重组H9N2禽流感病毒的构建及其在不同细胞中的复制特性

目的利用反向遗传学技术构建具有感染性的H9N2亚型禽流感病毒,并揭示其在不同细胞中的复制特性。方法以A/Quail/Hong Kong/G1/97(H9N2)禽流感病毒基因组RNA为模板,用RT-PCR技术获得该病毒的8条完整基因片段,并分别将它们插入到p HW2000载体上,最终在293T细胞中包装产生重组H9N2病毒。并将重组的H9N2病毒感染不同细胞,观察病毒在不同细胞上的增殖状况来揭示该病毒在不同细胞上的复制特性。结果应用流感病毒8质粒反向遗传学技术,成功获得重组病毒,并揭示了该毒株在不同细胞(A549,MDCK,Vero)中的复制特性,发现A549人肺腺癌细胞更适宜H9N2病毒的正常复制。此外还分析总结了该毒株的与病毒毒力相关的重要位点特征。结论成功建立了H9N2禽流感病毒的反向遗传系统,该系统的建立为在分子水平研究H9N2病毒以及制备H9N2禽流感疫苗提供了依据。     

Karyotype analysis of the acute fibrosarcoma from chickens infected with subgroup J avian leukosis virus associated with v-src oncogene

To understand the cytogenetic characteristics of acute fibrosarcoma in chickens infected with the subgroup J avian leukosis virus associated with the v-src oncogene, we performed a karyotype analysis of fibrosarcoma cell cultures. Twenty-nine of 50 qualified cell culture spreads demonstrated polyploidy of some macrochromosomes, 21 of which were trisomic for chromosome 7, and others were trisomic for chromosomes 3, 4, 5 (sex chromosome w), and 10. In addition, one of them was trisomic for both chromosome 7 and the sex chromosome 5 (w). In contrast, no aneuploidy was found for 10 macrochromosomes of 12 spreads of normal chicken embryo fibroblast cells, although aneuploidy for some microchromosomes was demonstrated in five of the 12 spreads. The cytogenetic mosaicism or polymorphism of the aneuploidy in the acute fibrosarcoma described in this study suggests that the analysed cells are polyclonal.     

Comparison of viral replication and IFN response in alpaca and bovine cells following bovine viral diarrhea virus infection

Alpacas develop diminished disease following bovine viral diarrhea virus (BVDV) infection compared to cattle. We hypothesized that alpaca and bovine cells have differential permissiveness and responses to BVDV infection. To characterize alpaca testicular (AT) and bovine turbinate (BT) cells BVDV infection permissiveness, viral replication and interferon (IFN) synthesis was evaluated. BVDV replicated 3-4 logs lower in AT cells with diminished antigen deposition compared to BT cells. BVDV infection inhibited IFN response in both AT and BT cells. Compared to BT cells, BVDV-infected AT cells had a 2-5 fold increase in IFN synthesis following dsRNA stimulation. The greater IFN response of AT cells compared to BT cells following poly I:C stimulation with or without ncp BVDV infection, may be the basis for the decreased BVDV permissiveness of AT cells and may contribute to the clinical differences following BVDV infection of alpacas and cattle.     

胞外高迁移率组蛋白1对HTLV-1感染的T细胞中病毒复制的影响

目的 探讨胞外的高迁移率组蛋白1(high mobility group box 1,HMGB1)对人T淋巴细胞白血病病毒1型(human T-cell leuk HTLV-1)感染的T细胞中病毒复制的影响.方法 ELISA检测HTLV-1病毒阴性T细胞系Jurkat、MOLT4细胞及HTLV-1病毒阳性的T细胞系MT2、MT4细胞培养上清中的HMGB1水平;将HTLV-1的长末端重复序列荧光素酶报告基因pHTLV-1-LTR-luc转染病毒阳性细胞MT2,随后加入终浓度为0.25、0.50、0.75 μg/ml的HMGB1多克隆抗体(PcAb)及其同型对照抗体,30、100、300 ng/ml的重组人HMGB1蛋白(rhHMGB1)及其对照PBS,48 h后检测荧光素酶活性;在病毒阳性细胞MT2中加入终浓度为0.25μg/ml的HMGB1 PcAb及同型对照抗体,300ng/ml的rhHMGB1及对照PBS,real-time PCR检测病毒编码基因tax、pol1、pol2、p19、gag、env.结果 HTLV-1病毒阴性T细胞系和HTLV-1病毒阳性细胞系培养上清中的HMGB1水平无明显差异;0.25μg/ml的HMGB1 PcAb可明显抑制HTLV-1-LTR的转录活性,而300 ng/ml的rhHMGB1可明显促进HTLV-1-LTR的转录活性;real-time PCR检测显示0.25 μg/ml的HMGB1 PcAb可明显抑制病毒编码基因pol1、pol2、gag、env的表达,300 ng/ml的rhHMGB1可明显促进pol1、pol2、gag、env的表达.结论 胞外的HMGB1可促进HTLV-1病毒感染T细胞的病毒复制.     

