Genomic Epidemiology of Imported Cases of COVID-19 in Guangdong Province, China, October 2020 - May 2021 / 生物医学与环境科学（英文）

Objective@#The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been engendering enormous hazards to the world. We obtained the complete genome sequences of SARS-CoV-2 from imported cases admitted to the Guangzhou Eighth People's Hospital, which was appointed by the Guangdong provincial government to treat coronavirus disease 2019 (COVID-19). The SARS-CoV-2 diversity was analyzed, and the mutation characteristics, time, and regional trend of variant emergence were evaluated.@*Methods@#In total, 177 throat swab samples were obtained from COVID-19 patients (from October 2020 to May 2021). High-throughput sequencing technology was used to detect the viral sequences of patients infected with SARS-CoV-2. Phylogenetic and molecular evolutionary analyses were used to evaluate the mutation characteristics and the time and regional trends of variants.@*Results@#We observed that the imported cases mainly occurred after January 2021, peaking in May 2021, with the highest proportion observed from cases originating from the United States. The main lineages were found in Europe, Africa, and North America, and B.1.1.7 and B.1.351 were the two major sublineages. Sublineage B.1.618 was the Asian lineage (Indian) found in this study, and B.1.1.228 was not included in the lineage list of the Pangolin web. A reasonably high homology was observed among all samples. The total frequency of mutations showed that the open reading frame 1a (ORF1a) protein had the highest mutation density at the nucleotide level, and the D614G mutation in the spike protein was the commonest at the amino acid level. Most importantly, we identified some amino acid mutations in positions S, ORF7b, and ORF9b, and they have neither been reported on the Global Initiative of Sharing All Influenza Data nor published in PubMed among all missense mutations.@*Conclusion@#These results suggested the diversity of lineages and sublineages and the high homology at the amino acid level among imported cases infected with SARS-CoV-2 in Guangdong Province, China.

Analysis of death causes of 345 cases with HIV/AIDS in Guangdong area / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the death causes of 345 cases with HIV/AIDS in Guangdong area.</p><p><b>METHODS</b>The situations of 345 hospitalized death cases with HIV/AIDS were conducted by retrospective analysis.</p><p><b>RESULTS</b>(1)There were total 3406 hospitalized cases with HIV/AIDS in a hospital from January 2001 to December 2011 and 345 cases died, the fatality rate was 10. 13%. Since 2005 the introduction of free anti-viral treatment, the fatality rate of HIV/AIDS declined. The fatality rate of the patients whose CD4+ T lymphocyte counts <200 cells/microl was 14.61% (299/2046) and it was significantly higher than that of patients whose CD4 T lymphocyte counts >or=200 cells/microl (P <0.01). (2) 99.42% of the death cases had more than one kind of opportunistic infections (OI) and there were 924 cases of OI totally. 84. 64% of OI related to the death directly. Fungal infection was the most common in OI, followed by bacterial infection. Most OI occurred in the lungs, mouth, other systemic disseminated diseases, gastrointestine, central nerver system, septicemia, skin. The AIDS defining opportunistic infections such as several pneumonia, disseminated penicilliosis marneffei and CNS infections accounted for 29.65%. Other factors that caused HIV/AIDS death included opportunistic tumors, HIV related disease and non AIDS-related disease accounted for 15.36%. No accepted effective highly active antiretroviral therapy (HARRT) also constituted factors of death. Among cases which accepted HARRT treatment, only 6.96% had the period of treatment over three months.</p><p><b>CONCLUSION</b>The fatality rate of end-stage AIDS patients was high and the opportunistic infections was the most important cause of death. Early diagnosis and treatment for opportunistic infections, timely effective HARRT were the key to improve the quality of life of AIDS patients.</p>

