Seroprevalence of Bartonella henselae,Toxoplasma gondii,FIV and FeLV infections in domestic cats in Japan

Seroprevalence of Bartonella henselae, Toxoplasma gondii, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was investigated in 1,447 domestic cats derived from the north (Hokkaido) to the south (Okinawa) prefectures in Japan. Of the cats investigated, 8.8% (128/1,447) were seropositive to B. henselae, 5.4% (78/1,447) to T. gondii, 9.8% (107/1,088) to FIV, and 2.9% (32/1,088) to FeLV, respectively. For B. henselae infection, the positive rate varied from 11.5% in cats of 1 to <2 years old to 7.2% in those over 3 years old. Outdoor cats showed higher positive rate (14.5%) than that (7.0%) in indoor ones. The rate (13.5%) in flea-infested cats was significantly higher than that (7.4%) in flea-negative cats. The positive rates in southern and urban sites were more likely to be higher than those in northern and suburban sites, suggesting that warm and humid environments, density of cat population, and raising status, including hygienic condition and flea infestation in cats may correlate to higher seroprevalence of B. henselae infection. For T. gondii, FIV and FeLV infections, the seroprevalence also tended to be higher in outdoor, flea-infested cats and advanced age groups. For FIV infection, the positive rates in male (14.3%) and outdoor cats (15.0%) were significantly higher than those in female (5.0%) and indoor cats (4.6%). On the other hand, no significant difference in seropositivities was observed in FeLV and T. gondii infections concerning to both genders and raising status.     

Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5

Human SERINC5 (SER5) protein is a recently described restriction factor against human immunodeficiency virus-1 (HIV-1), which is antagonized by HIV-1 Nef protein. Other retroviral accessory proteins such as the glycosylated Gag (glycoGag) from the murine leukemia virus (MLV) can also antagonize SER5. In addition, some viruses escape SER5 restriction by expressing a SER5-insensitive envelope (Env) glycoprotein. Here, we studied the activity of human and feline SER5 on HIV-1 and on the two pathogenic retroviruses in cats, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). HIV-1 in absence of Nef is restricted by SER5 from domestic cats and protected by its Nef protein. The sensitivity of feline retroviruses FIV and FeLV to human and feline SER5 is considerably different: FIV is sensitive to feline and human SER5 and lacks an obvious mechanism to counteract SER5 activity, while FeLV is relatively resistant to SER5 inhibition. We speculated that similar to MLV, FeLV-A or FeLV-B express glycoGag proteins and investigated their function against human and feline SER5 in wild type and envelope deficient virus variants. We found that the endogenous FeLV recombinant virus, FeLV-B but not wild type exogenous FeLV-A envelope mediates a strong resistance against human and feline SER5. GlycoGag has an additional but moderate role to enhance viral infectivity in the presence of SER5 that seems to be dependent on the FeLV envelope. These findings may explain, why in vivo FeLV-B has a selective advantage and causes higher FeLV levels in infected cats compared to infections of FeLV-A only.     

Optimal Packaging of FIV Genomic RNA Depends upon a Conserved Long-range Interaction and a Palindromic Sequence within gag

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5′ 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5′ and 3′ sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ.     

Structural, biochemical, and in vivo characterization of the first virally encoded cyclophilin from the Mimivirus

Although multiple viruses utilize host cell cyclophilins, including severe acute respiratory syndrome (SARS) and human immunodeficiency virus type-1(HIV-1), their role in infection is poorly understood. To help elucidate these roles, we have characterized the first virally encoded cyclophilin (mimicyp) derived from the largest virus discovered to date (the Mimivirus) that is also a causative agent of pneumonia in humans. Mimicyp adopts a typical cyclophilin-fold, yet it also forms trimers unlike any previously characterized homologue. Strikingly, immunofluorescence assays reveal that mimicyp localizes to the surface of the mature virion, as recently proposed for several viruses that recruit host cell cyclophilins such as SARS and HIV-1. Additionally mimicyp lacks peptidyl-prolyl isomerase activity in contrast to human cyclophilins. Thus, this study suggests that cyclophilins, whether recruited from host cells (i.e. HIV-1 and SARS) or virally encoded (i.e. Mimivirus), are localized on viral surfaces for at least a subset of viruses.     

Evaluation of the antiviral efficacy of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives against the nucelocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV infection

A diverse library of bis[1,2]dithiolo[1,4]thiazines and bis[1,2]dithiolopyrrole derivatives were prepared for evaluation of activity against the nucleocapsid protein of the Feline Immunodeficiency Virus (FIV) as a model for HIV, using an in vitro cell culture approach, yielding nanomolar active compounds with low toxicity.     

