Adaptation of feline immunodeficiency virus subtype B strain TM2 to a feline astrocyte cell line (G355-5 cells)

Based on receptor usage during infection, feline immunodeficiency virus (FIV) isolates can be divided into two groups; those that require feline CD134 (fCD134) as a primary receptor in addition to CXCR4 to enter the cells, and those that require CXCR4 only. Most primary isolates, including strain TM2, belong to the former group and cannot infect a feline astrocyte cell line (G355-5 cells) due to a lack of fCD134 expression. In a previous study, we found that G355-5 cells transduced with fCD134 (termed G355-5/fOX40 cells) were susceptible to strain TM2 and the inoculated cells became persistently infected. In this study, we examined the phenotype of the virus prepared from the persistently infected cells (termed strain TM2PI). Intriguingly, strain TM2PI replicated well in na?ve G355-5 cells and the inoculated G355-5 cells (termed G355-5/TM2PI cells) became persistently infected. The infection of TM2PI in G355-5 cells was inhibited by CXCR4 antagonist AMD3100 and TM2PI infected other fCD134-negative, CXCR4-positive cell lines, FeTJ and 3201 cells. Four amino acid substitutions were found in the Env protein of the strain TM2PI when compared with that of the parental strain TM2. Among the substitutions, the Env amino acid position at 407 of TM2PI was substituted to lysine which has been known to be responsible for the FIV tropism for Crandell feline kidney cells. The strain TM2PI will be useful for studying the receptor switching mechanism and FIV pathogenesis in cats.     

Detection of feline immunodeficiency virus infection in bone marrow of cats.

Natural or experimental feline immunodeficiency virus (FIV) infection in cats is often associated with hematologic abnormalities which are similar to those observed in human immunodeficiency virus (HIV) infected patients. To determine if cells in bone marrow are infected with FIV and whether severity of hematopoietic disorder is correlated with the level of viral infection, bone marrow tissues from ten experimentally and two naturally FIV infected cats were examined by in situ hybridization for presence of FIV RNA. Seven of the 12 FIV infected cats were also naturally or experimentally coinfected with feline leukemia virus (FeLV). FIV RNA was detected mainly in megakaryocytes and unidentified mononuclear cells in the bone marrow of cats that were sick and had marrow hypercellularity and immaturity. These included all cats in the acute phase of FIV infection and two of seven long term FIV infected cats. One long term FIV infected cat with lymphosarcoma was also positive for FIV RNA in bone marrow cells. The other four long term FIV infected cats were relatively healthy, with normal bone marrow morphology, and were negative for FIV infected cells. Bone marrow from three non-infected and two cats infected with FeLV alone were also negative for FIV RNA by in situ hybridization. We concluded that megakaryocytes and mononuclear cells were targets of the viral infection and that the presence of FIV RNA in cells of the bone marrow correlated with marrow hypercellularity and immaturity, and severity of illness.     

Restriction of the felid lentiviruses by a synthetic feline TRIM5-CypA fusion

Gene therapy approaches to the treatment of HIV infection have targeted both viral gene expression and the cellular factors that are essential for virus replication. However, significant concerns have been raised regarding the potential toxic effects of such therapies, the emergence of resistant viral variants and unforeseen biological consequences such as enhanced susceptibility to unrelated pathogens. Novel restriction factors formed by the fusion of the tripartite motif protein (TRIM5) and cyclophilin A (CypA), or "TRIMCyps", offer an effective antiviral defence strategy with a very low potential for toxicity. In order to investigate the potential therapeutic utility of TRIMCyps in gene therapy for AIDS, a synthetic fusion protein between feline TRIM5 and feline CypA was generated and transduced into cells susceptible to infection with feline immunodeficiency virus (FIV). The synthetic feline TRIMCyp was highly efficient at preventing infection with both HIV and FIV and the cells resisted productive infection with FIV from either the domestic cat or the puma. Feline TRIMCyp and FIV infection of the cat offers a unique opportunity to evaluate TRIMCyp-based approaches to genetic therapy for HIV infection and the treatment of AIDS.     

