企业“病毒”及其“免疫”机制研究

企业"病毒"对企业生产经营活动造成影响和破坏,而企业组织免疫系统则有助于预防和消灭企业"病毒",使企业不断适应环境的变化。本文借鉴免疫学和组织免疫系统的研究成果,从企业"病毒"的传染特性角度,对企业"病毒"的类型进行了分类,系统分析了企业"病毒"的传染机制和传染方式,最后,从非特异性免疫和特异性免疫两个角度,讨论了企业实施"病毒"免疫的路径。

Survey of HIV Infection among Injection Drug Users in Guangdong, China

Objective： To understand the prevalence and behavioral risk factors of HIV infection among injection drug users in the Pearl River Delta Region （PRDR） of Guangdong province, and to provide evidence for establishing effective intervention strategies. Methods： Face to face interviews were conducted and serum samples from injection drug users from detoxification centers and the community were collected for HIV screening. Results： 655 drug users were recruited and interviewed. The HIV seropositive rate was 29.0%. 99.5 % of subjects were injection drug users （IDUs）, of whom,75.4% reported sharing injection equipment. Conclusion： HIV prevalence among injection drug users is high in the PRDR of Guangdong. Injection drug use is the principal behavioral risk factor for HIV transmission. Pragmatic harm reduction programs should be implemented to prevent the spread of HIV infection.

Construction of Recombinant Adenovirus Vector Containing CEVB2L Gene

[Objective] Sheep contagious ecthyma virus B2L gene recombinant adenovirus was built by adenovirus vector system.[Method] Genome DNA extracted from sheep contagious ecthyma virus strain JLSY04 as a template,Gene fragments obtained from B2L by PCR amplification;B2L gene cloning was cloned into PDNR-CMD vector,screening positive clones and plasmid CTC572-6 was obtained;CTC572-6 plasmid for homologous was recombined with the adenoviral vector.Screening positive clones and bacilli PCR,digestion and sequencing and so on were identified.[Result] After identified by enzyme digestion and gene sequencing,recombinant adenovirus vector CTC572Ade-30 of carrying sheep contagious ecthyma virus B2L gene was constructed successfully.[Conclusion] Which laid the foundation for sheep contagious ecthyma genetically engineered vaccine.

Genetic Analysis of the VP2 Hypervariable Region of Thirty-six Infectious Bursal Disease Virus Isolates in China during 2009-2012

[Objective] Infectious bursal disease （IBD） is a highly contagious immuno- suppressive disease caused by infectious bursal disease virus （IBDV）. IBDV is ge- netically prone to mutation, which results in challenges to the disease prevention and control. Thus, it is necessary to continuously monitor the prevalence of IBDV. [Method] 36 IBDVs were identified from ten provinces in China from 2009 to 2012. Partial fragments of VP2, including the hypervariable region （HVR）, from new iso- lates were sequenced and analyzed through comparisons with published sequences of IBDV, including 18 strains isolated previously by our lab and 24 reference strains from China and around the world. [Result] Phylogenetic analysis showed a co-exis- tence of IBDV strains belonging to classic, variant, attenuated, and very virulent IB- DV （wlBDV） in China. wlBDVs remain the predominant strains in China and the new subgroup was emerging. Alignment analysis revealed several distinct amino acid mutations that might be involved in virulence or antigenicity variation. [Conclu- sion] The results offered evolutionary clues showing the emerging trend of obvious variations and diversity of IBDV in major poultry-producing regions of China particu- larly in recent years. These findings will contribute to a better understanding of the genetic evolution of IBDV.

Genomic Sequencing and Molecular Characteristics of A Very Virulent Strain of Infectious Bursal Disease Virus Isolated in China

[Objective] The paper was to determine the genomic sequence of a very virulent strain of infectious bursal disease virus（IBDV）,and study its molecular characteristics.[Method] A very virulent strain（vvIBDV）（HLJ-0504） of infectious bursal disease virus（IBDV） with special characters was isolated in China and its genome was sequenced.[Result] Sequence analysis showed that segment A of HLJ-0504 was derived from vvIBDV,while segment B was from a distinct ancestor.The morbidity and mortality of HLJ-0504 was 100% and 86.7%to SPF chickens,respectively.[Conclusion] vvIBDV with distinct segment B were still circulating and the evolution of IBDV was diversified in China.Besides,it is hard to imagine that the virulence of IBDV is determined solely by segment A or B.

