Eduardo Esteban

Degenerate and specific PCR assays for the detection of bovine leukaemia virus and primate T cell leukaemia/lymphoma virus pol DNA and RNA: phylogenetic comparisons of amplified sequences from cattle and primates from around the world

Degenerate and specific PCR assays were developed for bovine leukaemia virus (BLV) and/or primate T cell leukaemia/lymphoma viruses (PTLV). The degenerate assays detected all major variants of the BLV/PTLV genus at a sensitivity of 10-100 copies of input DNA; the specific systems detected 1-10 copies of input target. Sensitivity was 100% in specific DNA-PCR assays done on peripheral blood from seropositive BLV-infected cattle and HTLV-I- or HTLV-II-infected humans, and 62% in RNA/DNA-PCR assays on sera from BLV seropositive cattle. The pol fragments from 21 different BLV strains, isolated from cattle in North and Central America, were cloned and sequenced, and compared to other published BLV and PTLV pol sequences. BLV and PTLV sequences differed by 42%. Sequence divergence was up to 6% among the BLV strains, and up to 36% among the PTLV strains (with PTLV-I and PTLV-II differing among themselves by 15% and 8%, respectively). Some cows were infected with several BLV strains. Among retroviruses, BLV and PTLV sequences formed a distinct clade. The data support the interpretation that BLV and PTLV evolved from a common ancestor many millennia ago, and some considerable time before the PTLV-I and PTLV-II strains diverged from each other. The dissemination of the BLV strains studied probably resulted from the export of European cattle throughout the world over the last 500 years. The relatively similar mutation rates of BLV and PTLV, after their various points of divergence, suggest that there could be a much wider genetic range of BLV than has currently been defined.

Lack of BLV and PTLV DNA sequences in the majority of patients with large granular lymphocyte leukaemia

The primate T‐cell lymphoma/leukaemia viruses (PTLV) and bovine leukaemia virus (BLV) comprise a unique genus of retroviruses, infection with which induces seroreactivity in the host against conserved epitopes in their p24 gag and gp21 env cognate proteins. Herein, we have confirmed this serocrossreactivity. Patients with large granular lymphocyte (LGL) leukaemia have frequent seroreactivity to the p24 and gp21 env proteins of human T‐cell lymphoma/leukaemia virus I (HTLV‐I), one of the species in the genus. However, only a small minority of patients are actually infected with prototypic HTLV‐I or HTLV‐II, another species within the group. In an attempt to determine whether LGL leukaemia might be associated with other members of the PTLV/BLV genus, we examined the peripheral blood mononuclear cell DNA of 22 HTLV p24 and/or gp21 seropositive LGL leukaemia patients via PCR using degenerate and specific primer pair/probe systems capable of detecting all known members of the PTLV/BLV genus. None of the samples was positive. These data indicate that although HTLV‐II may be associated with some cases of LGL leukaemia most patients are not infected with a PTLV or BLV virus.

Induction and inhibition of bovine leukaemia virus expression in naturally infected cells

Bovine leukaemia virus (BLV) resides in infected lymphocytes in a latent, repressed state but becomes expressed a few hours after the cells are cultured in vitro. We have identified several conditions and factors affecting the expression of BLV in short-term cultures of naturally infected lymphoid cells. The presence of foetal calf serum in the culture medium greatly stimulates virus expression. This stimulation is not due to cellular proliferation. Transcription of BLV RNA and synthesis of p25 in the cultures of peripheral blood lymphocytes are preceded by a lag period of several hours. Synthesis of BLV p25 in these cultures takes place almost immediately after viral RNA synthesis. Extending previous results, we demonstrate that the plasma and lymphatic fluid of cattle contain factors that suppress and stimulate BLV expression. As a result of systematic examination of several parameters, we have developed reproducible assays for the detection of these factors. It is very likely that their relative concentration in the host is an important determinant of susceptibility and resistance to the development of lymphosarcoma and persistent lymphocytosis in BLV-infected cattle.

Hantavirus infection in people inhabiting a highly endemic region of the Gran Chaco territory, Paraguay: association with Trypanosoma cruzi infection, epidemiological features and haematological characteristics

Abstract The seroprevalences of anti-hantavirus antibodies were determined in 712 individuals (551 Indians, 140 Mennonites of German ancestry, and 21 Paraguayans of Spanish ancestry) inhabiting a region of western Paraguay in the Gran Chaco territory of South America. The overall seroprevalence of hantavirus infection among the 712 subjects, who were aged 2-80 years, was 42.7% (45.2% in the Indians and 34.2% in the non-Indians). Of the 672 subjects also checked for antibodies against Trypanosoma cruzi, 226 (33.6%) were seropositive for this protozoan parasite. The results of a multivariate regression analysis indicated that, after adjusting for age, sex, setting of residence (rural/urban) and infection with the human T-cell leukaemia/lymphoma virus type II (HTLV-II), a T. cruzi-seropositive individual was 1.73 times more likely to be hantavirus seropositive than a T. cruzi-seronegative individual. Living in a rural setting increased the risk of being hantavirus seropositive 2.17-fold. In both the Indians and non-Indian subpopulations, hantavirus seroprevalence increased with age in both sexes, but only in the non-Indian supopulation was this increase significantly greater in males than in females. Hantavirus seropositivity was significantly associated with thrombocytosis, even after adjusting for the relevant confounders.