Strain-specific viral distribution and neuropathology of feline immunodeficiency virus

Feline immunodeficiency virus (FIV) is a naturally occurring lentivirus of domestic cats, and is the causative agent of feline AIDS. Similar to human immunodeficiency virus (HIV), the pathogenesis of FIV involves infection of lymphocytes and macrophages, and results in chronic progressive immune system collapse and death. Neuropathologic correlates of FIV infection have not yet been elucidated, and may be relevant to understanding HIV-associated neurologic disease (neuroAIDS). As in HIV, FIV strains have been shown to express differential tendencies towards development of clinical neuroAIDS. To interrogate viral genetic determinants that might contribute to neuropathogenicity, cats were exposed to two well-characterized FIV strains with divergent clinical phenotypes and a chimeric strain as follows: FIV(PPR) (PPR, relatively apathogenic but associated with neurologic manifestations), FIV(C36) (C36, immunopathogenic but without associated neurologic disease), and Pcenv (a chimeric virus consisting of a PPR backbone with substituted C36 env region). A sham inoculum control group was also included. Peripheral nerve conduction velocity, CNS imaging studies, viral loads and hematologic analysis were performed over a 12 month period. At termination of the study (350 days post-inoculation), brain sections were obtained from four anatomic locations known to be involved in human and primate lentiviral neuroAIDS. Histological and immunohistochemical evaluation with seven markers of inflammation revealed that Pcenv infection resulted in mild inflammation of the CNS, microglial activation, neuronal degeneration and apoptosis, while C36 and PPR strains induced minimal neuropathologic changes. Conduction velocity aberrations were noted peripherally in all three groups at 63 weeks post-infection. Pcenv viral load in this study was intermediate to the parental strains (C36 demonstrating the highest viral load and PPR the lowest). These results collectively suggest that (i) 3' C36 genomic elements contribute to viral replication characteristics, and (ii) 5' PPR genomic elements contribute to CNS manifestations. This study illustrates the potential for FIV to provide valuable information about neuroAIDS pathogenesis related to genotype and viral kinetics, as well as to identify strains useful to evaluation of therapeutic intervention.     

The plasmid constructs producing shRNA corresponding to the conserved 3D polymerase of Foot and Mouth Disease virus protects guinea pigs against challenge virus

RNA interference (RNAi) has been used as an effective antiviral strategy for its specific silencing of viral gene expression in mammalian cells. In this study, shRNA targeting two regions of Foot and Mouth Disease Virus (FMDV) i.e. 3D and 5'UTR which are very essential in virus replication were evaluated. The constructs were made using h7K RNA polymerase III promoter. We investigated in vivo inhibitory effect of shRNA on FMDV replication in BHK-21 cells and guinea pigs. The results showed that transfection of 3D shRNA could reduce virus growth by three folds when cells were challenged with 10(2) TCID(50) of FMDV. Pretreated guinea pigs with 3DshRNA were protected 80% with 10(3) GPID(50) of FMDV. As a first report in guinea pigs which are recognized animal model for FMD vaccine potency testing, the study suggests that shRNA could be a viable therapeutic approach to control severity of FMD infection and spread.     

Shedding of feline immunodeficiency virus in semen of domestic cats during acute infection

OBJECTIVE: To examine shedding of cell-free and cell-associated feline immunodeficiency virus (FIV) in semen of domestic cats during acute infection. ANIMALS: 7 specific-pathogen-free sexually intact male cats. PROCEDURE: 6 cats were inoculated IV with 5 x 10(6) 50% tissue culture infective doses of FIV-NCSU1, and 1 cat served as an uninfected (control) cat. Infection was confirmed in the 6 cats. Periodically for up to 16 weeks after inoculation, cats were anesthetized and ejaculates obtained by use of electroejaculation. Virus was isolated from filtered seminal plasma and washed seminal cells by co-cultivation with a feline CD4+ T-cell line. Seminal cell lysates were also examined for a 582-base pair segment of FIV gag provirus DNA, using a nested polymerase chain reaction amplification. RESULTS: During the acute phase of FIV infection, virus was evident in semen of 5 inoculated cats. Five cats had virus-positive seminal plasma and 3 had virus-positive cellular constituents during the study. Virus was isolated from 8/22 (36%) seminal plasma samples and 2/17 (18%) seminal cell specimens. Provirus DNA was detected in 5/24 (21%) seminal cell lysates. Cell-free virus was isolated as early as 6 weeks after inoculation, whereas cell-associated virus was isolated as early as 12 weeks after inoculation. Provirus DNA was detected in seminal cells from one cat as early as 1 week after inoculation. CONCLUSIONS AND CLINICAL RELEVANCE: Cell-free and cell-associated FIV are shed in semen of cats early during the course of infection. Samples obtained before seroconversion may contain virus. Virus shedding in ejaculates varies between and within cats during acute infection.     

