To construct an HSV-1 vector vaccine carrying HIV-1 antigens, HIV-1 gp160, gag, protease and the expression elements were chained together, and then inserted into the internal inverted repeat sequence region of HSV-1 by bacterial artificial chromosome technology. Firstly, HIV-1 gp160 (including type B and C), gag and protease genes were cloned into pcDNA3 in series to generate the pcDNA/gBgp and pcDNA/gCgp, then the recombinant plasmids were transfected into 293FT cells, and HIV-1 antigen was detected from transfected cells by Western blotting. Then the expression cassettes from pcDNA/gBgp and pcDNA/gCgp, comprising HIV-1 antigen genes and expression elements, were cloned into pKO5/BN to generate the shuttle plasmids pKO5/BN/gBgp and pKO5/BN/gCgp. The shuttle plasmids were electroporated into E. coli cells that harbor an HSV-BAC, the recombinant bacteria were screened, and the recombinant DNA was extracted and transfected into Vero cells. The recombinant virus was purified through picking plaques, the virus' DNAs were identified by Southern blotting; HIV-1 antigen was detected from the recombinant HSV-1 infected cells by Western blotting, and the virus' replication competent was analyzed. As the results, gp160 and gag proteins were detected from 293FT cells transfected with pcDNA/gBgp and pcDNA/gCgp by Western blotting. The recombinant bacteria were generated from the E. coli electroporated with pKO5/BN/gBgp or pKO5/BN/gCgp. The recombinant HSV was purified from the Vero cells transfected with the recombinant DNA, the unique DNA fragment was detected from the genome of recombination HSV by Southern blotting; gp120 and gp41 were detected from the infected cells by Western blotting, and the recombinant HSV retained replication competent in mammalian cells. The results indicate that the recombinant HSV carrying HIV-1 gp160, gag and protease genes was generated, the virus retains replication competent in mammalian cells, and could be used as a replicated viral vector vaccine.
Animals ; Cercopithecus aethiops ; Chromosomes, Artificial, Bacterial ; DNA, Recombinant ; genetics ; DNA, Viral ; genetics ; Escherichia coli ; HIV Antigens ; genetics ; immunology ; HIV Envelope Protein gp160 ; genetics ; immunology ; HIV Protease ; genetics ; immunology ; Herpes Simplex Virus Vaccines ; immunology ; Herpesvirus 1, Human ; physiology ; Plasmids ; Transfection ; Vero Cells ; Virus Replication ; gag Gene Products, Human Immunodeficiency Virus ; genetics ; immunology

Background and purpose:The incidence of cervical cancer is rather high in Xinjiang, which is closely associated with the infection of human papilloma virus type 16 (HPV-16). The purpose of this study was to analyze the variants and function of HPV-16 upstream regulatory region (URR) in the tissues of cervical cancer biopsies from Xinjiang.Methods:The DNAs were extracted from the tissues of cervical epithelial atypical hyperplasia (CIN) and cervical cancer biopsies. HPV-16URR segments were ampliifed by PCR. Based on the sequence analysis of the URR, the representativeURR variants were selected and cloned into pGL3-Basic. The recombinant plasmids were transfected into Vero cell lines respetively. Luciferase activity of transfected cells was detected 48 h after transfection. Results:Fifty-ifve HPV-16URR DNA fragments were obtained through PCR, and 44 mutations were found from the URR fragments. 4 of these mutations, including nt7192(G→T) , nt7433(- →T), nt7435 (C→G) and nt7863 (A→-) occurred in all sequences. The mutation at nt7520 (G→A) occurred in 54URR sequences, and the 39 other mutations were present in different samples. Based on the location and frequency of the mutations in theURR fragments, 9URR variants were selected and cloned into pGL3-Basic. Then the luciferase activity of the cells transfected with pGL3-URR plasmids was detected respectively. Promoter activity ofURR mutants from cervical cancer are significantly higher than that ofURR mutants from CIN (P<0.01). Promoter activity ofURR fragments from some cervical cancer was signiifcantly higher than that of theURR fragments from SiHa and Caski cells.Conclusion:Multiple mutations occurred in HPV-16URR of cervical cancer patients from Xinjiang. The promoter activity and carcinogenicity of some URR mutants have been enhanced.