The trans-activating C-type retroviruses share a distinct epitope(s) that induces antibodies in certain infected hosts

Using sera from hosts infected with bovine leukaemia virus (BLV), human T cell lymphoma virus types I and II (HTLV-I and -II), or simian T cell lymphoma virus type I (STLV-I), we found that the major gag proteins of these viruses cross-react immunologically. The specificity of this cross-reactivity was demonstrated by absorption using purified viral proteins, virus lysates and extracts of infected cells. The data strongly suggested that the cross-reacting epitope(s), referred to as CE, differs from those responsible for cross-reactions between the major gag proteins of HTLV-I, HTLV-II and STLV-I, and between those of BLV and HTLV-I reported previously. The prevalence of antibodies to CE was low, even amongst infected hosts with high titres to other epitopes present in the major gag proteins of the homologous viruses. CE was not detected in any of the other C- or D-type retroviruses, or lentiviruses examined. Therefore, it is likely that CE can be used to define serologically a subgroup of C-type retroviruses, the genomes of which display unique features and functional activities.

Expansion of clonotypic T-cell populations in the peripheral blood of asymptomatic Gran Chaco Amerindians infected with HTLV-IIB

Peripheral blood mononuclear cells from asymptomatic HTLV-II-infected and uninfected Gran Chaco Amerindians were analyzed using polymerase chain reaction (PCR) for expansions of T-cell receptor (TCR) V-beta gene clonotypes. Analyses were performed using primer pairs designed to identify expanded T-cell familial clonotypes based on their unique TCR beta gene rearrangements. Of the 30 HTLV-IIB-positive samples tested, five showed evidence of V-beta clonotypic T-cell expansion. Of the five expansions, two were monoclonotypic and the remaining three were oligoclonotypic. In comparison, 30 HTLV-II-negative Amerindians showed no evidence of clonotypic T-cell expansion. Amplified DNA from one of the monoclonotypic samples was subsequently cloned and sequenced and was found to have uniform variable/ diversity/joining sequences confirming its unique monoclonal T-cell expansion. This method of detecting clonal TCR beta gene rearrangements has the advantage over traditional Southern blot techniques of being more sensitive and specific even with suboptimal specimens. The prognostic significance of clonotypic T-cell expansion in a group such as the HTLV-II-infected Gran Chaco Amerindians remains to be determined.

Perfil Proteico y Peptídico de una base Fluida para Bebidas Funcionales obtenida por Fermentación de Lactosuero

Proteins and peptides present in fresh whey and after fermentation to be transformed in a fluid base appropriate to prepare functional beverages were studied. With the SDS-PAGE technique (sodium dodecyl sulfate polyacrylamide gel electrophoresis) it was observed total digestion of high molecular weight proteins such as lactoferrin and serum albumin. Immunoglobulin heavy chains suffered a partial digestion and the light chains were not affected. The major proteins βlactoglobulin and α-lactalbumin did not experience any change. The peptide analysis of the cool whey and base fluid showed a profound change in the post-fermentation profile, with a distribution of 1.000 to 6.000 Dalton. The range of pre-fermentation peptides was 1.500 to 18.000 Dalton. The fermentation process caused a decrease in pH to 3.5 and increased the thermal stability of proteins. The results suggest that whey transformed by this method is a suitable fluid base for designing functional beverages.

Bovine leukemia virus: current perspectives

Fil: Juliarena, Marcela Alicia. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Tandil. Centro de Investigacion Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigacion Veterinaria de Tandil. Provincia de Buenos Aires. Gobernacion. Comision de Investigaciones Cientificas. Centro de Investigacion Veterinaria de Tandil; Argentina

Major Histocompatibility Complex-Associated Resistance to Infectious Diseases: The Case of Bovine Leukemia Virus Infection

The major histocompatibility complex (MHC) is a polymorphic gene cluster of about 150 genes, present in all vertebrates. Many of these genes contribute to immunity. Particularly, MHC‐encoded class I and class II molecules, which are typically highly polymorphic and polygenic, are central in defining the specificity of the adaptive immune response. Among the diversity of genes associated with disease resistance, MHC genes are particularly interesting as they are associated with resistance and susceptibility to a wide range of diseases, some of which produce important economic losses in livestock. Enzootic bovine leukosis is an infectious disease caused by the retrovirus bovine leuke‐ mia virus (BLV), with an important economic impact, mainly in dairy herds. In this chap‐ ter, MHC‐associated genetic resistance to BLV is revised. Certain alleles of the bovine MHC (BoLA) class II locus have been found strongly associated with resistance to viral dissemination. Genetic selection of resistant animals emerges as a natural strategy for the control of infectious diseases, especially when there is no other alternative of control or prevention, as vaccines. Founded on this knowledge, a BLV control program based on selection of genetically resistant cattle was designed. The proof of concept indicates that this strategy is feasible to implement in dairy herds.