Role of arginine-56 within the structural protein VP3 of foot-and-mouth disease virus (FMDV) O1 Campos in virus virulence

FMDV O1 subtype undergoes antigenic variation under diverse growth conditions. Of particular interest is the amino acid variation observed at position 56 within the structural protein VP3. Selective pressures influence whether histidine (H) or arginine (R) is present at this position, ultimately influencing in vitro plaque morphology and in vivo pathogenesis in cattle. Using reverse genetics techniques, we have constructed FMDV type O1 Campos variants differing only at VP3 position 56, possessing either an H or R (O1Ca-VP3-56H and O1Ca-VP3-56R, respectively), and characterized their in vitro phenotype and virulence in the natural host. Both viruses showed similar growth kinetics in vitro. Conversely, they had distinct temperature-sensitivity (ts) and displayed significantly different pathogenic profiles in cattle and swine. O1Ca-VP3-56H was thermo stable and induced typical clinical signs of FMD, whereas O1Ca-VP3-56R presented a ts phenotype and was nonpathogenic unless VP3 position 56 reverted in vivo to either H or cysteine (C).     

Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): Influence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C

HCV exists within its host as pools of related genetic variants referred to as quasispecies. The hypervariable region 1 (HVR1) of the E2 envelope gene is subjected to strong selective pressure from neutralizing antibodies. The genetic complexity of this region is defined as the total number of genetic variants within the quasispecies population. The genetic complexity of the HVR1 region was examined in patients with chronic hepatitis C and its relationship with the epidemiology of HCV infection, and its influence on liver disease and the response to interferon treatment were determined in 114 patients with chronic hepatitis C. The genetic complexity of the HVR1 major variants was measured before treatment by using a polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) technique, and was compared with epidemiological, clinical, virological and histological features. The patients were treated with 3 megaunits of interferon (IFN) alfa for 3 to 6 months and the response to treatment was assessed at 3, 6 and 12 months. The HVR1 could be studied in 101 of the 114 patients (89%). Genetic complexity was significantly higher in patients infected through blood transfusion than intravenous drug use (mean complexity index: 5.7 ± 2.3 vs. 4.7 ± 1.5, respectively; P = 0.04). This relationship was independent of age and the estimated time since infection. No significant relationship was found with other parameters of infection or liver disease. In univariate analysis, the genetic complexity of HVR1 major variants did not affect the rates of ALT normalization at months 3 and 6 of IFN treatment. HVR1 genetic complexity was lower in patients with a sustained virological response than in non-responders (4.0 ± 1.7 vs. 5.4 ± 2.0, respectively; P = 0.07). In multivariate analysis of pretreatment parameters associated with a sustained virological response to treatment, three parameters appeared to be independent predictors of such a response: a low viral load (P < 0.04), a low anti-HCV core IgM titer (P = 0.03) and a low genetic complexity of HVR1 major variants (P < 0.04). In conclusion, the HVR1 of HCV has a quasispecies distribution in infected individuals. Its genetic complexity is significantly higher in transfusion recipients than in intravenous drug users, suggesting that the size of the initial inoculum affects the later emergence and development of viral quasispecies. The genetic complexity of HVR1, together with viral load and the anti-HCV IgM titer, are independent predictors of a sustained virological response to IFN alfa in patients with chronic hepatitis C. J. Med. Virol. 54:256–264, 1998. © 1998 Wiley-Liss, Inc.     