Objective:To analyze phylogenetic structure and molecular characteristics of H5N6 avian influenza virus (AIVs) isolated from live poultry market (LPM).Methods:Oropharyngeal and cloacal swabs from poultry, and environmental samples were collected from LPM in Urumqi in December 2018, AIVs were isolated and identified by inoculation of chicken embryo, hemagglutination test and RT-PCR, the viral whole genome was amplified with the universal primers of influenza A virus, and then sequenced, pairwise sequence alignments, phylogenetic and molecular characteristics analysis were performed by BLAST, Clustal W, MEGA-X and DNAStar software.Results:Six strains of H5N6 AIVs were isolated from poultry samples, the identity between the viral genes was high (99.4%-100.0%), so the isolates were the same source. BLAST analysis revealed that the viral NP sequence had the highest identity (99.7%) with H5N6 AIVs isolated from poultry in Suzhou, while the sequence of the remaining 7 viral genes had the highest identity (99.0%-100.0%) with H5N6 AIVs isolated from environment in Guangdong during 2017 to 2018. Phylogenetic analysis showed that the viral HA belonged to Clade 2.3.4.4C, and the viral HA, NA, PB1, PA, NP, and MP were all clustered together with H5N6 AIVs isolated from mink in Eastern China in 2018, while the PB2 and NS were clustered together with H5N6 AIVs isolated from environment in Guangdong from 2017 to 2018. The HA cleavage site contained multiple basic amino acid residues, which was highly pathogenic AIVs (HPAIVs). S137A and T160A mutations of HA could increase binding to human-type receptor SAα2, 6-Gal. Additionally, the viral multiple mutations, including 59-69 deletion in NA, the L89V, G309D, R477G, I495V, I504V, D391E, and A661E in PB2, as well as the P42S, D92E, and 80-84 deletion in NS1, could enhance the viral virulence and pathogenicity to mammals. Conclusions:The 6 strains of H5N6 HPAIVs isolated from LPM have relatively close genetic relationship with H5N6 AIVs isolated from mink in Eastern China and environment in Guangdong during 2017 to 2018, the viral multiple mutations could increase its pathogenicity to mammals, which could pose a potential risk to public health.

Objective: To detect the mutation of epidermal growth factor receptor (EGFR) gene in peripheral blood of non-small cell lung cancer (NSCLC) patients in Yunnan area with Super-ARMS, and to explore its correlation with clinicopathological characteristics. Methods: A total of 222 blood samples from patients with NSCLC were collected between January 2017 to December 2018 in the Molecular Diagnostic Center of Yunnan Cancer Hospital. The EGFR gene mutation in peripheral blood samples was detected by SuperARMS, and the relationship between EGFR gene mutation and clinicopathological features was analyzed. Meanwhile, the independent risk factors influencing EFGR mutation were also analyzed. Results: In the peripheral blood of 222 NSCLC patients, there were 81 cases (36.5%) with EGFR gene mutation. Among them, exon 19 deletion and L858R gene point mutation were the most common (75.3% of total mutation); female patients had a higher mutation rate than male patients (45.9% vs 27.0%); patients <60 years old had a higher incidence of mutation than patients≥60 years old (43.2% vs 28.8%) (P<0.05 or P<0.01); moreover, patients with no history of smoking, no history of radical surgery, adenocarcinoma, advanced stage and no history of chemotherapy had higher incidence of EGFR mutation (43.9% vs 21.6%, 39.2% vs 21.2%, 43.9% vs 4.8%, 39.7% vs 23.3% and 44.0% vs 23.5%) (P<0.05 or P<0.01). Multivariate logistic analysis showed that young, no smoking history, adenocarcinoma and no surgical history were independent risk factors for EGFR gene mutation (all P<0.01). Conclusion: In the peripheral blood of patients with NSCLC in Yunnan, the mutation rate of EGFR gene is higher in patients with age<60 years old, adenocarcinoma and non-smoking. Super-ARMS method is more sensitive in the detection of EGFR mutation in peripheral blood of lung cancer patients.

