Apoptosis induced by tumor necrosis factor in cells chronically infected with feline immunodeficiency virus.

Tumor necrosis factor alpha (TNF-alpha) induced morphologic changes such as chromatin condensation and cell shrinkage in a feline fibroblastic cell line (CRFK) chronically infected with feline immunodeficiency virus (FIV) but not in uninfected CRFK cells. DNA extracted from TNF-alpha-treated CRFK cells infected with FIV showed a ladder of nucleosomal DNA, indicating that this cytocidal effect by TNF-alpha was due to programmed cell death, or apoptosis. These findings may have implications for understanding the pathogenesis of FIV infection and for the design of specific therapeutic strategies for AIDS in humans as well as cats.     

Serosurvey for selected virus infections of wild carnivores in Taiwan and Vietnam.

Serum samples from two leopard cats (Felis bengalensis) and four Formosan gem-faced civets (Paguma larvata taivana) in Taiwan, September 1995, and nine leopard cats in Vietnam, August and December 1997, were examined for the prevalence of antibodies against feline parvovirus, feline herpesvirus type 1, feline calicivirus and feline immunodeficiency virus. All civets and nine of 11 leopard cats were shown to have antibodies against feline parvovirus (FPV), and FPV's were isolated from mononuclear cells in the peripheral blood of the six leopard cats.     

Molecular cloning of a novel isolate of feline immunodeficiency virus biologically and genetically different from the original U.S. isolate.

The Japanese isolate (TM1 strain) of feline immunodeficiency virus (FIV) which replicates in a feline CD4 (fCD4)-positive lymphoblastoid cell line (MYA-1 cells) was molecularly cloned from extrachromosomal closed circular DNA. The restriction map of the clone, termed pFTM 191 complete genome (CG), showed a considerable difference from that of the U.S. isolate (Petaluma strain) of FIV. The sequence homology in the long terminal repeat between the TM1 and Petaluma strain was 82%. The pFTM 191 CG was biologically active after transfection into Crandell feline kidney cells which were permissive for replication of FIV Petaluma. However, the progeny virions could not reinfect fCD4-negative Crandell feline kidney cells but could infect fCD4-positive MYA-1 cells. When a specific-pathogen-free cat was inoculated with the virus derived from the pFTM 191 CG, the cat seroconverted within 8 weeks postinoculation and FIV was reisolated at 4, 8, and 20 weeks postinoculation. These results indicate the infectivity of the pFTM 191 CG in vivo.     

A severe dual infection by feline panleukopenia virus and feline calicivirus in an adult cat

A dual infection by feline panleukopenia virus (FPV) and feline calicivirus (FCV) in a 7 month-old cat is described. The animal developed a severe illness with depression, anorexia, fever, leucopoenia, nasal and ocular discharge and oral ulcers. Both FPV and FCV were isolated in cell cultures from a rectal swab and the presence of FCV was confimed by polymerase chain reaction. Antibodies to both the viruses were detected in the serum. The severity of the disease induced by the mixed viral infection highlights the need for intensifying FPV vaccination in cats.     

Canine and feline host ranges of canine parvovirus and feline panleukopenia virus: distinct host cell tropisms of each virus in vitro and in vivo.

Canine parvovirus (CPV) emerged as an apparently new virus during the mid-1970s. The origin of CPV is unknown, but a variation from feline panleukopenia virus (FPV) or another closely related parvovirus is suspected. Here we examine the in vitro and in vivo canine and feline host ranges of CPV and FPV. Examination of three canine and six feline cell lines and mitogen-stimulated canine and feline peripheral blood lymphocytes revealed that CPV replicates in both canine and feline cells, whereas FPV replicates efficiently only in feline cells. The in vivo host ranges were unexpectedly complex and distinct from the in vitro host ranges. Inoculation of dogs with FPV revealed efficient replication in the thymus and, to some degree, in the bone marrow, as shown by virus isolation, viral DNA recovery, and Southern blotting and by strand-specific in situ hybridization. FPV replication could not be demonstrated in mesenteric lymph nodes or in the small intestine, which are important target tissues in CPV infection. Although CPV replicated well in all the feline cells tested in vitro, it did not replicate in any tissue of cats after intramuscular or intravenous inoculation. These results indicate that these viruses have complex and overlapping host ranges and that distinct tissue tropisms exist in the homologous and heterologous hosts.     

