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Biomedical subjects

P M Zanotto

Publications and source records attributed to P M Zanotto.

At least 19 recordsLinked to original sources
PubMed2001

Construction of a recombinant Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV-2D) harbouring the beta-galactosidase gene.

We have constructed a transfer vector (pAgGal) containing the beta-galactosidase gene under control of the Escherichia coli gpt and AgMNPV polyhedrin (polh) promoters. The transfer vector was cotransfected with wild type Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) DNA into A. gemmatalis (UFL-AG-286) cells and a recombinant baculovirus (vAgGalA2) was isolated. The beta-galactosidase gene insertion was checked by polymerase chain reaction (PCR) using DNA from AgMNPV and vAgGalA2 and primers specific for regions upstream and downstream of the polh gene. Insect cells (UFL-AG-286) were infected with the recombinant vAgGalA2 and wild type AgMNPV viruses and the production of the heterologous protein analyzed by SDS-PAGE and Pulse-Chase. Beta-galactosidase was expressed at high levels late on infection as expected for a gene under the control of the polh promoter. The highly expressed beta-galactosidase protein was also shown to be biologically active by a beta-galactosidase assay.

Animals↗
PubMed2000

Viral diseases and human evolution.

The interaction of man with viral agents was possibly a key factor shaping human evolution, culture and civilization from its outset. Evidence of the effect of disease, since the early stages of human speciation, through pre-historical times to the present suggest that the types of viruses associated with man changed in time. As human populations progressed technologically, they grew in numbers and density. As a consequence different viruses found suitable conditions to thrive and establish long-lasting associations with man. Although not all viral agents cause disease and some may in fact be considered beneficial, the present situation of overpopulation, poverty and ecological inbalance may have devastating effects on human progress. Recently emerged diseases causing massive pandemics (e.g., HIV-1 and HCV, dengue, etc.) are becoming formidable challenges, which may have a direct impact on the fate of our species.

Biological Evolution↗
PubMed1999

Physical maps and virulence of Anticarsia gemmatalis nucleopolyhedrovirus genomic variants.

Seventeen plaque purified isolates of two viral preparations of Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), were analyzed in terms of the genomic changes after digestion of their DNAs with HindIII and PstI restriction enzymes. The 1979 AgMNPV wild type preparation (AgMNPV-'79) resulted in six different variants and the 1985 viral commercial preparation (AgMNPV-'85), in eleven. The genomic variation of all the isolates was mapped showing that those from 1985 presented more heterogeneity with changes mapped in additional sites in comparison to the AgMNPV-'79 variants. Their virulence was compared by infecting two Lepidopteran cell lines, Spodoptera frugiperda (IPLB-SF-21AE) and Anticarsia gemmatalis (UFL-AG-286). The results indicated that there was some difference in virulence within the AgMNPV-'85 variants. This commercial preparation had been applied in soybean fields in Brazil over several years to control the velvetbean caterpillar defoliation.

Animals↗
PubMed1999

Genealogical evidence for positive selection in the nef gene of HIV-1.

The pattern and process of evolution in the nef gene of HIV-1 was analyzed within and among patients. Using a maximum likelihood method that allows for variable intensity of selection pressure among codons, strong positive selection was detected in a hemophiliac patient over 30 mo of infection. By reconstructing the process of allele substitution in this patient using parsimony, the synapomorphic amino acid changes separating each time point were found to have high probabilities of being under positive selection, with selective coefficients of at least 3.6%. Positive selection was also detected among 39 nef sequences from HIV-1 subtype B. In contrast, multiple pairwise comparisons of nonsynonymous and synonymous substitution rates provided no good evidence for positive selection and sliding window analyses failed to detect most positively selected sites. These findings demonstrate that positive selection is an important determinant of nef gene evolution and that genealogy-based methods outperform pairwise methods in the detection of adaptive evolution. Mapping the locations of positively selected sites may also be of use in identifying targets of the immune response and hence aid vaccine design.

Acquired Immunodeficiency Syndrome↗
PubMed1999

High prevalence of GB virus C in Brazil and molecular evidence for intrafamilial transmission.