Precise gene editing of chicken Na+/H+ exchange type 1 (chNHE1) confers resistance to avian leukosis virus subgroup J (ALV-J)

Avian leukosis virus subgroup J (ALV-J), first isolated in the late 1980s, has caused economic losses to the poultry industry in many countries. As all chicken lines studied to date are susceptible to ALV infection, there is enormous interest in developing resistant chicken lines. The ALV-J receptor, chicken Na⁺/H⁺ exchange 1 (chNHE1) and the critical amino acid sequences involved in viral attachment and entry have already been characterized. However, there are no reported attempts to induce resistance to the virus by targeted genome modification of the receptor sequences. In an attempt to induce resistance to ALV-J infection, we used clustered regularly interspaced short palindromic repeats (CRISPR)-associated (CRISPR/Cas9)-based genome editing approaches to modify critical residues of the chNHE1 receptor in chicken cells. The susceptibility of the modified cell lines to ALV-J infection was examined using enhanced green fluorescent protein (EGFP)-expressing marker viruses. We showed that modifying the chNHE1 receptor by artificially generating a premature stop codon induced absolute resistance to viral infection, with mutations of the tryptophan residue at position 38 (Trp38) being very critical. Single-stranded oligodeoxynucleotide (ssODN)-mediated targeted recombination of the Trp38 region revealed that deletions involving the Trp38 residue were most effective in conferring resistance to ALV-J. Moreover, protein structure analysis of the chNHE1 receptor sequence suggested that its intrinsically disordered region undergoes local conformational changes through genetic alteration. Collectively, these results demonstrate that targeted mutations on chNHE1 alter the susceptibility to ALV-J and the technique is expected to contribute to develop disease-resistant chicken lines.     

高致病性禽流感病毒H5N1安徽株A/Anhui/1/2005感染人肺癌上皮细胞(A549)的差异表达分析

目的 筛选人细胞中与高致病性禽流感病毒致病相关的基因,探讨高致病性禽流感病毒的致病机理.方法 分别用高致病性禽流感病毒安徽株和普通人流感病毒H1N1株分别感染人肺癌上皮细胞,通过人类全基因表达谱芯片技术对不同时段感染细胞进行差异表达分析,筛选出与高致病性禽流感病毒感染相关的候选基因,以实时定量荧光PCR法验证差异表达基因.结果 获得了不同致病性流感病毒感染细胞后的差异表达谱,验证了细胞凋亡通路、mTOR通路中以及与免疫相关的16个基因的差异表达.结论 H5N1感染后与普通人流感病毒H1N1相比具有促进细胞凋亡的趋势.     

Ability of small animal cells to support the postintegration phase of human immunodeficiency virus type-1 replication

We examine the potential for a broad range of small animal cells, including rodent, mink, and avian cells, from multiple tissues to support postintegration steps of HIV-1 replication. These cells were engineered so as to support a stable expression of human cyclin T1 and were further transduced with HIV-1 gag and pol genes. Viral gene expression was activated by the presence of human cyclin T1, but, with the exception of mink cells, was not at the level seen in human cells. Furthermore, there were considerable defects in p24 CA release, in particular in the case of rodent cells. Fractionation of Gag proteins by sucrose floatation revealed that the Gag in human cells trafficked to membrane fractions and was processed to p24 CA and p17 MA efficiently. Confocal imaging demonstrated that Gag was localized in a punctate pattern at the plasma membrane as well as intracellular membrane trans-Golgi cisternae in these cells. In contrast, the majority of Gag in rodent cells was largely present in cytosolic complexes and remained unprocessed. Labeling with [9,10(n)-(3)H]myristic acid showed a similar degree of N-myristoylated Pr55(gag) in rodent and human cells, indicating that while N-myristoylation of Gag was essential for membrane binding, it was not sufficient to confer membrane targeting specificity. Remarkably, despite the reduced level of intracellular Gag processing, mink Mv.1.Lu cells did not appear to differ significantly from human cells in support of virion assembly and release. Analysis of reciprocal heterokaryons suggested that the cellular factor(s) required for efficient assembly and release of infectious virions is lacking in murine cells but appears to be functionally present in mink as well as human cells. Our findings confirm and extend previous reports of multiple blocks to HIV replication in nonhuman cells that are most profound in murine cells. They also raise the possibility that other small animals, such as mink, could serve as novel model systems for studying HIV-1 infection and disease.     