Bicyclams, Selective Antagonists of the Human Chemokine Receptor CXCR4, Potently Inhibit Feline Immunodeficiency Virus Replication

Bicyclams are low-molecular-weight anti-human immunodeficiency virus (HIV) agents that have been shown to act as potent and selective CXC chemokine receptor 4 (CXCR4) antagonists. Here, we demonstrate that bicyclams are potent inhibitors of feline immunodeficiency virus (FIV) replication when evaluated in Crandell feline kidney (CRFK) cells. With a series of bicyclam derivatives, 50% inhibitory concentrations (IC50s) against FIV were obtained in this cell system that were comparable to those obtained for HIV-1 IIIB replication in the human CD4(+) MT-4 T-cell line. The bicyclams were also able to block FIV replication in feline thymocytes, albeit at higher concentrations than in the CRFK cells. The prototype bicyclam AMD3100, 1-1'-[1,4-phenylene-bis(methylene)]-bis(1,4,8, 11-tetraazacyclotetradecane), was only fourfold less active in feline thymocytes (IC50, 62 ng/ml) than in CRFK cells (IC50, 14 ng/ml). AMD2763, 1,1'-propylene-bis(1,4,8, 11-tetraazacyclotetradecane), which is a less potent CXCR4 antagonist, was virtually inactive against FIV in feline thymocytes (IC50, >66.5 microgram/ml), while it was clearly active in CRFK cells (IC50, 0.9 microgram/ml). The CXC chemokine stromal-cell-derived factor 1alpha had anti-FIV activity in CRFK cells (IC50, 200 ng/ml) but not in feline thymocytes (IC50, >2.5 microgram/ml). When primary FIV isolates were evaluated for their drug susceptibility in feline thymocytes, the bicyclams AMD3100 and its Zn2+ complex, AMD3479, inhibited all six primary isolates at equal potency. The marked susceptibility of FIV to the bicyclams suggests that FIV predominantly uses feline CXCR4 for entering its target cells.     

虎源猫泛白细胞减少症病毒灭活疫苗的制备及初步应用

摘要：【目的】研制出虎源猫泛白细胞减少症病毒灭活疫苗并对其应用效果进行评价。【方法】以FPV-HLJ为种毒,按1×10-2接毒量,采用同步接毒的方法接于猫肾传代细胞系F81株,于37 ℃静置培养,待细胞病变（CPE）达到75 %以上时,进行收毒。病毒悬液经甲醛灭活24 h,加入佐剂氢氧化铝胶,制备成FPV-HLJ细胞培养灭活疫苗。【结果】皮下接种2月龄非免疫家猫,结果显示,实验组免疫猫FPV HI抗体水平随免疫次数的增加而呈上升趋势,三免后FPV HI抗体效价为1:1024～1：2048,且免疫猫能抵抗FPV强毒攻击,具有100 %的存活率。随后将该苗接种2月龄左右幼虎,三免后抗体效价多数能达到1：1024。【结论】表明此灭活苗可产生较为理想的免疫效果。     

The effect of bovine lactoferrin and lactoferricin B on the ability of feline calicivirus (a norovirus surrogate) and poliovirus to infect cell cultures