Mammalian zona pellucida 3(ZP3) plays an important role in the induction of capacitating sperm acrosome reaction. In this study, we obtained the soluble mZP3 fusion protein and identified its immunoreactivity. mZP3 cDNA was cloned into plasmid pMAL-p2x, and the recombinant plasmid was transformed into Escherichia coli BL21. To get the soluble mZP3 fusion protein, we tried to optimize the expression conditions, including additives, IPTG concentrations, temperatures and induction duration. Then, Western blotting and ELISA were used to identify the immunoreactivity of the purified protein. Based on the optimization experiments, we concluded that the best soluble expression conditions for the mZP3 fusion protein involved incubation to an A600 of 0.6, addition of glucose to a final concentration of 0.02 mol/L, addition of IPTG to a final concentration of 0.6 mmol/L and then further incubation for 4 h at 25 degrees C. Western blotting and ELISA showed that the mZP3 fusion protein retained immunoreactivity. The fusion protein can be used as solubility antigens for developing the immunocontraception vaccines of mZP3 and detecting the immune effects of the vaccine.
Animals ; Egg Proteins ; biosynthesis ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Membrane Glycoproteins ; biosynthesis ; genetics ; immunology ; Mice ; Receptors, Cell Surface ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Solubility ; Vaccines, Contraceptive ; immunology ; Zona Pellucida Glycoproteins

Objective: To explore the screening efficiency of colorectal cancer in urban residents of Kunming, China. Methods: Using the method of cluster sampling, from October 2014 to October 2017, residents of the three jurisdictions of Xishan, Guandu and Chenggong Districts of Kunming city were investigated. The inclusion criteria: (1) resident (for more than 3 years) population of Kunming city aged 40-74 years old; (2) voluntarily participating and receiving colonoscopy; (3) signing informed consent. Based on the Harvard Cancer Risk Index, the questionnaire was built on the consensus of more than 20 years of common cancer epidemiology in China. Through the consensus reached by the multidisciplinary expert panel discussion, a comprehensive evaluation system for cancer risk in China was designed. The high-risk group of colorectal cancer was determined by preliminary screening of the questionnaire, and a free colonoscopy was performed for the appointment to the gastrointestinal endoscopy department of the Yunnan Cancer Hospital. All polypoid lesions and ulcers found by colonoscopy must be biopsied to confirm the diagnosis. χ² test or Fisher exact probability method was used to compare the detection of colorectal cancer in 4 groups of 40-49 years old, 50-59 years old, 60-69 years old, and ≥70-years old. Detection of colonoscopy, compliance, pathological examination, pathological diagnosis, and morbidity of colorectal cancer were analyzed. Results: A total of 127 960 people from 40 to 74 years old of urban residents in Kunming city participated in the preliminary screening of the questionnaire, including 59 748 (46.7%) males and 68 212 females (53.3%) with mean age of (53.6±8.6) years old. The 40-49 years old group had the largest number of participants (48 044, 37.5%), followed by the groups of 50-59 years old (42 473, 33.2%), 60-69 years old (34 111, 26.7%), and ≥70 years old (3332, 2.6%). Till October 2017, a total of 14 971 people were screened as at high risk of colorectal cancer, with the high-risk detection rate of 11.7%, and the high-risk detection rate of women was significantly higher than that of men [13.4% (9 109/68 212) vs. 9.8% (5 862/59 748), χ²=386.947, P<0.001]. The highest high-risk detection rate was in the 50-59 years group in both gender [men: 11.1% (2202/19 831), women: 15.3% (3034/22 642)]. A total of 3449 people among the high-risk population received colonoscopy examination. The compliance rate of colonoscopy was 23.0% (3449/14 971), and the male compliance rate was 19.8% (1162/5862), which was significantly lower than that of females [25.1% (2287/9109), χ²=56.175, P<0.001]. The highest compliance was observed in the 50-59 years group [25.4% (1438/5668)], followed by 40-49 years and 60-69 year group [22.1%(1091/4931) and 22.0%(891/4048), respectively], and the compliance of ≥70 years old group was the lowest [9.0% (29/324)]. Colonoscopy examination revealed 606 cases with lesions, the detection rate of lesions was 17.6%, and the male detection rate was significantly higher than that of females [26.9% (313/1162) vs. 12.8% (293/2287), χ²=106.140, P<0.001]. The detection rate of lesions increased with age [40-49, 50-59, 60-69, ≥70: 10.9% (119/1091), 17.5% (252/1438), 25.0% (223/891) and 41.4% (12/29), respectively, χ²=79.010, P<0.001]. A total of 584 cases underwent endoscopic excision and pathological diagnosis, and 465 cases (13.5%) of precancerous lesions were detected. The prevalence of precancerous lesions in men was higher than that in women [21.3% (247/1162) vs. 9.5% (218/2287), χ²=90.801, P<0.001], the precancerous lesion detection rate increased with age [40-49, 50-59, 60-69, ≥70: 8.0% (87/1091), 14.3% (206/1438), 18.1% (161/891) and 37.9% (11/29); χ²=58.109, P<0.001]. A total of 4 patients with colorectal cancer were detected, including 3 males and 1 female. The detection rate of male colorectal cancer was 258.2/100 000, and the female was 43.7/100 000, whose difference was not statistically significant (χ²=1.488, P=0.223). There was no significant difference in the detection rate of colorectal cancer among 4 age groups [40-49, 50-59, 60-69, ≥70: 91.7/100 000 (1/1091), 69.5/100 000 (1/1438), 224.5/100 000 (2/891) and 0, respectively, P=0.696]. Conclusions Screening for colorectal cancer is an important measure to control the onset and death of colorectal cancer. Through the questionnaire risk assessment plus colonoscopy, two-step screening method can improve the screening efficiency and greatly reduce the screening cost.

