ABSTRACT
<p><b>OBJECTIVE</b>To construct and characterize recombinant adenoviruses containing glycoprotein (GP) gene from rabies virus CVS-N2C strain.</p><p><b>METHODS</b>To obtain the recombinant adenovirus by pAdEasy system, identify recombinant virus with cDNA sequencing, Northern blot, Dot blot, Western blot and challenge-protection experiment of mice.</p><p><b>RESULTS</b>Recombinant adenovirus showed typical adenovirus morphological characteristics; the viral genome was stable; GP specific mRNA and presence of glycoprotein were determined in rAdGPcvs and rAdGPcvs' infected cells. The glycoprotein produced by recombinant adenovirus had a molecular mass of 66,000, which was similar to that of natural glycoprotein. In the group of rAdGPcvs immunized mice, 87.5%-100% of mice survived from a 35.8LD50/38.0LD50 lethal rabies intracerebral challenge. Finally 73.3%-83.3% of the mice that had received eAdGPcvs survived, and all the Ad5 immunized mice succumbed to rabies.</p><p><b>CONCLUSION</b>Recombinant adenovirus rAdGPcvs and rAdGPcvs' hold great potential to be developed as recombinant rabies vaccines, and at the same time, it is actually the first study that on high neuropathogenicity rabies CVS-N2C glycoprotein based adenoviral recombinants.</p>
Subject(s)
Animals , Humans , Mice , Adenovirus E3 Proteins , Genetics , Allergy and Immunology , Adenoviruses, Human , Genetics , Antibodies, Viral , Genetics , Allergy and Immunology , Antigens, Viral , Base Sequence , Genetic Vectors , Glycoproteins , Genetics , Allergy and Immunology , Molecular Sequence Data , Nucleoproteins , Genetics , Allergy and Immunology , Rabies , Allergy and Immunology , Rabies Vaccines , Genetics , Allergy and Immunology , Rabies virus , Genetics , Allergy and Immunology , Vaccines, Synthetic , Genetics , Allergy and Immunology , Viral Envelope Proteins , Genetics , Allergy and Immunology , Virus ReplicationABSTRACT
<p><b>OBJECTIVE</b>To complete the full-length sequencing of the human adenovirus type 7 vaccine strain (Ad7v) for novel vector constructing.</p><p><b>METHODS</b>The Ad7v DNA was digested with SalI and the 17.5-68.0 map unit (mu) fragment was cloned and sequenced. The homology of encoding sequence of Ad7v hexon to those of group A,C,D,E,F and other numbers of group B was accomplished with the software CLUSTAL.V. The three-dimensional structure of the Ad7v hexon was predicted with the RasMo12.71.</p><p><b>RESULTS</b>The fragment contains 17,596 bp, part of E2 and late gene L1, L2 and L3 were encoded by this region. Polypeptide encoded by hexon gene lies in L3 region, which is composed of 934 amino acids. Multiple sequence alignment with the other nine known hexon protein sequences suggested that the variable sequences are mainly concentrated on seven regions, namely hypervariable regions (HVRs). The seven HVRs are related to type-specificity and group-specificity. The three-dimensional structure of the Ad7v hexon revealed that the variable regions are located in the I1 and I2 loops of the molecule mostly on the tower of the hexon.</p><p><b>CONCLUSION</b>The full-length genome sequencing of Ad7v was accomplished at last. Since the deduced amino acid sequence of Ad7v hexon was quite different from other adenoviral vectors such as Ad5 and Ad2, this virus can be potentially used for the construction of novel gene delivery vectors to counterpart the immunity to the vectors widely used at present.</p>
Subject(s)
Adenovirus E3 Proteins , Chemistry , Adenoviruses, Human , Genetics , Amino Acid Sequence , Base Sequence , Capsid Proteins , Chemistry , Genetic Vectors , Sequence Homology, Amino Acid , Viral Vaccines , ChemistryABSTRACT
Adenovirus genotype 7h was previously reported to be originated from a recombination event between adenovirus genotypes 7p and 3p. Based on those findings, further characterization of other adenovirus 7h strains become important to determine whether all adenovirus 7h strains arose from a single recombinational event. To explore such a possibility, 160 clinical isolates were studied after developing a PCR assay using a primer set designed to amplify the region corresponding to E3-7,7 Kd of adenovirus ADV 7p and E3-9 Kd of adenovirus 3p. The assay was able to differentiate most of the subgenus B strains from adeno 7h with the genotype 3d. The study of several adenovirus 7h clinical isolates revealed the existence of three variants of adeno7h. One of the variants, 7h3, shows a high degree of similarity with gene E3-9 Kd of ADV 3p, but lacks the corresponding AUG codon. Our results suggest that more than one recombination event may explain the detection of three different types of adenovirus 7h. The genotype variants of adeno 7h were detected in different years, indicating that the recombination events took place independently from each other. The study of the recombination region may allow further understanding of the function of several viral polypeptides in the immune response, and understanding the mechanism involved in virulence associated to adenovirus 7h.