Tese
Construção e manipulação de clone infeccioso de uma amostra brasileira do vírus da diarreia viral bovina
Fecha
2012-03-29Registro en:
ARENHART, Sandra. CONSTRUCTION AND MANIPULATION OF INFECTIOUS CLONE FROM A
BRAZILIAN BOVINE VIRAL DIARRHEA VIRUS ISOLATE. 2012. 119 f. Tese (Doutorado em Medicina Veterinária) - Universidade Federal de Santa Maria, Santa Maria, 2012.
Autor
Arenhart, Sandra
Institución
Resumen
Bovine viral diarrhea virus (BVDV) is a worldwide pathogen associated with
important losses to livestock production. Most of these losses come from reproductive
disorders and from the ability of the virus to produce persistent infections following in utero
infection of the fetus. A number of reverse genetics methodologies have been used for BVDV
in order to better understand the biology of the virus, which allowed the elucidation of a
number of biological features including virus replication, host-virus interaction, immune
response, and the pathogenesis of fetal infection. The present study describes the construction,
characterization and manipulation of an infectious clone out of a non-cytophatic Brazilian
BVDV strain IBSP4-ncp. The cDNA recombinant clone was constructed by yeast
homologous recombination with a low-copy vector, from three genomic fragments
comprising the open reading frame (ORF). The two untranslated regions (5' and 3' UTR) were
replaced by the respective UTRs of the reference strain NADL. The constructed vector was
transcribed in vitro and the resulting RNA was transfected on MDBK cells to rescue
infectious virus. The rescued viruses (IC-pBSC_IBSP4-ncp#2 and #3) were maintained for
ten passages in tissue culture and characterized in vitro, showing replication dynamics, focus
size and morphology similar to those of the parental IBSP-4. Genomic analysis revealed five
point mutations in the gene coding for Npro protein, resulting in amino acid changes. These
mutations probably reflect an adaptation of the virus to the heterologous UTRs. The infectious
clone IC-pBSC_IBSP4-ncp#2 was further used for the construction of a recombinant virus
expressing the Gaussia luciferase (Gluc) reporter gene. The reporter gene was inserted
between the Npro and Core genes, being flanked by an upstream linker and a downstream
sequence of the Foot and Mouth Disease virus protease (FMDV2Apro) for accurate protein
processing. The recombinant vector was in vitro transcribed and the RNA was transfected on
MDBK cells. Recombinant infectious viruses were rescued (IC-pBSC_IBSP4-ncpGluc#3 and
#4) and characterized in vitro, showing replication dynamics, focus size and morphology
similar to those of the parental IBSP-4 clone. The Gluc reporter gene was accurately
expressed and processed by the recombinant virus during 15 passages in tissue culture. These
studies revealed that the infectious clone constructed herein can be easily manipulated and is
able to carry in its genome heterologous genes up to 555 base pairs in length in a stable
fashion and without interference with its replication efficiency. Thus, the constructed clone
may be very useful for genetic manipulation towards studying different aspects of the BVDV
biology and its interactions with the host, and for the development of vaccine strains with
attenuated phenotype and/or with antigenic markers.