Involvement of cytolytic and non-cytolytic T cells in the control of feline immunodeficiency virus infection

The appearance of non-cytolytic T cells that suppressed feline immunodeficiency virus (FIV) replication in vitro, and FIV-specific cytotoxic T cell (CTL) responses was compared in a group of seven, specific pathogen free (SPF) domestic cats following primary infection with the Glasgow(8) isolate of FIV (FIV(GL-8)). FIV proviral burdens were quantified in the blood and lymphoid tissues by real-time PCR. Non-cytolytic T cell suppression of FIV replication was measured by co-cultivating lymphoblasts prepared from the cats at different time-points during infection with FIV-infected MYA-1 cells in vitro. Non-cytolytic suppressor activity was detected as early as 1 week after infection, and was evident in all the lymphoid tissues examined. Further, this activity was present in subpopulations of T cells in the blood with normal (CD8(hi)) or reduced (CD8(lo)) expression of the CD8 molecule, and temporal modulations in non-cytolytic suppressor activity were unrelated to the circulating CD8(+) T cell numbers. Virus-specific CTL responses, measured by (51)Cr release assays, were not detected until 4 weeks after infection, with the emergence of FIV-specific effector CTLs in the blood. Throughout infection the response was predominantly directed towards FIV Gag-expressing target cells, and by 47 weeks after infection CTL responses had become localised in the lymph nodes and spleen. The results suggest that both non-cytolytic T cell suppression of FIV replication and FIV-specific CTL responses are important cellular immune mechanisms in the control of FIV replication in infected asymptomatic cats.     

RNAi靶向GP5基因抑制猪繁殖与呼吸综合征病毒在MARC-145细胞中的复制

针对猪繁殖与呼吸综合征病毒(PRRSV)JL/07/SW株GP5基因设计了3个RNA干扰靶位,构建shRNA表达质粒;将转染干扰质粒6 h后的MARC-145细胞接种病毒,并通过Real-time RT-PCR、TCID50、CPE、间接免疫荧光检测(IFA)对所设计的shRNA表达质粒的干扰效果进行评价;结果表明,构建的干扰质粒可以高效抑制PRRSV在MARC-145细胞中的复制,说明GP5基因的这3个干扰靶位可能是PRRSV复制所必需的。本试验为PRRSV复制及基因组功能研究、抗病毒药物开发和转基因动物研究奠定了基础。     

慢病毒介导的水牛CD14基因shRNA序列的筛选

研究慢病毒载体介导的RNA干扰对水牛CD14基因表达的影响,设计并筛选具有抑制作用的靶序列。将真核表达载体pDsRed-N1-buffaloCD14转染293细胞株,建立稳定表达水牛CD14基因细胞系。同时设计5条水牛CD14 shRNA(319/421/755/970/1 041)以及1条阴性对照序列(NC-1864),构建慢病毒重组质粒pSicoR-GFP-shRNA。采用磷酸钙沉淀法将三质粒共转染293T细胞包装慢病毒,并进行滴度测定。最后,通过对慢病毒感染的pDsRed1-N1-buffalo CD14-293细胞进行QRT-PCR检测,筛选具有抑制效果的shRNA。结果显示:在各感染细胞组中,外源水牛CD14基因mRNA的表达均受到不同程度的抑制,其中,CD14 shRNA-970靶点的干扰效率最高,达到79.3%(P<0.01)。成功筛选出具有较高抑制效果的水牛CD14 shRNA靶序列,为研究CD14基因在布氏杆菌所致炎症反应中的作用,建立家畜布氏杆菌病防治新方法奠定了基础。     

水牛脂多糖结合蛋白基因表达载体构建及其shRNA片段的筛选

试验旨在构建水牛脂多糖结合蛋白(LBP)基因融合蛋白表达载体,探讨其在293细胞中的表达情况;筛选获得可抑制水牛LBP基因表达的shRNA干扰片段。采用RT-PCR方法,以水牛肝脏cDNA为模板,扩增水牛LBP开放阅读框(ORF),将其连接到pMD18-T载体中。序列测定并分析正确后,采用SalⅠ和BamHⅠ进行双酶切,将水牛LBP基因编码区定向克隆至pDsRed-N1载体中,构建其融合蛋白表达载体pDsRed-N1-LBP。设计2个针对LBP靶基因序列的shRNA(774-792、1212-1231),同时设计无关序列1864作为阴性对照。合成好的shRNA序列先退火连接到pUC 57载体上,再亚克隆到慢病毒表达载体pSicoR-GFP中,将重组质粒命名为pSicoR-GFP-shLBP774/1212/1864(N.C),采用PCR和测序方法鉴定阳性克隆。将LBP融合蛋白表达载体和其shRNA慢病毒表达载体经脂质体共转染293细胞,48 h后观测荧光蛋白表达情况。收集共转染细胞样品,采用实时定量PCR(QRT-PCR)检测LBP基因的表达,筛选有效抑制LBP基因表达的shRNA干扰序列。结果表明,成功构建水牛LBP融合蛋白表达载体pDsRed-N1-LBP,并在293细胞中瞬时表达。PCR和测序结果均证实所构建的shRNA慢病毒表达载体pSicoR-GFP-shLBP774/1212/1864(N.C)为阳性克隆。共转染48 h后观测,红色和绿色荧光蛋白均有表达。QRT-PCR检测结果显示,shRNA-774和shRNA-1212对293细胞中LBP基因mRNA的表达均有抑制效果,抑制效率分别为49.53%、29.27%。因此,设计合成的2条shRNA序列能有效抑制水牛LBP基因的表达,这为进一步探讨LBP基因在LPS诱导的革兰氏阴性菌跨膜机制和信号转导中的作用机理研究奠定了基础。     