Untying the FIV frameshifting pseudoknot structure by MS3D

The structure of the putative feline immunodeficiency virus (FIV) ribosomal frameshifting pseudoknot (PK) has been investigated by a mass spectrometric three-dimensional (MS3D) approach, which involves the application of established solvent-accessibility probes and chemical crosslinkers with detection by electrospray ionization (ESI) Fourier transform mass spectrometry (FTMS). Regardless of their size, probed substrates can be treated with ribonucleases and analyzed by ESI-FTMS to obtain the correct position of chemically modified nucleotides. Protection maps and distance information can be utilized to generate 3D models using the constraint satisfaction algorithm provided by MC-SYM and the energy minimization modules included in CNS. Control experiments were performed on a mutant of mouse mammary tumor virus pseudoknot (VPK), for which an NMR structure is available. Comparison between the MS3D model and the high-resolution structure provided a approximately 3A root-mean-square deviation calculated from all the atoms present in double-stranded regions. Applied to FIV-PK, the MS3D approach confirmed that the selected sequence could fold into an actual pseudoknot, supporting the sequence alignment predictions. Characteristic features of H-type pseudoknots were recognized immediately, but a putative A13-U30 pair was not observed at the stem junction, making FIV-PK resemble VPK more closely than the initially suggested simian retrovirus type-1 pseudoknot. In our model, the unpaired U30 protrudes into the medium, while the hinging A13 assumes a stacked conformation that enables the stems to form a approximately 60 degrees bend and relieve the strain caused by a short loop 1. The model provided the basis to explain the different alkylation patterns observed in the absence and presence of Mg(2+), suggesting the possible formation of a specific metal-binding site between loop 1 and stem 2. This instance illustrates how the MS3D model of FIV-PK can be utilized effectively to generate hypotheses and support functional observations in the absence of a high-resolution structure.     

FIV gp40七肽重复区基因的克隆表达及其产物的纯化

本研究利用Leam Coil-VMF程序预测到FIV Env蛋白gp40存在两个七肽重复区(Heptad repeat,HR1和HR2),对包括HR1和HR2在内的部分gp40胞外区基因(称为HRI-HR2)进行了人工合成,以此基因为模板获得了HR1和HR2基因的扩增产物,同时构建了用氨基酸连接子SGGRGG将HR1和HR2连接起来的串联基因(即HRIlinkerHR2,命名为2-Helix),采用人肠杆菌GST融合表达系统对HR1、HR2和2-Helix蛋白进行了表达,并对2-Helix进行了纯化。同时利用凝胶过滤层析证明2-Helix在PBS缓冲系统中以寡聚体的形式存在。     

Viral infections in wild-living European wildcats in Slovenia

Prevalence of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) was investigated in wild-living European wildcats (Felis silvestris) in Slovenia. Seventeen blood samples of 15 wildcats (13 males and two females, two recaptures—1 and 1.5 years after capture) were collected between August 1999 and April 2006. Wildcats were anesthetized using ketamine and medetomidine. Specific antibodies against FIV and FeLV antigens were detected using commercial virus antibody test kits or commercial antigen detection kits, respectively. All investigated sera were negative for presence of specific antibodies against FIV and all investigated animals were negative for presence of FeLV, showing that the highest expected prevalence of the diseases in the population is low. This contrasts with the data from the domestic cats, suggesting a low level of contact between both populations. Apart from addressing the obvious concerns about the impact of infectious diseases on a wild population, epidemiology can be a useful tool for detection of the level of contact in cases when introgression of genes of a common or domestic subspecies/variety might pose a problem for conservation of a threatened species/population.     

The secondary structure of the 5' end of the FIV genome reveals a long-range interaction between R/U5 and gag sequences, and a large, stable stem-loop

Feline immunodeficiency virus (FIV) is a lentivirus that infects cats and is related to human immunodeficiency virus (HIV). Although it is a common worldwide infection, and has potential uses as a human gene therapy vector and as a nonprimate model for HIV infection, little detail is known of the viral life cycle. Previous experiments have shown that its packaging signal includes two or more regions within the first 511 nucleotides of the genomic RNA. We have undertaken a secondary structural analysis of this RNA by minimal free-energy structural prediction, biochemical mapping, and phylogenetic analysis, and show that it contains five conserved stem–loops and a conserved long-range interaction between heptanucleotide sequences 5′-CCCUGUC-3′ in R/U5 and 5′-GACAGGG-3′ in gag. This long-range interaction is similar to that seen in primate lentiviruses where it is thought to be functionally important. Along with strains that infect domestic cats, this heptanucleotide interaction can also occur in species-specific FIV strains that infect pumas, lions, and Pallas' cats where the heptanucleotide sequences involved vary. We have analyzed spliced and genomic FIV RNAs and see little structural change or sequence conservation within single-stranded regions of the 5′ UTR that are important for viral packaging, suggesting that FIV may employ a cotranslational packaging mechanism.