Phylogenetic analyses of Texas isolates indicate an evolving subtype of the clade B feline immunodeficiency viruses

Rigorous phylogenetic analyses were used to compare the nucleotide sequences of feline immunodeficiency virus strains isolated from Texas and throughout the world. The envelope V3-V4 sequences and capsid gene of the Texas isolates formed a cluster between subtypes B and E. Statistical comparisons with other published sequences confirmed that the Texas group is a unique cluster, possibly a new subtype, arising from subtype B.     

Characterization of a highly pathogenic molecular clone of feline immunodeficiency virus clade C

We have derived and characterized a highly pathogenic molecular isolate of feline immunodeficiency virus subtype C (FIV-C) CABCpady00C. Clone FIV-C36 was obtained by lambda cloning from cats that developed severe immunodeficiency disease when infected with CABCpady00C (Abbotsford, British Columbia, Canada). Clone FIV-C36 Env is 96% identical to the noninfectious FIV-C isolate sequence deposited in GenBank (FIV-Cgb; GenBank accession number AF474246) (A. Harmache et al.) but is much more divergent in Env when compared to the subgroup A clones Petaluma (34TF10) and FIV-PPR (76 and 78% divergence, respectively). Clone FIV-C36 was able to infect freshly isolated feline peripheral blood mononuclear cells and primary T-cell lines but failed to productively infect CrFK cells, as is typical of FIV field isolates. Two-week-old specific-pathogen-free cats infected with FIV-C36 tissue culture supernatant became PCR positive and developed severe acute immunodeficiency disease similar to that caused by the uncloned CABCpady00C parent. At 4 to 5 weeks postinfection (PI), 3 of 4 animals developed CD4(+)-T-cell depletion, fever, weight loss, diarrhea, and opportunistic infections, including ulcerative stomatitis and tonsillitis associated with abundant bacterial growth, pneumonia, and pyelonephritis, requiring euthanasia. Histopathology confirmed severe thymic and systemic lymphoid depletion. Interestingly, the dam also became infected with a high viral load at 5 weeks PI of the kittens and developed a similar disease syndrome, requiring euthanasia at 11 weeks PI of the kittens. This constitutes the first report of a replication-competent, infectious, and pathogenic molecular clone of FIV-C. Clone FIV-C36 will facilitate dissection of the pathogenic determinants of FIV.     

Cytokine kinetics in the plasma of monkeys infected with pathogenic and nonpathogenic simian and human immunodeficiency chimeric viruses at an early stage of infection

To evaluate the pattern of cytokines as a result of pathogenic and nonpathogenic SHIV infections in monkeys, we analyzed the cytokines interleukin (IL)-2, IL-4, IL-10, IL-12, and interferon (IFN)-gamma in the plasma of 8 monkeys infected with either pathogenic 89.6P or nonpathogenic NM-3rN chimeric viruses. The cytokine kinetics in the 89.6P-infected monkeys was characterized by increases of IL-2, IL-10, and to some extent IFN-gamma and a decrease of IL-12. Although that of NM-3rN-infected monkeys was characterized by an increase of IFN-gamma, and a transient decrease of IL-12. IL-4 was not detected in any of the monkeys. The results, therefore, showed a mixture of Th-1 and Th-2 cytokine profiles implying these cytokines are not clear enough to use as an index of the pathogenicity of the viruses at an early stage of infection.     

Immunogenic comparison of chimeric adenovirus 5/35 vector carrying optimized human immunodeficiency virus clade C genes and various promoters

