion Norovirus GⅡ genotype were predominant in summer and fall acute gastroenteritis in 2010 for Guiyang regions,and the variants were diversity.

BACKGROUND:Open fracture of lower limb with severe soft tissue and bone defects also accompanies anterior tibial soft tissue defects and exposure of sclerotin and steel plate, which can be crucial y treated with strong fixation and use of skin flap to block the wound.
OBJECTIVE:To explore the clinical efficacy of a large area of soft tissue defects in the anterior tibia using vacuum sealing drainage combined with groin free flap.
METHODS:A total of 24 patients with a large area of soft tissue defects in the anterior tibia were included in this study and then divided into two groups, with 12 cases in each group. In vacuum sealing drainage group, the scope of soft tissue defects was ranged from 10 cm×15 cm to 15 cm×20 cm. After the debridement, the fracture was fixed with external fixation scaffold and the wound was covered with the vacuum sealing drainage dressing. The blood clot was rinsed with normal saline via T-tube, and 7-10 days later the vacuum sealing drainage was given. According to the growth of granulation tissue, the wound was secondarily sutured, fol owed by groin free skin flap of superficial iliac circumflex artery with medial knee arteriovenous anastomosis transplantation. In the non-vacuum sealing drainage group, the wound size was ranged from 10 cm×5 cm to 30 cm×20 cm, the period from injury to admission was 1-24 hours. They were given conventional debridement and secondary fixation or skin flap transplantation.
RESULTS AND CONCLUSION:The length of preoperative hospital stay and the skin flap are in vacuum sealing drainage group were significantly better than those in non-vacuum sealing drainage group (P<0.05). There was no significant difference in the length of postoperative stay and total length of hospital stay between the two groups (P>0.05). The wound infection rate was 0 in vacuum sealing drainage group and 75%in non-vacuum sealing drainage group at 8-14 days after treatment. The wound and donor area incision were healed at I stage, the skin grafts survived. Al the involved patients in two groups were fol owed up, for 6-36 months. During the fol ow-up process, the fracture healing time in non-vacuum sealing drainage group was significantly longer than that in vacuum sealing drainage group. The skin flap in two groups was similar to surrounding skin in color and texture, the flap exhibited no vessels, no ulceration, and no clumsy. The vacuum sealing drainage combined with groin free flap can timely control a large area of soft tissue defects post-trauma, improve wound blood supply, shorten preoperative preparation time, early close the wound, significantly promote the healing of wound and fracture. The skin flap is soft, flexible, wel-looking, and active functional, it significantly shortens the course of treatment and maximizes the recovery of limb function.

Objective:To report a case of McCune-Albright syndrome (MAS). Methods:Investigated one case's clinical data of McCune-Albright syndrome and reviewed related literatures. Analyzed the cause of disease,clinical manifestation, diagnosis, treatment and prognosis. Results: The diagnostic criteria of MAS are the fibrous dysplasia of bone (FD), in addition to at least a kind of typical hypercrinemia, and (or) special café-au-lait skin spots. A definite diagnosis can be made by discovering the mutation of Gs α-gene in cyst fluid from the ovarian follicle and exceptional bone tissue by gene diagnosis. Conclusion:MAS is a rare disease in clinic and the most of domestic doctors don't know it yet, so it is easy to be misdiagnosed or missed diagnosis. There is no specific treatment for MAS. We must consider it when encounter these patients with sexual precocity as well as with café-au-lait skin spots, and it will be helpful to treat by earlier discovery.

Objective The clinical effect of the lateral femoral cutaneous artery flap for soft tissue defects of leg wounds. Methods From October 2007 to January 2016, VSD was firstly used to promote the growth of granulation tissue. When the growth of granulation tissue became satisfactory, flaps were designed based on the anatomical characteristics of the lateral femoral cutaneous artery. We repaired 20 cases of wound defects by cutting flaps that coincide with the recipient vessels. Result 20 cases were followed up for 6 to 24 months, 12 months on average. All flaps were survived and only one case had small area of necrosis flap which was healed by replacing medicines. In all cases, wounds were healed and flaps showed good color and good texture. The strength of quadriceps muscle was good and the extension of knee flexion was 0° to 150°. Conclusion To The lateral femoral cutaneous artery flap is used for soft tissue defects of refractory wounds on leg , flap donor sites are sutured directly, the treatment period is shorten and the method is safe and effective. The lateral femoral cutaneous artery flap is one of ideal choices for wound tissue defects.

Objective To confirm the death of a child injured by a dog was due to rabies and to understand the molecular biologic features of rabies virus in Kaili,Guizhou province.Methods Brain tissue samples of patient and dog were collected to detect the rabies virus by direct immunofluorescence assay (DFA) and RT-nested PCR assay.Homology and phylogenetic tree were analyzed based on the whole nucleotide and deduced amino acid sequence of N gene of rabies virus followed by molecular epidemiological analysis.Results Both the human and dog brain tissue samples were confirmed positive by DFA and RT-nested PCR assay.The homology analysis of N gene sequences among GZH,GZD and other epidemic and vaccine rabies strains isolated from other provinces and other countries indicated that the detected samples shared the highest homology with the strain detected in Anlong prefecture in Guizhou in the year of 2006,and the homology between GZH and GZD was as high as 100％.Besides,among the vaccine strains,GZH and GZD showed the highest homology with strain CNT.Phylogenetic analysis indicated that the two samples were very close and belonged to genetype 1 lyssavirus,with the closest relationship between samples reported in Guizhou and Beijing.Conclusion It was confirmed on the viral molecular level that both the human and dog in Kaili were suffered from rabies,and the pathogens were genetype 1 lyssavirus.The prevalent strains in Kaili city was probably imported from other prefectures of Guizhou province,suggesting that prevention and control measures on rabies in Guizhou province should be strengthened.