Background: The H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China. Objectives: This study aimed to analyze the origin, reassortment, and mutations of the AIV isolates. Methods: AIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software. Results: Nine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016–2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds. Conclusions These results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

Background: The H5 avian influenza viruses (AIVs) of clade 2.3.4.4 circulate in wild and domestic birds worldwide. In 2017, nine strains of H5N6 AIVs were isolated from aquatic poultry in Xinjiang, Northwest China. Objectives: This study aimed to analyze the origin, reassortment, and mutations of the AIV isolates. Methods: AIVs were isolated from oropharyngeal and cloacal swabs of poultry. Identification was accomplished by inoculating isolates into embryonated chicken eggs and performing hemagglutination tests and reverse transcription polymerase chain reaction (RT-PCR). The viral genomes were amplified with RT-PCR and then sequenced. The sequence alignment, phylogenetic, and molecular characteristic analyses were performed by using bioinformatic software. Results: Nine isolates originated from the same ancestor. The viral HA gene belonged to clade 2.3.4.4B, while the NA gene had a close phylogenetic relationship with the 2.3.4.4C H5N6 highly pathogenic avian influenza viruses (HPAIVs) isolated from shoveler ducks in Ningxia in 2015. The NP gene was grouped into an independent subcluster within the 2.3.4.4B H5N8 AIVs, and the remaining six genes all had close phylogenetic relationships with the 2.3.4.4B H5N8 HPAIVs isolated from the wild birds in China, Egypt, Uganda, Cameroon, and India in 2016–2017, Multiple basic amino acid residues associated with HPAIVs were located adjacent to the cleavage site of the HA protein. The nine isolates comprised reassortant 2.3.4.4B HPAIVs originating from 2.3.4.4B H5N8 and 2.3.4.4C H5N6 viruses in wild birds. Conclusions These results suggest that the Northern Tianshan Mountain wetlands in Xinjiang may have a key role in AIVs disseminating from Central China to the Eurasian continent and East African.