Hepatitis B virus (HBV) mutation in enhancer I (HBV Enh I)/X-promoter and the relationship between chronic HBV-related disease spectrum / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the hepatitis B virus (HBV) mutation in the Enhancer I (HBV Enh I)/X-promoter and to analysis the relationship between chronic HBV-related disease spectrum.</p><p><b>METHODS</b>275 patients were enrolled in this study, including 100 cases of chronic hepatitis B (CHB), 74 cases of liver cirrhosis (LC), 101 cases of hepatocellular carcinoma (HCC), grouping by different HBV genotypes, using semi-nested PCR amplification of HBV Enh I/X-promoter and sequencing DNA, the mutations were determined by alignment to HBV reference sequence, the data was compared by chi2 test and analyzed by multivariate logistic regression.</p><p><b>RESULTS</b>(1) Genotyping results: 61.48% (158/257) were infected with HBV genotype B, including 70 cases of CHB, 36 cases of LC and 52 cases of HCC; 38.52% (117/257) were infected with HBV genotype C, including 30 cases of CHB, 38 cases of LC and 49 cases of HCC. (2) In the patients were infected with HBV genotype B, A1123Y mutation in LC was significantly higher than in CHB (30.56% vs. 8.58%, chi2 = 8.533, P = 0.005, A = 4.693, 95% CI [1.567-14.056]), HCC was significantly higher than in CHB (28.85% vs. 8.58%, chi2 = 8.607, P = 0.003, A = 4.324,95% CI [1.544-2.109]); A1317G mutation in HCC was significantly higher than in CHB (30.77% vs. 7.14%, chi2 = 11.687, P = 0.001, A = 5.778, 95% CI [1.955-17.076]). In the patients were infected with HBV genotype C, T1323C mutation in HCC was significantly higher than in CHB (30.61% vs. 6.67%, chi2 = 6.318, P = 0.12, A = 6.176, 95% CI [1.301-29.331]). (3) Multivariate regression analyses showed that A1317G (OR = 5.706, 95% CI [1.770-18.837], P = 0.004) and T1323C (A = 5.810, 95% CI [1.114-30.306], P = 0.037) mutation were risk factors for HCC.</p><p><b>CONCLUSION</b>HBV Enh I/X-promoter mutations were associated with the development of LC and HCC, the mutations can help to predict the occurrence of LC and HCC.</p>

Inhibitory effect of anluohuaxianwan on experimental hepatic fibrosis in rats / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effects of anluohuaqianwan on experimental hepatic fibrosis induced by dimethyl nitrosamine (DMN) in rats.</p><p><b>METHODS</b>36 male SD rats were randomly dividied into three groups: model group, normal group, anluohuaqianwan group. The rats in the three groups were treated with DMN daily for 4 weeks. The liver function was detected using auto biochemistry analyzer, the serum HA, LN, IV-C, PIIIP were detected by immunoradiometry, the histopathology was observed in the left liver lobe after HE staining, the expression of matrix metalloproteinase-2 (MMP-2) in liver tissue were detected by immunohistochemistry.</p><p><b>RESULTS</b>The serum levels of ALT, AST, ALP, TP, ALB and the contents of HA, LN, IV-C in model group were significantly increased compared to these in the normal group (P less than 0.01). The serum levels of ALT, AST and the contents of HA in anluohuaqianwan group were significantly lower than those in the model group (P less than 0.01). The liver MMP-2 in the model group was significantly increased compared to that in the normal group (P less than 0.05). The expression of MMP-2 in liver tissue of model group was lower than that in the anluohuaqianwan group (P less than 0.05).</p><p><b>CONCLUSION</b>Anluohuaqianwan can inhibit liver fibrosis in rats induced by DMN.</p>

The infection of dendritic cells by recombinant adenoviral vector carrying HBsAg-HSP70 chimeric gene and its biological characteristics observations / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To test the infeciton efficiency of recombinant adenoviral vector carrying HBsAg-HSP70 chimeric gene and to abserve its biological characteristics.</p><p><b>METHODS</b>Peripheral blood mononuclear cells (PBMC) were separated from healthy blood donor and they were infected by Ad-HSP70-HBsAg on the first day of isolation. DCs were induced in medium with cytokines IL-4, GM-CSF and TNF-alpha in vitro. The biological characteristics of DC induced were analyzed by inverted fluorecent microscope, RT-PCR, flow cytometer (FACS), and mixed lymphocyte reaction (MLR).</p><p><b>RESULTS</b>The traced gene-GFP were abserved in DCs by inverted fluorecent microscope and HSP70-HBsAg gene mRNA expression was detected by RT-PCR after the Ad-HSP70-HBsAg infection. FACS analysis shown that the expression of CD1a, CD80, CD86 and HLA-DR on surfece of two groups of DCs were similar. MLR showed that there are not a statitic difference of stimulated index (SI) between two groups.</p><p><b>CONCLUSION</b>Results indicated that Ad-HSP70-HBsAg can effectively infected DCs without affecting its biological characteristics.</p>