IgG from acutely infected cats blocks mucosal feline immunodeficiency virus infection

We have previously shown an absence of detectable systemic or local infection in cats exposed to an infectious (100 TCID(50)) feline immunodeficiency virus (FIV) plasma inoculum via either the rectal or vaginal mucosa. In contrast, this same plasma inoculum was infectious via parenteral inoculation. Moreover an equivalent dose of cell-free tissue culture-origin virus inoculum infected 100% of cats by either the rectal or vaginal exposure route. To evaluate this phenomena, we used a tissue culture system to identify a heat-stable factor in the plasma of cats acutely (3 weeks) infected with FIV that blocked infection of naive peripheral blood mononuclear cells (PBMC) by either cell-free or cell-associated FIV in vitro. A single application of as little as a 1:200 dilution of either heparinized or Alsevier's anticoagulated plasma effectively inhibited production of FIV p26 in culture over a 21-day co-culture period. Depletion of antibody using a protein A column abrogated the inhibitory effect of FIV plasma against in vitro FIV infection. Co-inoculation of heat-inactivated plasma with 400 TCID(50) FIV-B-2542 cell-free supernatant virus onto the vaginal mucosa of two cats resulted in complete inhibition of infection in one cat and increased time to infection in the second. Thus, antibody found in the plasma of cats acutely infected with FIV blocks cell-associated and cell-free infection, inhibits virus production in previously infected cells, and reduces mucosal transmission efficiency in vivo. Extrapolation may help explain the relatively inefficient mucosal transmission of human immunodeficiency virus-1 (HIV) and other lentiviruses.     

Induction of feline immunodeficiency virus from a chronically infected feline T-lymphocyte cell line

The infection of the feline T-lymphocyte cell line FeT-J with the feline immunodeficiency virus (FIV) Petaluma strain led to the establishment of nonvirus-producing cells. One clone (C15) obtained by limiting dilution was found to express FIV in response to chemical inducers of retroviruses. The chemical treatment of C15 cells led to not only FIV protein synthesis but also an augmentation of viral production. Examination of the C15 cell derivatives obtained by recloning revealed that 10-40% of treated cells constitutively expressed FIV antigens, whereas 100% with expressed FIV antigen in response to the inducer. Chemical induction resulted in more than a 100-fold increase in infectious viral production. The results suggest that a majority of FeT-J cells that are infected with FIV exist in a non-productive state. Establishing a cell line that can be non-productively infected by FIV may help determine the mechanisms of FIV latency.     

转录靶向猪瘟病毒3''''-UTR shRNA细胞株的初步建立

利用质粒载体在细胞内转录并加工成siRNA的方法，设计3对针对猪瘟病毒3＇-UTR不同位点的干扰片段，分别与干扰载体pGenesil-1连接，转化DH5a，筛选阳性克隆得到重组干扰质粒pGene-1，pGene-2和pGene-3，脂质体介导转染重组干扰质粒于PK-15细胞，G418抗性筛选得到转录靶向猪瘟病毒3＇-UTRshRNA的PK-15细胞株，为今后应用RNAi研究猪瘟病毒Y-UTR调控病毒复制的功能以及抑制猪瘟病毒增殖的研究奠定了基础。     

In vitro inhibition of feline coronavirus replication by small interfering RNAs

Infection with virulent biotypes of feline coronavirus (FCoV) can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. In this study we demonstrate the ability of small interfering RNA (siRNA) mediated RNA interference (RNAi) to inhibit the replication of virulent FCoV strain FIPV WSU 79-1146 in an immortalised feline cell line. A panel of eight synthetic siRNAs targeting four different regions of the FCoV genome were tested for antiviral effects. Efficacy was determined by qRT-PCR of intracellular viral genomic and messenger RNA, TCID50 infectivity assay of extracellular virus, and direct IFA for viral protein expression. All siRNAs demonstrated an inhibitory effect on viral replication in vitro. The two most effective siRNAs, targeting the untranslated 5' leader sequence (L2) and the nucleocapsid gene (N1), resulted in a >95% reduction in extracellular viral titre. Further characterisation of these two siRNAs demonstrated their efficacy when used at low concentrations and in cells challenged with high viral loads. Taken together these findings provide important information for the potential therapeutic application of RNAi in treating FIP.