Development of TaqMan-based Real-time PCR Assay for Detecting Transmissible Gastroenteritis Virus and Its Application in Vaccine Evaluation

[Objective] This study aimed to establish a TaqMan-based real-time PCR assay for detecting transmissible gastroenteritis virus （TGEV）. [Method] Primers and a probe were designed according to the conserved sequence of N gene in TGEV genome. After gradient dilution, the recombinant plasmid harboring the N gene was used as a standard for real-time PCR assay to establish the standard curve. [Re- sult] The results showed that the established real-time PCR assay exhibited a good linear relationship within the range of 102-10^10 copies/ul; the correlation coefficient was above 0.99 and the amplification efficiency ranged from 90% to 110%. The de- tection limit of real-time PCR assay for TGEV was 10 copies/μl, suggesting a high sensitivity; there was no cross reaction with other porcine viruses, indicating a good specificity; coefficients of variation within and among batches were lower than 3%, suggesting a good repeatability. The established real-time PCR method could be ap- plied in quantitative analysis and evaluation of the immune efficacy of TGEV vac- cines and detection of TGEV in clinical samples. [Conclusion] The TaqMan-based real-time PCR assay established in this study is highly sensitive and specific, which can provide technical means for the epidemiological survey of TGEV, development of TGEV vaccines and investigation of the pathogenesis of TGE.

Study on the Knockout and the Soluble Prokaryotic Expression of VP5 Protein Transmembrane Region of IBDV

[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.

Immune Effect of Combination of Universal Molecular Adjuvant and IBDV Subunit

In order to evaluate the immune effect of the protein expressed by the universal vector pET-mLTA-CTLA-4 plus IBDV subunit, the fusion protein mLTA-CTLA-4 was expressed and purified. Protein toxicity tests were carried out on rabbits.The VP2 gene of infectious bursal virus was amplified by RT-PCR, and lately used for pET-VP2 construction. Ten-day-old free healthy chickens were chosen for a grouped test, including the mLTA-CTLA-4（at different doses） plus VP2 groups, IBDV living vaccine group and control group. Serum and mucosal samples were collected regularly and the neutralization titers of IgG and IgA were assayed, while an animal protection test was conducted to determine the protection rate. The results showed that the protein m LTA-CTLA-4 was non-toxic and its protection rate was100%. IgG or IgA levels in the IBDV vaccine group were slightly higher than those in recombinant protein groups. These results indicated that the recombinant protein mLTA-CTLA-4 could be applied with IBDV subunit vaccine to protect chickens from infection.

Impact of parietal cell autoantibodies and non-organ-specific autoantibodies on the treatment outcome of patients with hepatitis C virus infection: A pilot study

AIM: Various side effects have been reported in patients infected with hepatitis C virus (HCV) who were treated with interferon-alpha (IFN-α), including the appearance or exacerbation of underlying autoimmune diseases and the development of a variety of organ and non-organ specific autoantibodies (NOSA). However, very few studies in adults have been strictly designed to address: whether the prevalence and the titre of organ and NOSA in serial samples of HCV-treated patients were affected by IFN-α, and the impact of these autoantibodies on the treatment outcome of HCV patients. METHODS: We investigated whether parietal cell autoantibodies (PCA) and/or NOSA were related with treatment-outcome in 57 HCV-treated patients (19 sustained-responders, 16 relapsers, 22 non-responders). Serum samples from patients were studied blindly at three time-points (entry, end of treatment and end of followup). For the detection of autoantibodies we used indirect immunofluorescence, commercial and in-house ELISAs. RESULTS: Sustained biochemical response was associated with ANA-negativity at the entry or end of follow up. Sustained virological response was associated with the absence of PCA at the entry. Combined virological and biochemical sustained response (CVBSR) was associated with the absence of antinuclear antibodies (ANA) at the end of follow up and PCA-negativity at the entry. Sustained virological and CVBSR were associated with a reduction of ANA and SMA titers during therapy. CONCLUSION: Although PCA and/or NOSA seropositivity should not affect the decision to treat HCV patients, the presence of some of them such as ANA, PCA and SMA before treatment or their increase during therapy with IFN- a may predict a worse response, indicating the need for a closer monitoring during treatment of HCV patients positive for these autoantibodies.