Case-controlled study of entecavir treatment for chronic severe hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the efficacy and safety of entecavir (ETV) treatment for chronic severe hepatitis B.</p><p><b>METHODS</b>78 patients with chronic severe hepatitis B and positive HBV DNA were divided into ETV group and control group, each group had 39 patients. ETV group was given the same conventional therapy as control group, and was treated with ETV. The change of liver function, PTA, HBV DNA level were observed, and adverse events were recorded. The effective rate of treatment between ETV group and control group, the baseline characteristics between the effective cases and non-responsive cases after ETV treatment were compared at week 12.</p><p><b>RESULTS</b>The baseline characteristics were well balanced between ETV group and control group. The effective rate of ETV group was 56.41% versus 33.33% of control group at week 12 (P = 0.0405). The effective rate of ETV group was higher than that of control group, in the early stage of chronic severe hepatitis B (P = 0.0275), but there was no statistically significant in the middle or late stage (P = 0.4687). The comparison result of baseline characteristics between the effective and non-responsive cases after ETV treatment showed: there were statistically different in age, bilirubin level, HBV DNA level and stage of the severe hepatitis, proportion of cirrhosis, but no statistically different in cholinesterase level, alpha-fetoprotein level and sex ratio, the proportion of ascites, positive HBeAg (P > 0.05). No serious adverse events occurred.</p><p><b>CONCLUSIONS</b>ETV improves the curative effect when used in the early stage of chronic severe hepatitis B, and may not in the middle and late stage. The curative effect of ETV may be affected by age, bilirubin level, HBV DNA level and stage of the severe hepatitis, cirrhosis. ETV has good security in the treatment for chronic severe hepatitis B.</p>

Construction of replication-deficient recombinant adenoviral vector carrying HBsAg and HSP70 chimeric gene and its expression in vitro / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To construct a recombinant adenoviral vector carrying HBcAg-HSP70 chimeric gene by homologous recombination in bacteria and to detect its expression in vitro.</p><p><b>METHODS</b>Heat shock protein 70 gene from Mycobacterium tuberculosis were amplified by PCR and were cloned to adenoviral shuttle plasmid pAdTrack-CMV-HBsAg. Then the resultant pAdTrack-CMV-HBsAg-HSP70 was cotransfected into BJ5183 bacteria with the plasmid pAdeasy-1. The adenoviral plasmid carrying HBsAg-HSP70 gene (pAd-HBsAg-HSP70) was generated with homologous recombination in bacteria and the adenoviruses were produced in 293 cells. Several kinds of mammal cells (293 cells and Vero cells) were infected with adenoviruses and the expression of HBsAg-HSP70 was detected by RT-PCR and ELISA in vitro.</p><p><b>RESULTS</b>The adenoviral plasmids pAd-HBsAg-HSP70 were obtained by selection for kanamycin resistance and confirmed by restriction endonuclease Pac analyses. The recombinant adenoviruses Ad-HBsAg-HSP70 were packaged successfully in 293 cells. The titer of Ad-HBsAg-HSP70 was up to 2 x 10(12) pfu/L after the second passage of proliferation in 293 cells. HBsAg and HSP70 were expressed efficiently in mammal cells after infection.</p><p><b>CONCLUSION</b>The recombinant adenoviruses expressing HBsAg and HSP70 were constructed successfully which can be used further in study of gene therapy for HBV.</p>