AIMS: To characterize the effect of bovine lactoferrin and lactoferricin B against feline calicivirus (FCV), a norovirus surrogate and poliovirus (PV), as models for enteric viruses. METHODS AND RESULTS: Crandell-Reese feline kidney (CRFK) cells were used for the propagation of FCV and monkey embryo kidney (MEK) cells for PV. The assays included visual assessment of cell lines for cytopathic effects and determination of the percentage cell death using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] dye reduction assay. Incubation of bovine lactoferrin with CRFK cells either prior to or together with FCV inoculation substantially reduced FCV infection. In contrast, the interference of lactoferrin with the infection of cells with PV was demonstrated only when lactoferrin was present with cell lines and virus for the entire assay period. Using indirect immunofluorescence, lactoferrin was detected on the surface of both CRFK and MEK cells, suggesting that the interference of viral infection may be attributed to lactoferrin binding to the surfaces of susceptible cells, thereby preventing the attachment of the virus particles. Lactoferricin B, a cationic antimicrobial peptide derived from the N-terminal domain of bovine lactoferrin, reduced FCV but not PV infection. CONCLUSION: Lactoferrin was shown to interfere with the infection of cells for both FCV and PV. However, lactoferricin B showed no interference of infection with PV and interference with infection for FCV required the presence of lactoferricin B together with the cell line and virus. SIGNIFICANCE AND IMPACT OF THE STUDY: An in vitro basis is provided for the effects of bovine lactoferrin and lactoferricin B in moderating food-borne infections of enteric viruses.     

Canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells

Canine parvovirus (CPV) enters and infects cells by a dynamin-dependent, clathrin-mediated endocytic pathway, and viral capsids colocalize with transferrin in perinuclear vesicles of cells shortly after entry (J. S. L. Parker and C. R. Parrish, J. Virol. 74:1919-1930, 2000). Here we report that CPV and feline panleukopenia virus (FPV), a closely related parvovirus, bind to the human and feline transferrin receptors (TfRs) and use these receptors to enter and infect cells. Capsids did not detectably bind or enter quail QT35 cells or a Chinese hamster ovary (CHO) cell-derived cell line that lacks any TfR (TRVb cells). However, capsids bound and were endocytosed into QT35 cells and CHO-derived TRVb-1 cells that expressed the human TfR. TRVb-1 cells or TRVb cells transiently expressing the feline TfR were susceptible to infection by CPV and FPV, but the parental TRVb cells were not. We screened a panel of feline-mouse hybrid cells for susceptibility to FPV infection and found that only those cells that possessed feline chromosome C2 were susceptible. The feline TfR gene (TRFC) also mapped to feline chromosome C2. These data indicate that cell susceptibility for these viruses is determined by the TfR.     

Development,characterization, and viral susceptibility of a feline (Felis catus) renal cell line (CRFK)

Summary Cell line CRFK, derived from kidney tissue of a normal domestic kitten, was initiated in 1964. With intermittent periods of storage in the frozen state, it has been grown in vitro during more than 200 passages, without apparent loss of susceptibility to selected viruses. Various herpesviruses and feline viruses belonging to differnet virus groups grow readily and with distinct, cytopathic features. The cells now grow as a smooth monolayer of epithelial-like cells; most have 37 chromosomes (2n−1) and are thus aneuploid for cat karyotype. Three distinct marker chromosomes are identified. The cell line, which is free of mycoplasmal contaimination, is useful in feline virus research and diagnostic medicine and has become of particular interest in cancer research. Supported in part by Contract E73-2001-NO1-CP-3-3237 within the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Public Health Service and the Morris Animal Foundation and General Research Support funds of the New York State Veterinary College. CRFK cells from stock provided by C. G. Fabricant are available for distribution to investigators from the Cell Culture Laboratory, Naval Biomedical Research Laboratory.     

Roles of the auxiliary genes and AP-1 binding site in the long terminal repeat of feline immunodeficiency virus in the early stage of infection in cats.