To study the characteristic of glycoprotein gene sequence of rabies virus in Guizhou Province in recent years and provide scientific basis for effective control and prevention of rabies,RT-PCR assay were used to detect human and dog brain tissues derived from different prefectures of Guizhou.The amplification products were sequenced and analyzed with bioinformatics software.The results showed that the full-length G gene sequences of 8 positive samples were obtained by RT-PCR amplification,sequencing and splicing.The homology of eight G gene sequences from Guizhou Province were between 87.4％ -99.9％ and 83.3.％- 100％ on nucleotide and deduced amino acid level,respectively,and the highest homology were found with the genotype 1 strains ( 86.5 ％- 87.0％ for nucleotide and 83.3 ％- 100 ％ for amino acid) among genotype 1- 7 representative strains.Besides,phylogenetic analysis based on the G gene indicated that the relationship of 8 strains derived from Guizhou were closest to genotype 1 Lyssavirus,and the strains of Guizhou were very close to the strains come from Hubei,Hunan,Anhui,Guangxi,Jiangsu provinces and Shanghai,except for GZ01 and GZ09.Moreover,GZ09 were evolutionarily closed to the strains come from Malaysia and Thailand,while the remaining sequences were closed to the strain of Indonesia.This study confirmed on the G gene level that rabies virus epidemic strains circulated in Guizhou Province in recent years belonged to rabies virus genotype 1 and the evolutionary relationship with reported strains come from different provinces of China and different countries were revealed in this study.It will provide scientific basis for effective control and prevention of human and animal rabies in Guizhou Province.

Aim:Pertussis is a respiratory tract infection,the vaccine for which was introduced in the Expanded Programme on Immunization (EPI) in Guizhou Province of China (hereafter referred as Guizhou) in the 1980s.This vaccine rapidly decreased incidence rates of pertussis in the province,however,despite the wide high coverage of the diphtheria,tetanus and acellular pertussis (DTaP) combined vaccine,there has been a resurgence of pertussis since 2014.Even with this recent increase in disease transmission,risk factors for pertussis infection have not been evaluated in Guizhou.We aimed to provide information on pertussis risk factors and insight on designing targeted pertussis control policies and measures through this study.Methods:A 1∶2 matched retrospective case-control study was conducted between 2016 and 2017,involving infants and children younger than 6 years old and parents of the participants.The enrolled cases included clinical and laboratory confirmed pertussis cases according to the WHO-recommended pertussis definition.Controls were selected from children in the same neighborhood who were not diagnosed with pertussis prior to our investigation and did not exhibit any clinical manifestations of pertussis.Results:The household size[Matched odds ratio (ORm) =1.4,95％ confidence interval (CI):1.1-1.7]and house-hold members with antecedent cough (ORm =3.6,95％ CI:1.8-7.2) were significantly associated with their child's pertussis onset.The parents' occupations were significantly associated with their child's pertussis onset(ORm =9.4,95％ CI:1.6-54.8,for mother side;ORm =4.5,95％ CI:1.2-16.5,for father side),when they worked in the business and/or service industry.Having family members with a history of cough was an independent risk factor for pertussis[Adjusted odds ratio (AOR) =43.6,95％ CI:2.7-694.0].Besides,the parents' demographic characteristics and DTaP doses were not found to be independent factors.Conclusion:Household exposure is an important risk factor for pertussis infection in infants and young children and should therefore be considered as a major factor during formulation of pertussis control policies and measures.

OBJECTIVEThis study aims to investigate the feasibility of tongue reconstruction by a rectus abdominis musculoperitoneal flap with neurovascular pedicled in a canine model.

METHODSTwelve Beagle dogs were enrolled to the experiment. The animals were randomly divided into thee groups, two of which (group A and B) had nerve anastomosis. The left sides were experimental sides, whereas the right sides were control sides. Twelve weeks after operation, electrophysiological test was performed to detect hypoglossal nerve latency amplitude and conduction velocity as well as to evaluate the reinnervation of the rectus abdominis musculoperitoneal flap.

RESULTSAmong the 12 Beagle dogs, nine animal tongue reconstruction models by rectus abdominis musculoperitoneal flap with neurovascular pedicled were successful, whereas one male Beagle dog died from ventral hemia 3 d after the operation, two female rectus peritoneal flaps were abandoned because their arterial anatomy differed from the male, which was not ideal. Hypoglossal nerve conduction velocity of group A and B were restored to the normal side of the 40%, 30%.

CONCLUSIONAnimal models of tongue reconstruction can be established by a rectus abdominis musculoperitoneal flap with neurovascular pedicled in Beagle dogs. Denervated rectus abdominis musculoperitoneal flap can regain hypoglossal nerve innervation. Hypoglossal nerve functions partly recover.

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.