Detection of HBsAg, HBcAg, and HBV DNA in ovarian tissues from patients with HBV infection

AIM: To investigate the presence of HBsAg, HBcAg, and HBV DNA in ovarian tissues from patients with HBV infection.METHODS: HBsAg and HBcAg were examined in ovarian biopsy tissues from 26 patients with HBV infection by immunocytochemistry, and HBV DNA was detected in ovarian tissues by PCR.RESULTS: HBsAg and HBcAg were present with the same positive rate of 34.6% (9/26). The total positive rate was 46.2% (12/26). HBsAg and HBcAg were positive in 6 (23.1%) of the 26 patients. Brown positive particles were diffusely distributed in ovarian cells. The positive rate of HBV DNA was 58.3% (7/12).CONCLUSION: HBsAg, HBcAg, and HBV DNA can be detected in ovarian tissues from patients with HBV infection. The presence of HBsAg and HBcAg in ovarian tissues does not correlate with the HBV markers in serum.

Relationship of hepatitis B virus infection of placental barrier and hepatitis B virus intra-uterine transmission mechanism

AIM: To explore the mechanism of intra-uterine transmission,the HBV infection status of placental tissue and in vitro cultured placental trophoblastic cells was tested through in vivo and in vitro experiments. METHODS: A variety of methods,such as ELISA,RT- PCR,IHC staining and immunofluorescent staining were employed to test the HBV marker positive pregnant women's placenta and in vitro cultured placental trophoblastic cells. RESULTS: The HBV DNA levels in pregnant women's serum and fetal cord blood were correlated. For those cord blood samples positive for HBV DNA,their maternal blood levels of HBV DNA were at a high level. The HBsAg IHC staining positive cells could be seen in the placental tissues and the presence of HBV DNA detected. After co-incubating the trophoblastic cells and HBV DNA positive serum in vitro,the expressions of both HBsAg and HBV DNA could be detected. CONCLUSION: The mechanism of HBV intra-uterine infection may be due to that HBV breaches the placental barrier and infects the fetus.

Screening of Chinese Herbal Medicines Resistant to Chicken Escherichia coli and Infectious Laryngotracheitis Virus

[Objective] This study aimed to screen Chinese herbal medicines resistant to Chicken Escherichia coli and infectious laryngotracheitis virus. [Methed] Conven- tional punch method, test tube method and plate dilution method were adopted for in vitro susceptibility test of chicken E, coil strains O5 and O8 using 13 kinds of Chi- nese herbal medicines including Sanguisorba officinalis, Coptis chinensis, Anemar- rhena asphodeloides, Strobilanthes cusia, Agastache rugosa, etc.; chicken embryo inoculation experiment was adopted to screen Chinese herbal medicines resistant to chicken infectious laryngotracheitis virus. [Result] Sanguisorba officinalis, Fructus mume, Rheum officinale, Coptis chinensis, Herba Taraxaci, Anemarrhena asphode- Ioides, Scutellaria baicalensis and Rhizoma Fagopyri Cymosi had ideal antibacterial effect against chicken E. coil strain O5; Sanguisorba officinalis, Fructus mume, Rheum officinale, Coptis chinensis, Herba taraxaci and Rhizoma Fagopyri Cymosi had ideal antibacterial effect against chicken E. coil strain 08; other Chinese herbal medicines showed relatively poor or no antibacterial effect. Results of chicken embryo inoculation experiment showed that nine kinds of Chinese herbal medicines showed relatively strong anti-lLTV effect, including Forsythia suspensa, Radix Isatidis, Fofium isatidis, Flos Ionicerae, Radix codonopsis, Radix astragali, Atractylodes, Radix gly- cyrrhizae, and Pericarpium granati. [Conclusion] The study laid the foundation for fur- ther development of Chinese herbal compound preparations to treat chicken cofibacil- Iosis, infectious laryngotracheitis and other bacterial, viral diseases.

PRODUCTION IN PICHIAPASTORISAND CHARACTERIZATION OF GENETIC ENGINEERED CHIMERIC HBV/HEV VIRUS-LIKE PARTICLES

Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichiapastorisunder the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualiza-tion. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoriswith an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immuno-genicity on surface-displayed HEnAg. Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Subfulminant hepatitis B after infliximab in Crohn's disease:Need for HBV-screening?