Induction of specific cytotoxic T lymphocyte response against hepatitis B virus by hepatitis B virus S gene-modified dendritic cells / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore whether hepatitis B virus (HBV) S gene-modified dendritic cells (DCs) might induce a specific cytotoxic T lymphocyte (CTL) response.</p><p><b>METHODS</b>The recombinant adenoviruses carrying HBsAg genes were prepared and used to transfect DCs generated from cord blood. The efficacy of transfection was observed through the expression of enhanced green fluorescent protein (EGFP) in DCs and the expression of HBsAg was detected by ELISA. HBV S gene-modified DCs were co-cultured with T cells from cord blood and T cells stimulating activities were detected using mixed lymphocyte reaction (MLR). The CTL assay was carried out to assess the ability of CTL lines to lyse target cells of HepG(2)22.1.5 by measuring lactate dehydrogenase (LDH) release.</p><p><b>RESULTS</b>The results showed that HBV S genes were expressed in DCs with high efficacy by recombinant adenoviral vector. DCs had a normal shape after transfection. The result of MLR showed that HBV S gene-modified DCs could effectively stimulate naive T cells to proliferate. The induced specific CTL lines could lyse target cells of HepG(2)22.1.5.</p><p><b>CONCLUSIONS</b>HBV S gene-modified DCs enhanced the function to induce a specific CTL effect, showing its promise for developing anti-viral vaccine in future.</p>

Diammonium glycyrrhizinate does not affect efficacy of adefovir dipivoxil therapy in patients with HBeAg-positive chronic hepatitis B / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate the effect of diammonium glycyrrhizinate on the antiviral therapy with adefovir dipivoxil (ADV) in patients with HBeAg-positive chronic hepatitis B.</p><p><b>METHODS</b>Patients with HBeAg-positive chronic hepatitis B enrolled in this study were randomized to receive either ADV 10 mg/d fir 48 weeks or placebo for 24 weeks followed by ADV 10 mg/d for 24 weeks. Antiviral activities of diammonium glycyrrhizinate administered during the trial were studied with respect to virological and serologic response, and ALT normalization.</p><p><b>RESULTS</b>Twenty-one of 142 patients in ADV group vs. 11 of 68 patients in placebo group were treated with diammonium glycyrrhizinate. There was no significant difference in virological, serological and biochemical responses between patients with or without diammonium glycyrrhizinate in both therapy groups. During double-blind period, virological response was significantly worse in patients only receiving diammonium glycyrrhizinate than those combined with ADV.</p><p><b>CONCLUSION</b>Diammonium glycyrrhizinate had no antiviral activity and exerts no influence on the efficacy of ADV treatment in patients with HBeAg-positive chronic hepatitis B.</p>

Efficacy of adefovir dipivoxil was not related to genotypes B and C of hepatitis B virus: a randomized, double-blind, multicenter clinical study / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To determine the relationship between the response to adefovir dipivoxil (ADV) treatment in patients with HBV genotypes B and C of HBeAg positive chronic hepatitis B.</p><p><b>METHODS</b>This clinical trial was a randomized, double-blind, placebo-controlled, multicenter study. A total of 226 eligible patients with HBeAg positive chronic hepatitis B were randomized (in a ratio of 2:1) receiving ADV 10 mg/d for 48 weeks (ADV+ADV group) or placebo for 24 weeks followed by ADV 10 mg/d for 24 weeks (PLB+ADV group). The primary efficacy was virologic response. The genotypes of HBV were determined by PCR-restricted fragment length polymorphism (RFLP) method using serum samples before therapy. rtN236T and rtA181V mutations were confirmed by sequencing.</p><p><b>RESULTS</b>In this study, HBV genotype C was 66.7%, genotype B was 25.2%. Genotype B was more common in Guangzhou. Patients with genotype B were much younger than those with the genotype C. Patients with genotype B previously received less anti-HBV therapy. There were no significant difference in virological response (including mean reduction in HBV DNA level from baseline, serum HBV DNA load after treatment and HBV DNA undetectable rate) and serological response (the rate of HBeAg loss and HBeAg seroconversion) between patients infected with genotypes B and C in both treatment arms.</p><p><b>CONCLUSION</b>There were no significant difference in virological and serological response to ADV therapy between patients infected with HBV genotype B and C.</p>

Expression of HBV preS2/S gene in mammalian cells transferred with adenoviral vector / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study HBV preS2/S gene expression effects in mammalian cells transferred with recombinant adenoviral vector.</p><p><b>METHODS</b>The replication-deficient recombinant adenoviral vector (Ad-HBs) carrying HBV preS2/S gene were constructed by homologous recombination in bacteria. The 293 cells, Vero cells, HepG2 cells and mesenchymal stem cells (MSCs) were infected with adenoviruses. The expressions of enhanced green fluorescent protein (EGFP) were observed with fluorescence microscope and the expressions of HBsAg were detected by RT-PCR and ELISA in vitro.</p><p><b>RESULTS</b>More than 90% of 293 cells, Vero cells, HepG2 cells or MSCs expressed EGFP after transfection at the MOI of 20 and the titers of HBsAg were more than 3.229 (A value) in culture supernatant.</p><p><b>CONCLUSION</b>The HBV preS2/S gene was not only expressed efficiently in immortalized cells, but also expressed efficiently in stem cells with the recombinant adenoviruses vector.</p>