To examine the roles of auxiliary genes and the AP-1 binding site in the long terminal repeat of feline immunodeficiency virus (FIV) in vivo, three mutant viruses, which are defective in the vif gene ([delta]vif), ORF-A gene (deltaORF-A), and AP-1 binding site (deltaAP-1), and wild-type virus as a positive control were separately inoculated into three specific-pathogen-free cats. These cats were assessed by measuring the number of proviral DNA copies in peripheral blood mononuclear cells (PBMCs), the CD4/CD8 ratio and antibody responses to FIV for 16 weeks and then examining histological changes at necropsy. Although viral DNAs were detected in PBMCs from all 12 cats to various degrees until 16 weeks postinoculation, no virus was recovered from PBMCs of cats infected with (delta)vif virus during the observation period. However, a very weak antibody response was induced in one cat infected with the (delta)vif virus. In contrast, despite the successful recovery of virus from both groups of cats infected with deltaORF-A and deltaAP-1 virus, antibody responses and decrease in the CD4/CD8 ratio in the groups were milder than those in cats infected with wild-type virus. Furthermore, the numbers of proviral DNA copies in PBMCs from the two groups were not able to reach the level in cats infected with wild-type virus during the observation period. From these results, we conclude that these mutant viruses are still infectious for cats but failed in efficient viral replication and suggest that these auxiliary genes and enhancer element are important or essential to full viral replication kinetics and presumably to full pathogenicity during the early stage of infection in vivo.     

Feline immunodeficiency virus decreases cell-cell communication and mitochondrial membrane potential.

The in vitro effects of viral replication on mitochondrial membrane potential (MMP) and gap junctional intercellular communication (GJIC) were evaluated as two parameters of potential cellular injury. Two distinct cell types were infected with the Petaluma strain of feline immunodeficiency virus (FIV). Primary astroglia supported acute FIV infection, resulting in syncytia within 3 days of infection, whereas immortalized Crandell feline kidney (CRFK) cells of epithelial origin supported persistent FIV infection in the absence of an obvious cytopathic effect. An examination of cells under conditions that included an infection rate of more than 90% for either population revealed that the astroglia produced about four times more virus than the CRFK cells. The mitochondrial uptake of the cationic fluorescent dye rhodamine 123 in infected astroglia was less than 45% of that of normal control cells, whereas the MMP of the CRFK cells, which produced about one-fourth as much virus, was 80.8% of that of the normal cells. Cell-cell communication between adjacent cells was determined by the recovery of fluorescence following photobleaching of a single cell. In spite of the lower level of innate cell-cell communication among cultured CRFK cells than among astroglia, viral replication resulted in a 30% decrease in the GJIC of both astroglia and CRFK cells. These studies indicate that cell injury, as defined by an inhibition of MMP and GJIC, can occur as a result of persistent and acute infection with the Petaluma strain of FIV.     

First evidence of feline herpesvirus, calicivirus, parvovirus, and Ehrlichia exposure in Brazilian free-ranging felids

Serum samples from 18 pumas (Puma concolor), one ocelot (Leopardus pardalis), and two little spotted cats (Leopardus tigrinus) collected from free-ranging animals in Brazil between 1998 and 2004 were tested by indirect immunofluorescence (IFA) for antibodies to feline herpesvirus 1 (FHV 1), calicivirus (FCV), coronavirus (FCoV), parvo-virus (FPV), Ehrlichia canis, Anaplasma pha-gocytophilum, and Bartonella henselae. Serum samples also were tested, by Western blot and ELISA, for feline leukemia virus (FeLV) specific antibodies and antigen, respectively, by Western blot for antibodies to feline immunodeficiency virus (FIV), and by indirect ELISA for antibodies to puma lentivirus (PLV). Antibodies to FHV 1, FCV, FCoV, FPV, FeLV, FIV, PLV or related viruses, and to B. henselae were detected. Furthermore, high-titered antibodies to E. canis or a closely related agent were detected in a puma for the first time.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

动物细胞系的染色体组型与遗传变异率分析

在建立国内首家犬,猫,猴,鼠传代细胞库,即7种动物肾细胞系（F－81,CRFK,MDCK,Vero,Vero-2,MA-104,BHK-21)的种子库和工作库的基础上,通过细胞染色体组型,G带核型,染色体数目变异率,结构畸变率分析,了解7种细胞系传代培养不同代次的染色体变异情况,以相应的细胞株皮下接种褐 形成肿瘤实验,软琼脂细胞克隆一苦恼经与植物凝集素作用下细胞凝集实验为对照,筛选出无致癌／致瘤性,符合细胞遗传学要求,无传染因子污染的细胞系（F－81,CRFK,Vero,Vero-2)或极低致癌性的MDCK细胞系用于制苗,发现肿瘤细胞系高变异率株可在裸鼠体内快速选育成功,细胞系染色体遗传特征决定致性质并具有种属特异性,得到一些100％成瘤和100％不成瘤的细胞株并了与染色体组型的关系,对于肿瘤的发病机理及实验治疗,都是非常好的模型,一些细胞系不仅成瘤而且还可转移（致恶性横纹肌样瘤的BHK－21和Vero 细胞株）,其他致瘤细胞株只成瘤不转移或不明显转移。     