Adenovirus vector-based vaccine is a promising approach to protect HIV infection. However, a recent phase IIb clinical trial using the vector did not show its protective efficacy against HIV infection. To improve the vaccine, we explored the transgene protein expression and its immunogenicity using optimized codon usage, promoters and adaptors. We compared protein expression and immunogenicity of adenovirus vector vaccines carrying native or codon usage-optimized HIV-1 clade C gag and env genes expression cassettes driven by different promoters (CMV, CMVi, and CA promoters) and adapters (IRES and F2A). The adenovirus vector vaccine containing optimized gag gene produced higher Gag protein expression and induced higher immune responses than the vector containing native gag gene in mice. Furthermore, CA promoter generated higher transgene expression and elicited higher immune responses than other two popularly used promoters (CMV and CMVi). The second gene expression using F2A adaptor resulted in higher protein expression and immunity than that of using IRES and direct fusion protein. Taken together, the adenovirus vector containing the expression cassette with CA promoter, optimized HIV-1 clade C gene and an F2A adaptor produced the best protein expression and elicited the highest transgene-specific immune responses. This finding would be promising for vaccine design and gene therapy.     

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.     

Vaginal transmission of chimeric simian/human immunodeficiency viruses in rhesus macaques.

Chimeric simian/human immunodeficiency viruses (SHIVs) that express the env genes derived from distinct HIV type 1 (HIV-1) isolates were tested for the ability to infect rhesus macaques following intravaginal inoculation. SHIVs containing either the HIV-1 HXBc2 or the HIV-1 89.6 envelope glycoproteins were capable of replicating in intravenously inoculated rhesus macaques. However, intravaginal inoculation of animals with these two SHIVs resulted in infection only with the SHIV containing the HIV-1 89.6 glycoprotein. Thus, properties conferred by the envelope glycoproteins in the chimeric virus affect the ability of particular SHIVs to initiate a systemic infection following vaginal inoculation. These results provide indirect support for the hypothesis that the selection of specific viral variants occurs in the genital tracts of individuals exposed to HIV by sexual contact.     

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.     

First case of peritoneal cystic echinococcosis in a domestic cat caused by Echinococcus granulosus sensu stricto (genotype 1) associated to feline immunodeficiency virus infection

A new cystic echinococcosis case in a cat in Uruguay is reported herein. The cat was taken to a veterinary clinic in Rocha city, Uruguay, due to dyspnea, constipation and abdominal enlargement. During surgery a large quantity of cysts was retrieved from the abdominal cavity. The cysts were morphologically studied and confirmed as Echinococcus granulosus sensu stricto (genotype 1) by molecular tools using cytochrome oxidase submit 1 and small subunit ribosomal RNA gene as target genes. Moreover, for the first time a coinfection with feline immunodeficiency virus (FIV) was detected. FIV-induced immunosuppression could be a determining factor in the development of cystic echinococcosis in cats.     

Yellow fever/Japanese encephalitis chimeric viruses: construction and biological properties

A system has been developed for generating chimeric yellow fever/Japanese encephalitis (YF/JE) viruses from cDNA templates encoding the structural proteins prM and E of JE virus within the backbone of a molecular clone of the YF17D strain. Chimeric viruses incorporating the proteins of two JE strains, SA14-14-2 (human vaccine strain) and JE Nakayama (JE-N [virulent mouse brain-passaged strain]), were studied in cell culture and laboratory mice. The JE envelope protein (E) retained antigenic and biological properties when expressed with its prM protein together with the YF capsid; however, viable chimeric viruses incorporating the entire JE structural region (C-prM-E) could not be obtained. YF/JE(prM-E) chimeric viruses grew efficiently in cells of vertebrate or mosquito origin compared to the parental viruses. The YF/JE SA14-14-2 virus was unable to kill young adult mice by intracerebral challenge, even at doses of 10(6) PFU. In contrast, the YF/JE-N virus was neurovirulent, but the phenotype resembled parental YF virus rather than JE-N. Ten predicted amino acid differences distinguish the JE E proteins of the two chimeric viruses, therefore implicating one or more residues as virus-specific determinants of mouse neurovirulence in this chimeric system. This study indicates the feasibility of expressing protective antigens of JE virus in the context of a live, attenuated flavivirus vaccine strain (YF17D) and also establishes a genetic system for investigating the molecular basis for neurovirulence determinants encoded within the JE E protein.     

Transmission of feline immunodeficiency virus in domestic cats via artificial insemination.