Infections are a major adverse effect during the treatment with anti-TNF-α. While exclusion of any bacterial infection and screening for tuberculosis are mandatory before initiating a therapy with anti-TNF- α-antibodies, there are no guidelines whether to screen for or how to deal with chronic viral infections such as hepatitis B. In this case report, we have described a patient with Crohn＇s disease who developed subfulminant hepatitis B after the fourth infusion of infliximab due to an unrecognized HBs-antigen carrier state. He recovered completely after lamivudine therapy was started, but this severe adverse event could have been prevented if screening for HBV and pre-emptive therapy with lamivudine would have been started prior to infliximab. We therefore strongly argue in favor of extended screening recommendations for infectious diseases including viral infections before considering a therapy with infliximab.

TT virus infection and pancreatic cancer: Relationship or accidental coexistence

TT virus (TTV) was first isolated in 1997 from the patient with acute post-transfusion hepatitis. This fact led to the conclusion that the virus was hepatotropic and could be one of the causative agents of acute hepatitis. Afterwards, however, the virus was found in other human tissues and serological studies revealed that it was widespread. Multiple tropisms of TTV and the fact of its high incidence in general population are considered to indicate no medical significance of TTV in human pathology. Here we present a report of two cases of TTV infection in patients who developed pancreas cancer. The patients were hospitalized at the Department of Infectious Diseases due to hepatitis of unknown origin. Since serological and virological markers of common primary and secondary hepatotropic viruses were negative, TTV-DNA was found in serum and was believed to be the only causative agent with probable hepatotropic action. The patients later developed pancreas cancer and they underwent operation. The relationship is difficult to confirm, however the cases we present should be treated as a preliminary report and a comment on the real role of TTV in human pathology.

Distribution of hepatitis B virus genotypes:Phylogenetic analysis and virological characteristics of Genotype C circulating among HBV carriers in Kolkata,Eastern India

AIM: To evaluate the genotype distribution of hepatitis B virus (HBV) in Eastern India and to clarify the phyloge- netic origin and virological characteristics of the recently identifi ed genotype C in this region. METHODS: Genotype determination, T1762/A1764 mutation in the basal core promoter (BCP) and A1896 mutation in the precore region of 230 subjects were de- termined by restriction fragment length polymorphism method (RFLP) and the result was confi rmed by direct sequencing. RESULTS: The predominant genotypes D (HBV/D) and A (HBV/A) were detected in 131/230 (57%) and 57/230 (25%) samples. In addition, genotype C (HBV/C) was detected in 42/230 (18%) isolates. Surface gene region was sequenced from 45 isolates (27 HBV/C, 9 HBV/A and 9 HBV/D). Phylogenetic analysis revealed that all of the HBV/C sequences clustered with South East Asian subgenotype (HBV/Cs). The sequence data showed re- markable similarity with a Thai strain (AF068756) (99.5% ± 0.4% nucleotide identities) in 90% of the genotype C strains analyzed. T1762/A1764 mutation in BCP re- gion, associated with high ALT was signifi cantly higher in HBeAg negative isolates than HBeAg positive isolates. Frequency of A1896 mutation leading to HBeAg negativ- ity was low.CONCLUSION: The present study reports the genotypic distribution and the characteristics of partial genome sequences of HBV/C isolates from Eastern India. Low genetic diversity and confi nement of HBV/C in Eastern India possibly indicate a recent, limited, spread in this region. Genotype C with T1762/A1764 mutation has been reported to increase the risk for hepatocellular car- cinoma; therefore genotype C carriers in Eastern India should be carefully monitored.

Modeling of Malicious Code Propagations in Internet of Things

Nowadays, the main communication object of Internet is human-human. But it is foreseeable that in the near future any object will have a unique identification and can be addressed and con- nected. The Internet will expand to the Internet of Things. IPv6 is the cornerstone of the Internet of Things. In this paper, we investigate a fast active worm, referred to as topological worm, which can propagate twice to more than three times faster tl^an a traditional scan-based worm. Topological worm spreads over AS-level network topology, making traditional epidemic models invalid for modeling the propagation of it. For this reason, we study topological worm propagation relying on simulations. First, we propose a new complex weighted network mod- el, which represents the real IPv6 AS-level network topology. And then, a new worm propagation model based on the weighted network model is constructed, which descries the topological worm propagation over AS-level network topology. The simulation results verify the topological worm model and demonstrate the effect of parameters on the propagation.