A putative thiamine transport protein is a receptor for feline leukemia virus subgroup A

Feline leukemia virus (FeLV) is a horizontally transmitted virus that causes a variety of proliferative and immunosuppressive diseases in cats. There are four subgroups of FeLV, A, B, C, and T, each of which has a distinct receptor requirement. The receptors for all but the FeLV-A subgroup have been defined previously. Here, we report the identification of the cellular receptor for FeLV-A, which is the most transmissible form of FeLV. The receptor cDNA was isolated using a gene transfer approach, which involved introducing sequences from a feline cell line permissive to FeLV-A into a murine cell line that was not permissive. The feline cDNA identified by this method was approximately 3.5 kb, and included an open reading frame predicted to encode a protein of 490 amino acids. This feline cDNA conferred susceptibility to FeLV-A when reintroduced into nonpermissive cells, but it did not render these cells permissive to any other FeLV subgroup. Moreover, these cells specifically bound FeLV-A-pseudotyped virus particles, indicating that the cDNA encodes a binding receptor for FeLV-A. The feline cDNA shares approximately 93% amino acid sequence identity with the human thiamine transport protein 1 (THTR1). The human THTR1 receptor was also functional as a receptor for FeLV-A, albeit with reduced efficiency compared to the feline orthologue. On the basis of these data, which strongly suggest the feline protein is the orthologue of human THTR1, we have named the feline receptor feTHTR1. Identification of this receptor will allow more detailed studies of the early events in FeLV transmission and may provide insights into FeLV pathogenesis.     

Altered cell tropism and cytopathicity of feline immunodeficiency viruses in two different feline CD4-positive, CD8-negative cell lines.

Two interleukin-2-dependent feline CD4-positive and CD8-negative cell lines, MYA-1 and the newly established FeL-039, were used as host cells for infection with feline immunodeficiency virus (FIV). All FIV strains used, the Petaluma strain and several new isolates, were highly cytopathic to MYA-1. In contrast, the kinetics of FIV replication in FeL-039 differed greatly depending on the strain tested, i.e., noninfectious strain, highly cytopathic strain, and less cytopathic strain producing a persistent state for a long period. It appears, therefore, that cell tropism for FIV differed with each FIV strain tested even in T-cell lines showing similar cell surface phenotypes. Cytopathicity of FIV is evidently due to both the FIV strain and the host T cell.     

Isolation and partial characterization of infectious molecular clones of feline immunodeficiency virus obtained directly from bone marrow DNA of a naturally infected cat.

Replication-competent molecular clones of feline immunodeficiency virus (FIV) were isolated directly from the DNA of bone marrow cells of a naturally FIV-infected cat. After transfection in a feline kidney cell line (CrFK) and subsequent cocultivation with peripheral blood mononuclear cells (PBMC), the viral progeny of the clones was infectious for PBMC but not for CrFK cells. PBMC infected with these clones showed syncytium formation, a decrease in cell viability, and gradual loss of CD4+ cells. The restriction maps of these clones differed from those obtained for previously described molecular clones of FIV derived from cats in the United States. The predicted amino acid sequence similarity of the envelope genes of the two clones was 99.3%, whereas the similarities of the sequences of the clones to those of two molecular clones from the United States, Petaluma and PPR, were 86 and 88%, respectively. Most of the differences between the amino acid sequences of the two clones and those of the clones from the United States were found in five different hypervariable (HV) regions, HV-1 through HV-5. The viral progeny of one of these clones was inoculated into two specific-pathogen-free cats. The animals seroconverted, and the virus could be reisolated from their PBMC.