Clinical trial of sequential antiviral therapy for patients with chronic hepatitis B in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To establish a sequential antiviral regime and evaluate its efficacy in patients with chronic hepatitis B using a controlled trial.</p><p><b>METHODS</b>Seventy-four patients with chronic hepatitis B were divided into 3 groups: 30 cases were enrolled in the sequential antiviral group in which patients received eight-week treatment with thymosin alpha1 (1.6 mg/time, subcutaneous injection, 2 times/week), six-month treatment with interferon (500 MU/ times, muscle inject, every other day) begun in the fifth week of the therapeutic course, and lamivudine treatment (100 mg/days) begun 2 months later after HBeAg seroconversion or just after the withdrawal of interferon to more than eighteen months. Fourteen cases were enrolled in combination group in which patients received six-month treatment with interferon and thymosin alpha1 simultaneously in the same manner as in sequential antiviral group. Thirty cases were enrolled in lamivudine group in which patients received more than eighteen-month treatment with lamivudine.</p><p><b>RESULTS</b>The temporary rates of HBeAg seroconversion and normalization of alanine aminotransferase (effective rate) in sequential antiviral group, combination group and lamivudine group were 76.7%, 78.6% and 13.3%, respectively. The effective rates of sequential group and combination group were very similar, and significantly higher than that of lamivudine group (P less than 0.01). Long-term efficacy rates were 76.7%, 57.1% and 16.7% among the three groups, respectively. The long-term effective rate of sequential group was relatively higher. The rate of liver damage sensitive period in sequential antiviral group and combination group was 47.7%. The time of onset was from 2 to 8 weeks after the treatment begun, earlier than that from 6 to 8 weeks after the beginning of interferon alone in the literature.</p><p><b>CONCLUSION</b>Sequential antiviral therapy had much higher rates of long-term HBeAg seroconversion, undetectable HBV DNA and normalization of alanine aminotransferase with good cost-effectiveness. Its mechanism to promote the antiviral effect might be dependent on the immunoregulatory action of thymosin alpha1 in the earlier period and the specific inhibition of HBV DNA replication by lamivudine in the later period of the therapeutic course.</p>

Initiative factors of the damage sensitive stage of hepatocytes induced by interferon in patients with chronic hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To probe into the initiative factors of the damage sensitive stage of hepatocytes induced by interferon in patients with chronic hepatitis B (CHB).</p><p><b>METHODS</b>Forty-four CHB patients with positive HBeAg and HBV DNA were treated with interferon. Serum ALT and viral markers levels of HBsAg, HBeAg, anti-HBc and HBV DNA were examined regularly. Liver biopsy was carried out just before the treatment.</p><p><b>RESULTS</b>The rate of HBeAg seroconversion was 75% at the sixth month, and 68.2% after one year of follow up. The rate of damage sensitive stage of hepatocytes was 47.7%. The average onset time was (3.14+-1.49) weeks after the treatment, and lasted for (8.24+-3.52) weeks. The ALT level raised (1.73+-1.13) times. The occurrence of damage sensitive stage of hepatocytes was indicator for good curative effect (Fisher exact probability, P=0.028). Damage sensitive stage of hepatocytes was more often developed in patients with moderate inflammation, overexpression of HBcAg in liver and higher level of HBeAg in blood stream before treatment. HBeAg and anti-HBc levels in peripheral blood decreased in the onset period of damage sensitive stage of hepatocytes.</p><p><b>CONCLUSIONS</b>The initiative factors of the damage sensitive stage of hepatocytes may be: HBeAg decreasing in peripheral blood induced by interferon may dismiss immune lutation of HBeAg and anti-HBc to cytotoxic T lymphocyte (CTL), which recognize HBcAg as target, thus activates the cytotoxicity of HBV-infected hepatocytes mediated by CTL.</p>