H5N1禽流感病毒NS1蛋白与干扰素诱导蛋白10表达的相关性研究

目的 研究高致病性禽流感(HPAI)H5N1病毒NS1蛋白对干扰素诱导蛋白10(IP-10)的影响.方法 分别将禽流感病毒A/Anhui/1/2005(H5N1)的NS1基因、插入80-84位缺失氨基酸的NS1突变基因及流感病毒A/Puerto Rico/8/1934(H1N1)的NS1基因克隆至真核表达载体pEGFP-N1,转染人支气管上皮细胞BEAS-2B,流式细胞仪检测转染细胞内IP-10的表达情况.结果 与pEGFP-N1对照组相比,三种NS1蛋白均能下调BEAS-2B细胞IP-10的表达(P<0.01),但三者之间下调程度差异无统计学意义(P>0.01).结论 A/Anhui/1/2005(H5N1)禽流感病毒单一NS1蛋白能够抑制BEAS-2B细胞IP-10表达,但这并不能完全阐明其与病毒致病性之间的关系.     

Composition of the sequence downstream of the dengue virus 5' cyclization sequence (dCS) affects viral RNA replication

RNA replication of dengue virus (DENV) requires an RNA-RNA mediated circularization of the viral genome, which includes at least three sets of complementary RNA sequences on both ends of the genome. The 5′ and the 3′ untranslated regions form several additional RNA elements that are involved in regulation of translation and required for RNA replication. Communication between the genomic termini results in a structural reorganization of the RNA elements, forming a functional RNA panhandle structure. Here we report that the sequence composition downstream of the 5′ CS element in the capsid gene, designated as downstream CS (dCS) sequence - but not the capsid protein - also influences the ability of the viral genome to circularize and hence replicate by modulating the topology of the 5′ end. These results provide insights for the design of reporter sub-genomic and genomic mosquito-borne flavivirus constructs and contribute to the understanding of viral RNA replication.     

In vitro replication phenotype of a novel (-1G) hepatitis B virus variant associated with HIV co-infection

The -1G mutant HBV is more prevalent in individuals co-infected with HIV/HBV than in individuals infected with HBV alone and in some cases is the dominant virus in circulation. This mutant is created by the deletion of a dGMP (-1G) from the guanine rich homopolymer sequence located at nts 2,085-2,090 (numbering from EcoRI site as position 1) in the HBV core gene. This deletion causes a frameshift generating a premature stop codon at (64) Asn in the HBV core gene (codon 93 in the precore gene), that truncates the precore protein, precursor of the secreted hepatitis B "e" antigen (HBeAg), and the core protein which forms the viral nucleocapsid. However, the replication phenotype of the -1G mutant HBV is unknown. An in vitro cell culture model in which hepatoma cells were transiently transfected with infectious cDNAs was used to show that the -1G mutant HBV is incapable of autonomous replication and, as expected, replication was restored to wild-type (wt) levels by supplying HBV core protein in trans. Although the -1G mutation had no deleterious effect on intracellular HBV-DNA levels, high levels of -1G mutant HBV relative to wt HBV reduced virus secretion and HBeAg secretion relative to empty vector controls. Importantly, the -1G mutant HBV also caused intracellular retention of truncated precore protein in the endoplasmic reticulum (ER) and Golgi apparatus. Together, these effects may be contributing to the increased pathology observed in the setting of HIV/HBV co-infection.     

Evolution of highly pathogenic avian influenza (H5N1) virus populations in Vietnam between 2007 and 2010

We report on the genetic analysis of 213 highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry in Vietnam between 2007 and 2010. Phylogenetic analyses of the viral genomes revealed 38 distinct viral genotypes, 29 were novel and 9 were reported in Vietnam or neighboring countries in recent years. Viruses from only six genotypes persisted beyond one season or year. Thus, most reassortant viruses were transient, suggesting that such genotypes lacked significant fitness advantages. Viruses with clade 2.3.2.1 HA were re-introduced into Vietnam in 2009 and their prevalence rose steeply towards the end of 2010. Clade 2.3.4-like viruses (genotype V) were predominant in northern Vietnam and caused the majority of zoonotic infections, whereas clade 1.1 (genotype Z) viruses were only detected in the Mekong delta region, in southern Vietnam. Antigenic analysis of representative viruses from the four clades indicated substantial drift.     