The objective of this study was to determine whether semen from male domestic cats infected with feline immunodeficiency virus (FIV) can transmit virus to females. Twelve inseminations were performed by an intrauterine laparoscopic technique with fresh or cryopreserved electroejaculates from asymptomatic males chronically infected with the NCSU1 strain of FIV. Of six inseminations performed with fresh semen, three resulted in infection of queens, as indicated by seroconversion, expression of FIV gag provirus in peripheral blood leukocytes, and reduced peripheral CD4+/CD8+ T-lymphocyte ratios. None of the six inseminates with thawed cryopreserved semen resulted in infection. Two infected queens and one uninfected queen became pregnant. Virus was not evident in the seven offspring. We conclude that FIV can be transmitted horizontally by artificial insemination with fresh semen.     

Immunoglobulin V(H) usage during primary infection of rhesus monkeys with chimeric simian-human immunodeficiency viruses.

It has been suggested that naive immunoglobulins encoded by the V(H)3 gene family interact aberrantly with human immunodeficiency virus type 1 (HIV-1) gp120 via a superantigenic epitope, causing initial expansion and eventual depletion of V(H)3-expressing B cells. However, this possibility has not been prospectively assessed during an AIDS virus infection. We determined V(H) family usage in rhesus monkeys during primary infection with chimeric viruses expressing HIV-1 envelopes on a simian immunodeficiency virus (SIVmac) backbone (SHIVs). Four SHIVs with different envelopes and pathogenicities were studied. V(H) family usage was prospectively assessed in peripheral blood mononuclear cells and lymph node cells of these monkeys by a semiquantitative PCR technique. In the first months following SHIV infection, a period of intense viral antigenemia, representation of various V(H) families increased or decreased for individual monkeys, but no single V(H) family was consistently altered. In particular, the average representation of V(H)3-bearing B lymphocytes did not change. This observation suggests that the envelope glycoprotein of HIV-1 does not selectively expand or deplete the V(H)3 repertoire of primate B cells during acute AIDS virus infection, contrary to predictions of the gp120 superantigen hypothesis.     

Replication and clearance of Venezuelan equine encephalitis virus from the brains of animals vaccinated with chimeric SIN/VEE viruses

Venezuelan equine encephalitis virus (VEEV) is an important, naturally emerging zoonotic pathogen. Recent outbreaks in Venezuela and Colombia in 1995, involving an estimated 100,000 human cases, indicate that VEEV still poses a serious public health threat. To develop a safe, efficient vaccine that protects against disease resulting from VEEV infection, we generated chimeric Sindbis (SIN) viruses expressing structural proteins of different strains of VEEV and analyzed their replication in vitro and in vivo, as well as the characteristics of the induced immune responses. None of the chimeric SIN/VEE viruses caused any detectable disease in adult mice after either intracerebral (i.c.) or subcutaneous (s.c.) inoculation, and all chimeras were more attenuated than the vaccine strain, VEEV TC83, in 6-day-old mice after i.c. infection. All vaccinated mice were protected against lethal encephalitis following i.c., s.c., or intranasal (i.n.) challenge with the virulent VEEV ZPC738 strain (ZPC738). In spite of the absence of clinical encephalitis in vaccinated mice challenged with ZPC738 via i.n. or i.c. route, we regularly detected high levels of infectious challenge virus in the central nervous system (CNS). However, infectious virus was undetectable in the brains of all immunized animals at 28 days after challenge. Hamsters vaccinated with chimeric SIN/VEE viruses were also protected against s.c. challenge with ZPC738. Taken together, our findings suggest that these chimeric SIN/VEE viruses are safe and efficacious in adult mice and hamsters and are potentially useful as VEEV vaccines. In addition, immunized animals provide a useful model for studying the mechanisms of the anti-VEEV neuroinflammatory response, leading to the reduction of viral titers in the CNS and survival of animals.     

New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor

Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position.     

A third hypotrichosis in the domestic cat

Redcar hairless (symbol hd) differs from previous cases of hypotrichosis in the cat. Little or no hair is produced and the condition is a postnatal lethal.     

Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses.

