Lev S. Sandakhchiev

Genes of variola and vaccinia viruses necessary to overcome the host protective mechanisms

Analysis of variola virus nucleotide sequence revealed proteins belonging to several families which provide the virus with the possibility of overcoming the barriers of specific and non‐specific host defence against viral infection. The complement‐binding proteins, lymphokine‐binding proteins, and serine protease inhibitors can be assigned to this type, as can the proteins providing the orthopoxviruses with resistance to interferon. The revealed differences between the genes (proteins) of variola and vaccinia viruses under study are discussed.

Human monkeypox and smallpox viruses: genomic comparison

Monkeypox virus (MPV) causes a human disease which resembles smallpox but with a lower person‐to‐person transmission rate. To determine the genetic relationship between the orthopoxviruses causing these two diseases, we sequenced the 197‐kb genome of MPV isolated from a patient during a large human monkeypox outbreak in Zaire in 1996. The nucleotide sequence within the central region of the MPV genome, which encodes essential enzymes and structural proteins, was 96.3% identical with that of variola (smallpox) virus (VAR). In contrast, there were considerable differences between MPV and VAR in the regions encoding virulence and host‐range factors near the ends of the genome. Our data indicate that MPV is not the direct ancestor of VAR and is unlikely to naturally acquire all properties of VAR.

ANALYSIS OF THE NUCLEOTIDE SEQUENCE OF 23.8 KBP FROM THE LEFT TERMINUS OF THE GENOME OF VARIOLA MAJOR VIRUS STRAIN INDIA-1967

Sequencing and computer analysis of a variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region has been carried out. Fifty-nine potential open reading frames (ORFs) of over 60 amino acid residues were identified. Structure-function organization of the VAR-IND DNA segment was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple differences between VAR-IND and the strains of VAC but the high identity of VAR-IND with VAR-HAR in the genetic maps are revealed. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.

Comparison of the genetic maps of variola and vaccinia viruses

The complete genetic map of the variola major virus strain India‐1967 is built basing on the sequence data. The suggested map is compared with the maps of the sequenced genomic regions of Copenhagen and Western Reserve strains of vaccinia virus and Harvey strain of variola major virus. The principle differences revealed in the genomic organization of these viruses are discussed.

Ankyrin-like proteins of variola and vaccinia viruses

Computer analysis of full coding sequences of variola major virus strain India‐1967 genome and vaccinia virus strain Copenhagen genome have been carried out. A wide set of proteins containing ankyrin‐like repeats have been identified for both viruses. Only three proteins of this family of the studied viruses are highly homologous. The rest of the proteins are different. The possible role of such proteins in determination of virus tissue tropism is discussed.

Design of immunogens as components of a new generation of molecular vaccines

Three new approaches to design effective immunogens are considered. At first, we derived an expression vector from bacteriophage M13 allowing the exposure of short peptides on the virion surface. EIA demonstrates that antibodies against a recombinant phage carrying the antigenic determinant of the HIV-1 gag protein reacted with the 17-kDa core protein of the virus and also with its polyprotein precursor p55 in immunoblotting. In another approach, we chose the hepatitis B core antigen (HBcAg) particle as a vehicle for the presentation of foreign antigenic determinants to the immune system. Chimerical particles of HBcAg containing epitope of the VEE virus were obtained. A vector system for insertion of foreign antigenic determinants and production of both hybrid and wild HBcAg proteins were also obtained. The third approach relies on construction of immunogens from different T- and B-cell epitopes of the HIV-1. We suggested to construct HIV-1 vaccines in a form of the TBI (T- and B-cell epitopes containing Immunogen) with a predetermined tertiary structure, namely, a four-alpha-helix bundle. The gene of the TBI protein consisting of nine HIV-1 epitopes was synthesized and expressed in Escherichia coli cells. Mice immunized with TBI showed humoral and cellular immune responses to HIV-1. Anti-TBI antibodies displayed HIV-1 neutralizing activity. These new approaches offer promise in the development of new effective vaccines.

Analysis of the nucleotide sequence of 48 kbp of the variola major virus strain India-1967 located on the right terminus of the conservative genome region

Computer analysis of a variola major virus (VAR) genomic fragment bounded by the open reading frames (ORFs) D1R and A33L, which is 47,961 bp long, revealed 46 potential ORFs. The VAR proteins were compared to the analogous proteins of vaccinia virus strain Copenhagen. The subunits of DNA-dependent RNA polymerase, as well as the transcription factors, mRNA-capping enzymes, and proteins necessary for the virion morphogenesis proved to be highly conservative within orthopoxviruses. The most pronounced differences between the VAR genome fragment under study and the corresponding vaccinia virus fragment were revealed in the vicinity of the gene encoding the A-type inclusion bodies protein. Possible functions of the analysed viral proteins are discussed.

Analysis of the nucleotide sequence of a 43 kbp segment of the genome of variola virus India-1967 strain

Sequencing and computer analysis of the nucleotide sequence of the variola virus strain India-1967 (VAR) genome segment (43069 bp) from the region of HindIII C, E, R, Q, K, H DNA fragments has been carried out. Forty-three potential open reading frames (ORFs) have been identified, and the polypeptides encoded by them have been compared with the analogous proteins of vaccinia virus strain Copenhagen (COP). ORF E7R of VAR is much shorter than the COP analog. The other polypeptides coded by the potential ORFs of VAR are highly conserved in comparison with COP. Possible functions of the predicted viral polypeptides are discussed.

Phylogenetic Comparison of the Genomes of Different Strains of Variola Virus

Variola virus (VAR) belonging to the family Poxviridae causes smallpox, one of the most hazardous human diseases. It is known that VAR strains of different geographic origin may cause forms of disease differing in severity and progress. The forms of variola with mild clinical manifestations are traditionally termed variola minor; and those with severe manifestations, variola major. Variola major was widespread in Asia; in Africa, VAR strains causing both types of the disease existed [1, 2]. Variola minor virus strains found in America are usually called alastrim. Biological tests have shown distinct differences of alastrim strains from both variola major and the African isolates of variola minor studied to date in several parameters, such as the limiting temperature of virus reproduction on the chorioallantoic membrane of chicken embryos and hemadsorption at 40°C [3, 4].

Multiple genetic differences between the monkeypox and variola viruses.

The elimination of variola, an extremely dangerous human epidemic disease, was the first and still remains the only example of a successful fight of the international community against an infectious disease under the aegis of World Health Organization [1]. The success of this campaign was to a large extent due to the fact that variola virus, which belongs to the genus Orthopoxvirus of the family Poxviridae and causes variola, has a very narrow host spectrum (only humans). Longterm medical practice has demonstrated the effectiveness of protection against variola based on the use of live vaccine designed on the basis of closely related vaccinia virus. Mass vaccination of people and thorough epidemiological control allowed variola elimination all over the world by 1977. Thereupon, vaccination against variola and other infections induced by closely related orthopoxviruses was stopped everywhere [1].