Attenuation of interferon regulatory factor 7 activity in local infectious sites of trachea and lung for preventing the development of acute lung injury caused by influenza A virus

The excessive activation of interferon regulatory factor 7 (IRF7) promotes the development of acute lung injury (ALI) caused by influenza A virus (IAV). However, the deficiency of IRF7 increases the susceptibility to deadly IAV infection in both humans and mice. To test whether the attenuation rather than the abolishment of IRF7 activity in local infectious sites could alleviate IAV-induced ALI, we established IAV-infected mouse model and trachea/lung-tissue culture systems, and designed two IRF7-interfering oligodeoxynucleotides, IRF7-rODN M1 and IRF7-rODN A1, based on the mouse and human consensus sequences of IRF7-binding sites of Ifna/IFNA genes, respectively. In the model mice, we found a close relationship between the IAV-induced ALI and the level/activity of IRF7 in local infectious sites, and also found that the reduced IRF7 level or activity in the lungs of mice treated with IRF7-rODN M1 led to decreased mRNA levels of Ifna genes, reduced neutrophil infiltration in the lungs and prolonged survival of mice. Furthermore, we found that the effects of IRF7-rODN M1 on alleviating IAV-induced ALI could be correlated to the reduced translocation of IRF7, caused by the IRF7-rODN M1, from cytosol to nucleus in IAV-infected cells. These data suggest that the proper attenuation of IRF7 activity in local infectious sites could be a novel approach for treating IAV-induced ALI.     

Synergistic antiviral activity of Sofosbuvir and type‐I interferons (α and β) against Zika virus

Zika virus (ZIKV) is transmitted by mosquitoes and causes Dengue‐like illness, neurological symptoms such as Guillain‐Barré Syndrome and microcephaly in children born to infected pregnant mothers. Recently, the World Health Organization (WHO) declared ZIKV infection as a Global Health Emergency. However, there are no known prophylactic or therapeutic measures against this virus. As a proof of concept toward combination therapeutic strategy against ZIKV, combinations of host‐targeted (Interferon‐α and Interferon‐β) and direct acting (Sofosbuvir) antivirals were evaluated in a hepatic cell line (Huh7) using a Cytoprotection (CP) assay. The combination of these antivirals resulted in synergistic inhibition of ZIKV infection in the in vitro CP assay. Additional testing in a ZIKV yield assay demonstrated that combination treatment of these antivirals conferred >2‐log reduction in the release of viral RNA. Measurement of ZIKV proteins in the cells infected with multiple ZIKV strains isolated from different geographical regions (Americas, Asia, and Africa) using an immunofluorescence assay confirmed the effective antiviral activity of this combination against ZIKV. These results demonstrate the in vitro proof of concept (POC) for using a combination approach utilizing the strengths of both virus and host‐targeted antivirals. These results suggest the effectiveness of the combination strategy in combating ZIKV, in the in vitro systems. Further evaluation of such combination therapies in vivo might provide an impetus for the development of effective ZIKV therapeutic strategies.     

Infectious salmon anemia virus (ISAV) replication is transiently inhibited by Atlantic salmon type I interferon in cell culture

Infectious salmon anemia virus (ISAV) is a piscine orthomyxovirus, which causes multisystemic disease in farmed Atlantic salmon that may result in large losses. Previous work has suggested that ISAV is able to resist the antiviral state induced in cells by type I interferon (IFN). These studies were, however, mainly based on cytopathic effect (CPE) reduction assays. Here we have investigated the antiviral activity of Atlantic salmon IFNa1, IFNb and IFNc against ISAV using quantitative PCR (qPCR) of segment 6, Western blot analysis of ISAV proteins and viral yield reduction assays, in addition to CPE reduction assays. Antiviral effects of IFNs were tested against the high virulent strain ISAV4 and the low virulent strain ISAV7 both at the optimum growth temperature 15 °C and at 20 °C. As expected, IFNa1 showed little protection against CPE development in cells after infection with both strains at 15 °C. However, the qPCR and Western blot analysis clearly showed strong inhibition of replication of the virus strains by IFNa1 between 24 and 72 h after infection. The inhibitory effect declined four to five days post-infection, which explains the low protection against CPE development 7–10 days later. At 20 °C, IFNa1 showed strong protection against CPE development, probably due to slower virus growth. IFNc showed similar antiviral activity as IFNa1 against ISAV4 while IFNb showed lower activity. There were observed differences between ISAV4 and ISAV7 both with respect inhibition by IFNa1 and ability to induce the two IFN-inducible antiviral effector proteins, Mx and ISG15, which may be related to differences in virulence properties and/or adaption to growth in cell culture.