Feline immunodeficiency virus (FIV) induces a disease state in the domestic cat that is similar to AIDS in human immunodeficiency virus (HIV)-infected individuals. As with HIV, FIV can be divided into primary and cell culture-adapted isolates. Adaptation of FIV to replicate and form syncytia in the Crandell feline kidney (CrFK) cell line is accompanied by an increase in the net charge of the V3 loop of the envelope glycoprotein, mirroring the changes observed in the V3 loop of HIV gp120 with the switch from a non-syncytium-inducing phenotype to a syncytium-inducing phenotype. These data suggest a common mechanism of infection with FIV and HIV. In this study, we demonstrate that cell culture-adapted strains of FIV are able to use the alpha-chemokine receptor CXCR4 for cell fusion. Following ectopic expression of human CXCR4 on nonpermissive human cells, the cells are able to fuse with FIV-infected feline cells. Moreover, fusion between FIV-infected feline cells and CXCR4-transfected human cells is inhibited by both anti-CXCR4 and anti-FIV antibodies. cDNAs encoding the feline CXCR4 homolog were cloned from both T-lymphoblastoid and kidney cell lines. Feline CXCR4 displayed 94.9% amino acid sequence identity with human CXCR4 and was found to be expressed widely on cell lines susceptible to infection with cell culture-adapted strains FIV. Ectopic expression of feline CXCR4 on human cells rendered the cells susceptible to FIV-dependent fusion. Moreover, feline CXCR4 was found to be as efficient as human CXCR4 in supporting cell fusion between CD4-expressing murine fibroblast cells and either HIV type 1 (HIV-1) or HIV-2 Env-expressing human cells. Previous studies have demonstrated that feline cells expressing human CD4 are not susceptible to infection with HIV-1; therefore, further restrictions to HIV-1 Env-dependent fusion may exist in feline cells. As feline and human CXCR4 support both FIV- and HIV-dependent cell fusion, these results suggest a close evolutionary link between FIV and HIV and a common mechanism of infection involving an interaction between the virus and a member of the seven-transmembrane domain chemokine receptor family of molecules.     

Phylogenetic analysis of feline immunodeficiency virus in feral and companion domestic cats of New Zealand

Nested PCR was used to amplify envelope V3-V6 gene fragments of feline immunodeficiency virus (FIV) from New Zealand cats. Phylogenetic analyses established that subtypes A and C predominate among New Zealand cats, with clear evidence of intersubtype recombination. In addition, 17 sequences were identified that were distinct from all known FIV clades, and we tentatively suggest these belong to a novel subtype.     

Neuropathogenesis of chimeric simian human immunodeficiency virus infection in rhesus macaques

Comparative studies were performed to determine the neuropathogenesis of infection in macaques with simian human immunodeficiency virus (SHIV)89.6P and SHIV_KU. Both viruses utilize the CD4 receptor and CXCR4 co-receptor. However, in addition, SHIV89.6P uses the CCR5 co-receptor. Both agents are dual tropic for CD4⁺ T cells and blood-derived macrophages of rhesus macaques. Following inoculation into macaques, both caused rapid elimination of CD4⁺ T cells but they varied greatly in mechanisms of neuropathogenesis. Two animals infected with SHIV89.6P developed typical lentiviral encephalitis in which multinucleated giant cell formation, nodular accumulations of microglial cells, activated macrophages and astrocytes, and perivascular accumulations of mononuclear cells were present in the brain. Many of the macrophages in these lesions contained viral RNA. Three macaques infected with SHIV_KU and killed on days 6, 11 and 18, respectively, developed a slowly progressive infection in the CNS but macrophages were not productively infected and there were no pathological changes in the brain. Two other animals infected with this virus and killed several months later showed minimal infection in the brain even though one of the two developed encephalitis of unknown etiology. The basic difference in the mechanisms of neuropathogenesis by the two viruses may be related to co-receptor usage. SHIV89.6P, in utilizing the CCR5 co-receptor, caused neuropathogenic effects that are similar to other neurovirulent primate lentiviruses.