Genetic diversity of Brazilian isolates of feline immunodeficiency virus

We isolated Feline immunodeficiency virus (FIV) from three adult domestic cats, originating from two open shelters in Brazil. Viruses were isolated from PBMC following co-cultivation with the feline T-lymphoblastoid cell line MYA-1. All amplified env gene products were cloned directly into pGL8MYA. The nucleic acid sequences of seven clones were determined and then compared with those of previously described isolates. The sequences of all of the Brazilian virus clones were distinct and phylogenetic analysis revealed that all belong to subtype B. Three variants isolated from one cat and two variants were isolated from each of the two other cats, indicating that intrahost diversity has the potential to pose problems for the treatment and diagnosis of FIV infection

Modulation of the virus-receptor interaction by mutations in the V5 loop of feline immunodeficiency virus (FIV) following in vivo escape from neutralising antibody

<b>BACKGROUND:</b> In the acute phase of infection with feline immunodeficiency virus (FIV), the virus targets activated CD4+ T cells by utilising CD134 (OX40) as a primary attachment receptor and CXCR4 as a co-receptor. The nature of the virus-receptor interaction varies between isolates; strains such as GL8 and CPGammer recognise a "complex" determinant on CD134 formed by cysteine-rich domains (CRDs) 1 and 2 of the molecule while strains such as PPR and B2542 require a more "simple" determinant comprising CRD1 only for infection. These differences in receptor recognition manifest as variations in sensitivity to receptor antagonists. In this study, we ask whether the nature of the virus-receptor interaction evolves in vivo.<p></p> <b>RESULTS:</b> Following infection with a homogeneous viral population derived from a pathogenic molecular clone, a quasispecies emerged comprising variants with distinct sensitivities to neutralising antibody and displaying evidence of conversion from a "complex" to a "simple" interaction with CD134. Escape from neutralising antibody was mediated primarily by length and sequence polymorphisms in the V5 region of Env, and these alterations in V5 modulated the virus-receptor interaction as indicated by altered sensitivities to antagonism by both anti-CD134 antibody and soluble CD134.<p></p> <b>CONCLUSIONS:</b> The FIV-receptor interaction evolves under the selective pressure of the host humoral immune response, and the V5 loop contributes to the virus-receptor interaction. Our data are consistent with a model whereby viruses with distinct biological properties are present in early versus late infection and with a shift from a "complex" to a "simple" interaction with CD134 with time post-infection.<p></p&gt

Expression of in situ biomarkers in striped bass

The proposed project has the following objectives : 1) Identify and sequence the striped bass (BB) glutathione Stransferase(s) (GSTs) gene(s); 2) Use the identified sequences as a probe to compare GST mRNA levels in control versus laboratory and/or selected field exposed SB.The goal of this research is to develop a biomonitoring model for specific California surface waters utilizing SB, the popular game fish which is present in this environment. An approach based on the increased/decreased expression of GSTs will be used to screen the effect of experimental exposures of SB to toxic chemicals including polycyclic aromatic hydrocarbons, rice field herbicides and pesticides for their hepatoxicity. GSTs are a multigene family of enzymes catalyzing the conjugation of numerous electrophiles with reduced glutathione (GSH) by formation of the thioether bond. These reactive electrophiles include metaboli tes and endogenous compounds, drugs and pollutants. For the majority of substrates, the GSH conjugate is less toxic than the parent compound providing the organism protection against chemical insult. The level of expression of GST isoforms is extremely important in determining exposure to pollutant xenobiotics.We have already isolated 3 GST proteins from SB following standard protein purification techniques and a GSH affinity column. These proteins were digested with CNBr, purified with HPLC and N-terminal sequenced. We plan to utilize the oligos derived from the back translated peptide sequences as well as conserved sequences (obtained from the literature) to screen a SB cDNA library for SB GSTs. Initially, hepatic mRNA will be isolated from control SB and cDNA synthesized using standard protocols. A cDNA library will be prepared in a lambda gtlO bacteriophage (Stratagene gigapack II cloning kit) and probed for the presence of glutathione (GST) clones using the previously described probes. positive clones will be isolated and the data used to further PCR amplify specific sequences of striped bass GST cDNA of exposed and control fish. It should be possible to determine biomarkers of host mediated protection (Phase II, GSTs) resulting from specific chemicals