Objective:To analyze the complete genome sequence and phylogenetic structure of a wild bird-derived H1N1 avian influenza virus (AIV) in the northern Tianshan Mountain.Methods:In November 2018, 320 samples of fresh wild bird feces were collected from several reservoirs in the middle part of northern Tianshan Mountain. Chicken embryo inoculation test, hemagglutination inhibition test and RT-PCR with PB1 universal primer were used to isolate and identify AIVs. Eight fragments of the viral genome were amplified with the universal primers of influenza A virus and the whole viral genome was sequenced. Pairwise sequence alignments and analysis of phylogenetic and molecular characteristics were performed by BLAST, Clustal W, MEGA7.0 and MegAlign software. Results:Influenza viruses were isolated and identified from six samples of wild bird feces with a positive rate of 1.88%. One of them was H1N1 AIV, named A/wild bird/Xinjiang/010/2018 (H1N1) (XJ-H1N1). The eight gene segments of XJ-H1N1 were all derived from AIVs isolated from wild ducks of Anseriformes. The surface genes of HA and NA were Eurasian lineages and derived from H1N1 isolated from Mongolian Anas platyrhynchos and H3N1 isolated from Bangladesh wild duck, respectively. The six internal genes were derived from H6N8 isolated from Anas strepera in Siberia, H7N3 isolated from Anas clypeata and teal in Egypt, and H7N5 isolated from wild birds such as Anas platyrhynchos in the Netherlands. The HA cleavage site of XJ-H1N1 contained only one basic amino acid, suggesting that it was a low pathogenic AIV. Amino acids at positions 190 and 225 of HA receptor binding sites were E and G (H3 count), which could bind both α2, 3 galactoside sialic acid (SAα2, 3Gal) and SAα2, 6Gal receptors. T200A and E227A mutations in HA amino acid sequences and P42S mutation in NS1 amino acid sequences could all enhance the replication ability and pathogenicity of the virus in mammalian cells. Conclusions:A low pathogenic H1N1 AIV, XJ-H1N1, was isolated from wild birds in the northern Tianshan Mountain, resulting from multiple reassortments of AIVs carried by migrating wild ducks. The replication capacity and pathogenicity of XJ-H1N1 in mammalian cells might be enhanced. Moreover, the virus could bind both SA 2-3gal and SA 2-6gal receptors.

Objective:To analyze the genetic evolution and molecular characteristics of H5N8 avian influenza viruses (AIVs) isolated from the poultry in a live poultry market (LPM) in Urumqi, Xinjiang.Methods:Oropharyngeal and cloacal swabs of poultry were collected from a LPM in Urumqi in 2016. AIVs were isolated by inoculating swab samples into chicken embryos. Hemagglutination test and RT-PCR were used to identify the AIVs. The genes of isolated AIVs were amplified with the universal primers of AIV and whole-genome sequencing was also performed. Pairwise sequence alignment and analysis of phylogenetic and molecular characteristics were performed using BLAST, Clustal W, MEGA-X and DNAStar software.Results:Five H5N8 AIVs were isolated from poultry. These strains shared a nucleotide identity of 99.70%-100.00%, which indicated that they were from the same source, and were named XJ-H5N8/2016. Phylogenetic analysis based on hemagglutinin( HA), NS and PB2 genes showed that these isolates were clustered together with H5N8 AIVs isolated from the migratory swans in Hubei, Shanxi and Sanmenxia, and the ducks in India during 2016 to 2017. Moreover, they were also clustered together with H5N6 AIVs isolated from minks in China and the first case of human infection in Fujian. The phylogenetic tree of neuraminidase( NA) gene indicated the five isolates clustered together with H5N8 AIVs isolated from ducks in India in 2016, and the phylogenetic trees of PB1, MP, PA and NP genes showed that they were clustered together with H5N8 AIVs isolated from wild birds and poultry in Egypt, Cameroon, Uganda, Congo and other African countries in 2017. The HA cleavage sites of XJ-H5N8/2016 contained five consecutive basic amino acids, indicating high pathogenicity. Multiple mutations in the genes of XJ-H5N8/2016 could enhance its virulence and pathogenicity to mammals. Conclusions:The five strains of H5N8 AIVs isolated from the LPM were highly pathogenic and closely related to the H5N8 AIVs isolated from migratory birds and poultry in Hubei, Shanxi, Sanmenxia area, Africa and India during 2016 to 2017. Meanwhile, some of the viral genes were also closely related to the H5N6 AIVs isolated from the minks and human in China. Multiple mutations could increase the virulence and pathogenicity of AIVs to mammals, which could pose a potential threat to public health.