Human bocavirus: Current knowledge and future challenges

Human bocavirus(HBoV) is a parvovirus isolated about a decade ago and found worldwide in both respiratory samples, mainly from early life and children of 6-24 mo of age with acute respiratory infection, and in stool samples, from patients with gastroenteritis. Since then, other viruses related to the first HBoV isolate(HBoV 1), namely HBoV 2, HBoV 3 and HBoV 4, have been detected principally in human faeces. HBo Vs are small nonenveloped single-stranded DNA viruses of about 5300 nucleotides, consisting of three open reading frames encoding the first two the non-structural protein 1(NS1) and nuclear phosphoprotein(NP1) and the third the viral capsid proteins 1 and 2(VP1 and VP2). HBoV pathogenicity remains to be fully clarified mainly due to the lack of animal models for the difficulties in replicating the virus in in vitro cell cultures, and the fact that HBo V infection is frequently accompanied by at least another viral and/or bacterial respiratory and/or gastroenteric pathogen infection. Current diagnostic methods to support HBoV detection include polymerase chain reaction, real-time PCR, enzymelinked immunosorbent assay and enzyme immunoassay using recombinant VP2 or virus-like particle capsid proteins, although sequence-independent amplification techniques combined with next-generation sequencing platforms promise rapid and simultaneous detection of the pathogens in the future. This review presents the current knowledge on HBoV genotypes with emphasis on taxonomy, phylogenetic relationship and genomic analysis, biology, epidemiology, pathogenesis and diagnostic methods. The emerging discussion on HBoV s as true pathogen or innocent bystander is also emphasized.

Comparative Studies on Detection of Antibodies against Infectious Bursal Disease Virus with Test Strips and Agar Gel Immunodiffusion Method

[Objective] This study aimed to compare the detection results of antibodies against infectious bursal disease virus with test strips and agar gel immunodiffusion method. [Method] Antibodies against infectious bursal disease virus in chicken serum were detected using test strips developed in our laboratory, and the results were comparad^with that using traditional agar diffusion method. [Result] The comparative study of the two methods showed that the sensitivity of test strips was eight times over agar gel immunodiffusion; test strips showed higher detection rate in the deter- mination test of 216 clinical samples, with high specificity, easy preservation, and simple and rapid operation, thereby being more suitable for the monitoring of clinical antibodies. [Conclusion] Test strips could replace the existing serological methods, having great promotion and application value in antibody monitoring.

Occult persistence and lymphotropism of hepatitis C virus infection

Recent discovery of occult hepatitis C virus (HCV) infection persisting after spontaneous or antiviral therapy-induced resolution of hepatitis C was made possible by the introduction of nucleic acid amplification assays capable of detecting HCV RNA at sensitivities superseding those offered by clinical tests. Although individuals with this seemingly silent HCV infection are usually anti-HCV antibody reactive and have normal liver function tests, occult HCV infection has also been reported in anti-HCV-negative individuals with persistently elevated liver enzymes of unknown etiology. Studies have shown that HCV RNA can persist for years in serum, lymphomononuclear cells and liver in the absence of clinical symptoms, although histological evidence of a mild inflammatory liver injury can be occasionally encountered. Furthermore, while HCV RNA can be detected in circulating lymphoid cells in approximately 30% of cases, a short-term culture under stimulatory conditions augments HCV replication in these cells allowing detection of virus in otherwise HCV-negative cases. HCV infects different immune cell subsets, including CD4+ and CD8+ T lymphocytes, B cells and monocytes. Studies employing clonal sequencing and single-stranded conformational polymorphism analyses have revealed unique HCV variants residing in immune cells, further strengthening the notion of HCV lymphotropism. Overall, the data accumulated suggest that occult HCV infection is a common consequence of resolution of symptomatic hepatitis C and that examination of the cells of the immune system is an effective approach to diagnosis of HCV infection and its long-term persistence. Further work is required to fully realize pathogenic and epidemiological consequences of occult HCV persistence.