The prevalence of GB virus C (GBV-C) in candidate Brazilian blood donors with normal and elevated alanine aminotransferase levels was found to be 5.2% (5 of 95) and 6.5% (5 of 76), respectively. Among Brazilian patients, GBV-C was found in 9.5% (13 of 137) of cases of hepatitis not caused by hepatitis A virus (HAV), HBV, HCV, HDV, or HEV (non-A-E hepatitis) and in 18.2% (8 of 44) of individuals infected with HCV. Molecular characterization of GBV-C by partial sequencing of the NS3 region showed clustering between members of a single family, implying intrafamilial transmission. In conclusion, these results together suggest that contagion mechanisms which facilitate intrafamilial transmission of GBV-C may partially explain the high prevalence of viremic carriers worldwide.

Base Sequence↗
PubMed1999

Genetic reassortment of Rift Valley fever virus in nature.

Rift Valley fever virus (RVFV), a phlebovirus of the Bunyaviridae family, is an arthropod-borne virus which emerges periodically throughout Africa, emphasizing that it poses a major threat for animal and human populations. To assess the genetic variability of RVFV, several isolates from diverse localities of Africa were investigated by means of reverse transcription-PCR followed by direct sequencing of a region of the small (S), medium (M), and large (L) genomic segments. Phylogenetic analysis showed the existence of three major lineages corresponding to geographic variants from West Africa, Egypt, and Central-East Africa. However, incongruences detected between the L, M, and S phylogenies suggested that genetic exchange via reassortment occurred between strains from different lineages. This hypothesis, depicted by parallel phylogenies, was further confirmed by statistical tests. Our findings, which strongly suggest exchanges between strains from areas of endemicity in West and East Africa, strengthen the potential existence of a sylvatic cycle in the tropical rain forest. This also emphasizes the risk of generating uncontrolled chimeric viruses by using live attenuated vaccines in areas of endemicity.

Genetic Variation↗
PubMed1998

Prion's progress: patterns and rates of molecular evolution in relation to spongiform disease.

Modification of the cellular prion protein has been correlated with the acquisition of several neurodegenerative diseases, including kuru, scrapie, bovine spongiform encephalopathy (BSE), and Creutzfeldt-Jakob disease (CJD). Sequence conservation and amino acid identity are known to influence the efficacy of interspecific transmission. We analyzed patterns of interspecific genetic variation with a view toward identifying features related to disease transmission. The reconstructed gene trees and amino acid tree were compared with the species tree, and all discordances observed were related to the species barrier of disease transmission. The rates of synonymous substitution, nonsynonymous substitution, and nucleotide content were determined for the protein-coding gene. Substitutions implicated in each of the prion diseases were found to occur in regions of the protein that are least variable across all species-opposite to the pattern of variability expected from interaction with an infectious pathogen. Amino acid residues related to the species barrier form a single cluster associated with the first alpha-helical domain of the protein. Residues related to sporadic and hereditary human prion disease form two separate clusters, associated with the second and third alpha-helical domains. Taken together, these results are consistent with the view that prion diseases arise from accidents in protein folding, rather than infection with an undiscovered virus-like particle. We speculate that the differences in disease phenotype between transmissable and hereditary forms could result from interactions between different parts of the protein during propagation.

Animals↗
PubMed1998

The complete sequence of four major structural proteins of African horse sickness virus serotype 6: evolutionary relationships within and between the orbiviruses.

The amino acid sequences of four major capsid proteins of African horse sickness virus (serotype 6, AHSV-6) have been determined from analyses of cDNA clones representing the L2, L3, M6 and S7 RNA segments. The AHSV-6 L3 RNA segment has an open reading frame of 2715 base pairs and encodes the inner capsid protein VP3 which comprises 905 amino acids. The VP3 layer forms the subcore of the virion and is surrounded by the VP7 protein which is encoded by the S7 gene. The AHSV-6 S7 gene was found to be 1047 nucleotides in length with a coding capacity for the VP7 protein of 349 amino acids. These core proteins are encapsulated by the outer capsid proteins VP5 and VP2 which are encoded by the M6 and L2 genes respectively. The M6 gene of AHSV-6 was determined to be 1564 nucleotides in length and encoded a protein product of 504 amino acids while the L2 gene comprised 3203 nucleotides which encoded a predicted protein product of 1051 amino acids. Comparison of these four sequences with the core protein sequences of other serotypes of African horse sickness virus, Bluetongue virus which infects sheep, and Epizootic haemorrhagic disease virus of deer, demonstrated that despite the pathobiological properties and host range of these distinct orbiviruses, extreme conservation is evident within the capsid genes. Sequence analyses also suggested that the similarity levels between serogroups depict the structure and function of the individual capsid proteins. The data indicated that the evolution of the capsid genes of gnat transmitted orbiviruses is strongly influenced by functional and structural constraints.

African Horse Sickness Virus↗
PubMed1997

Complete sequence of two tick-borne flaviviruses isolated from Siberia and the UK: analysis and significance of the 5' and 3'-UTRs.

The complete nucleotide sequence of two tick-transmitted flaviviruses, Vasilchenko (Vs) from Siberia and louping ill (LI) from the UK, have been determined. The genomes were respectively, 10928 and 10871 nucleotides (nt) in length. The coding strategy and functional protein sequence motifs of tick-borne flaviviruses are presented in both Vs and LI viruses. The phylogenies based on maximum likelihood, maximum parsimony and distance analysis of the polyproteins, identified Vs virus as a member of the tick-borne encephalitis virus subgroup within the tick-borne serocomplex, genus Flavivirus, family Flaviviridae. Comparative alignment of the 3'-untranslated regions revealed deletions of different lengths essentially at the same position downstream of the stop codon for all tick-borne viruses. Two direct 27 nucleotide repeats at the 3'-end were found only for Vs and LI virus. Immediately following the deletions a region of 332-334 nt with relatively conserved primary structure (67-94% identity) was observed at the 3'-non-coding end of the virus genome. Pairwise comparisons of the nucleotide sequence data revealed similar levels of variation between the coding region, and the 5' and 3'-termini of the genome, implying an equivalent strong selective control for translated and untranslated regions. Indeed the predicted folding of the 5' and 3'-untranslated regions revealed patterns of stem and loop structures conserved for all tick-borne flaviviruses suggesting a purifying selection for preservation of essential RNA secondary structures which could be involved in translational control and replication. The possible implications of these findings are discussed.

Animals↗
PubMed1997

Molecular variation, evolution and geographical distribution of louping ill virus.

Following the demonstration that the tick-borne encephalitis (TBE) subgroup viruses are distributed as a cline across the Northern Hemisphere (Zanotto et al., 1995), we have analyzed the dispersal pattern of louping ill (LI) virus, the most westerly located member in the cline. A total number of 21 LI or LI-related virus E gene sequences have been used for a detailed molecular analysis of the evolution, phylogeny and geographical distribution of LI virus in the British Isles and Ireland. The results show that LI virus is genetically stable in general but minor differences enable its separation into four genetically distinct subtypes (genotypes) with clear geographical correlation, designated Type 1 in Scotland and England, Type 2 in Scotland, Type 3 in Wales and Type 4 in Ireland. These data demonstrate that geographically independent evolution of LI viruses has occurred. The molecular systematics and substitutional parameters analyses combined with the clinal distribution of the TBE virus complex allow the assignment of the origin for both Negishi (NEG) virus and a Norwegian isolate to the British Isles. Moreover, proposals for the classification of LI and LI-like viruses which cause encephalomyelitis in sheep, goat or cattle are presented.

Animals↗
PubMed1996

A tick homologue of the human DNA helicase II 70-kDa subunit.

A clone isolated from a Rhipicephalus appendiculatus salivary gland cDNA library encodes a homologue of the 70-kDa subunit of the mammalian Ku protein, an ATP-dependent DNA helicase. The tick homologue appears to be more closely related to the mammalian protein than to the only other p70 homologue reported in arthropods, the inverted repeat binding protein (IRBP) in the fruitfly, Drosophila melanogaster.

Adenosine Triphosphatases↗
PubMed1996

Population dynamics of flaviviruses revealed by molecular phylogenies.

The phylogeny of 123 complete envelope gene sequences was reconstructed in order to understand the evolution of tick- and mosquito-borne flaviviruses. An analysis of phylogenetic tree structure reveals a continual and asymmetric branching process in the tick-borne flaviviruses, compared with an explosive radiation in the last 200 years in viruses transmitted by mosquitoes. The distinction between these two viral groups probably reflects differences in modes of dispersal, propagation, and changes in the size of host populations. The most serious implication of this work is that growing human populations are being exposed to an expanding range of increasingly diverse viral strains.

Animals↗
PubMed1996

A reevaluation of the higher taxonomy of viruses based on RNA polymerases.

In order to assess the validity of classifications of RNA viruses, published alignments and phylogenies of RNA-dependent RNA and DNA polymerase sequences were reevaluated by a Monte Carlo randomization procedure, bootstrap resampling, and phylogenetic signal analysis. Although clear relationships between some viral taxa were identified, overall the sequence similarities and phylogenetic signals were insufficient to support many of the proposed evolutionary groupings of RNA viruses. Likewise, no support for the common ancestry of RNA-dependent RNA polymerases and reverse transcriptases was found.

Animals↗
PubMed1995

Identification and preliminary characterization of a chitinase gene in the Autographa californica nuclear polyhedrosis virus genome.

A functional chitinase gene (chiA) has been identified in the genome of the Autographa californica nuclear polyhedrosis virus (AcMNPV). It is expressed in the late phase of virus replication in insect cells. High levels of both endo- and exochitinase activity were detected by 12 hr p.i. and remained stable throughout infection. An AcMNPV chiA protein-specific antibody was prepared using recombinant material prepared in bacteria. This was used to demonstrate that a product of approximately 58 kDa was synthesised in virus-infected cells. Immunofluorescence analysis of virus-infected cells showed that most chitinase was located in the cytoplasm. Primer extension analysis of mRNA from AcMNPV-infected cells confirmed that transcription initiated from a baculovirus late start site (TAAG), 14 nucleotides upstream from the putative translation initiation codon. The predicted protein sequence of the AcMNPV chiA shares extensive sequence similarity with chitinases from bacteria and, in particular, the Serratia marcescens chitinase A (60.5% identical residues). Phylogenetic analyses indicate that AcMNPV, or an ancestral baculovirus, acquired the chitinase gene from a bacterium via horizontal gene transfer.

Amino Acid Sequence↗
PubMed1995

An arbovirus cline across the northern hemisphere.

The mode and tempo of arbovirus evolution and dispersal can help to explain the dynamics of pandemics, viral outbreaks, and emerging viruses. By comparing nucleotide and deduced amino acid sequences of their envelope proteins, we describe the continuous distribution of the tick-borne encephalitis (TBE) complex viruses, the most important flaviviruses in Europe, across major geographical areas and the conditions under which mutations occur. The analyses reveal a correlation between the geographical and genetic distances of these viruses. The arthropod host appears to be a key factor for the formation and maintenance of this cline by constraining TBE dispersal and evolution. This is also illustrated by comparisons with mosquito-borne flaviviruses.

Animals↗
PubMed1995

Phylogeny of TYU, SRE, and CFA virus: different evolutionary rates in the genus Flavivirus.

The nucleotide and deduced amino acid sequence of the envelope (E) gene of Tyuleniy (TYU) and Saumarez Reef (SRE) virus have been determined and the data used to classify these viruses in relation to the other tick- and mosquito-borne viruses in the genus Flavivirus. The phylogenetic trees obtained by maximum parsimony and distance methods for 22 flavivirus E genes showed that TYU and SRE virus are a sister group of the TBE virus complex. The trees were consistent with the Flavivirus serological classification and are compatible with the proposition that Cell Fusing Agent could be another genus in the family Flaviviridae. Comparison of the phylogeny and mutational regime of the E gene with the RNA-dependent RNA polymerase (NS5) gene shows the validity of the E gene as a phylogenetic marker and suggests that the mosquito-borne flaviviruses are evolving twice as fast as the tick-borne flaviviruses. The implications of these observations are discussed.

Amino Acid Sequence↗