Genetic variability of wild-type measles viruses, circulating in the Russian Federation during the implementation of the National Measles Elimination Program, 2003-2007.

Genetic characterization of wild-type measles viruses (MVs) is an important component of laboratory surveillance of measles. In this study, a phylogenetic analysis was performed of the nucleoprotein gene sequences of 228 MVs isolated in the Russian Federation between 2003 and 2007. Five genotypes, D4, D5, D6, D8, and H1, were detected. From 1999 through the first 6 months of 2003, the most prevalent genotype in the European part of Russia was D4. All genotype D4-type viruses were closely related to each other (with overall sequence diversity of

Genetic variability and mRNA editing frequencies of the phosphoprotein genes of wild-type measles viruses.

The sequences of the nucleoprotein (N) and hemagglutinin (H) genes are routinely used for molecular epidemiologic studies of measles virus (MV). However, the amount of genetic diversity contained in other genes of MV has not been thoroughly evaluated. In this report, the nucleotide sequences of the phosphoprotein (P) genes from 34 wild-type strains representing 15 genotypes of MV were analyzed and found to be almost as variable as the H genes but less variable than the N genes. Deduced amino acid sequences of the three proteins encoded by the P gene, P, V and C, demonstrated considerably higher variability than the H proteins. Phylogenetic analysis showed the same tree topography for the P gene sequences as previously seen for the N and H genes. RNA editing of P gene transcripts affects the relative ratios of P and V proteins, which may have consequences for pathogenicity. Wild-type isolates produced more transcripts with more than one G insertion; however, there was no significant difference in the use of P and V open reading frames, suggesting that the relative amounts of P and V proteins in infected cells would be similar for both vaccine and wild-type strains.

Identification and characterization of the neutralization epitope(s) of the hepatitis E virus.

The neutralization epitope(s) of the hepatitis E virus (HEV) was studied by an in vitro neutralization assay using antibodies obtained by immunization of mice with 51 overlapping 30-mer synthetic peptides spanning the region 221-660 amino acids (aa) of the HEV open reading frame 2 encoded protein (pORF2) and 31 overlapping recombinant proteins of different sizes derived from the entire pORF2 of the HEV Burma strain. Antibodies against synthetic peptides and short recombinant proteins of approximately 100 aa did not neutralize HEV, suggesting the HEV neutralization epitope(s) is conformation-dependent. However, one recombinant protein of approximately 400 aa in length comprising the pORF2 sequence at position 274-660 aa as well as all truncated derivatives of this protein containing region 452-617 aa elicited antibodies, demonstrating HEV neutralizing activity. These findings establish for the first time that the minimal size fragment, designated pB166, that can efficiently model the neutralization epitope(s) is 166 aa in length and is located at position 452-617 aa of the HEV pORF2. Additionally, antibodies against pB166 were found to cross-neutralize three different HEV genotypes, suggesting that a common neutralization epitope(s) may exist within the different HEV genotypes. Thus, recombinant proteins constructed in this study may be considered as potential candidates for the development of an HEV subunit vaccine as well as for the development of highly sensitive and specific diagnostic tests.

Artificial NS4 mosaic antigen of hepatitis C virus.

An artificial antigen composed of 17 small antigenic regions derived from the NS4-protein of hepatitis C virus (HCV) genotypes 1 through 5 was designed and constructed. Eleven antigenic regions were derived from the 5-1-1 region, and 6 others were derived from the C-terminus of the NS4-protein of different genotypes. The gene encoding for this artificial antigen was assembled from synthetic oligonucleotides by a new approach designated as restriction enzyme-assisted ligation (REAL). The full-length synthetic gene was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. By the use of site-specific antibodies raised against synthetic peptides, it was shown that all regions for which sequence-specific antibodies were obtained were accessible to antibody binding. The diagnostic relevance of the NS4 artificial antigen was demonstrated by testing this antigen with 4 HCV seroconversion panels and a panel of previously tested and stored serum specimens. The artificial antigen was found to specifically detect anti-NS4 antibodies in a number of specimens that were previously found to be anti-NS4 negative. Furthermore, this antigen detected anti-NS4 activity earlier in 2 of 4 seroconversion panels than did the antigen used in a commercially available supplemental assay. Equally important is the observation that the artificial NS4 antigen demonstrated equivalent anti-NS4 immunoreactivity with serum specimens obtained from patients infected with different HCV genotypes, whereas the NS4 recombinant protein derived from genotype 1, used in the commercial supplemental test, was less immunoreactive with serum specimens containing HCV genotypes 2, 3, and 4. Collectively, these data support the significant diagnostic potential of the NS4 mosaic antigen. The strategy employed in this study may be applied to the design and construction of other artificial antigens with improved diagnostically pertinent properties. J. Med. Virol. 59:437-450 1999.

Antigenic domains of the open reading frame 2-encoded protein of hepatitis E virus.

The antigenic composition of the hepatitis E virus (HEV) protein encoded by open reading frame 2 (ORF2) was determined by using synthetic peptides. Three sets of overlapping 18-, 25-, and 30-mer peptides, with each set spanning the entire ORF2 protein of the HEV Burma strain, were synthesized. All synthetic peptides were tested by enzyme immunoassay against a panel of 32 anti-HEV-positive serum specimens obtained from acutely HEV-infected persons. Six antigenic domains within the ORF2 protein were identified. Domains 1 and 6 located at the N and C termini of the ORF2 protein, respectively, contain strong immunoglobulin G (IgG) and IgM antigenic epitopes that can be efficiently modeled with peptides of different sizes. In contrast, antigenic epitopes identified within the two central domains (3 and 4) were modeled more efficiently with 30-mer peptides than with either 18- or 25-mers. Domain 2 located at amino acids (aa) 143 to 222 was modeled best with 25-mer peptides. A few 30-mer synthetic peptides derived from domain 5 identified at aa 490 to 579 demonstrated strong IgM antigenic reactivity. Several 30-mer synthetic peptides derived from domains 1, 4, and 6 immunoreacted with IgG or IgM with more than 70% of anti-HEV-positive serum specimens. Thus, the results of this study demonstrate the existence of six diagnostically relevant antigenic domains within the HEV ORF2 protein.

Antigenic epitopes of the hepatitis A virus polyprotein.

Forty-two antigenic domains were identified across the hepatitis A virus (HAV) polyprotein by using a set of 237 overlapping 20-mer synthetic peptides spanning the entire HAV polyprotein and a panel of serum samples from acutely HAV-infected patients. The term "antigenic domain" is used in this study to define a protein region spanned with consecutive overlapping immunoreactive peptides. Nineteen antigenic domains were found within the structural proteins, and 22 were found within the nonstructural proteins, with 1 domain spanning the junction of VP1 and P2A proteins. Five of these domains were considered immunodominant, as judged by both the breadth and the strength of their immunoreactivity. One domain is located within the VP2 protein at position 57-90 aa. A second domain, located at position 767-842 aa, contains the C-terminal part of the VP1 protein and the entire P2A protein. A third domain, located at position 1403-1456 aa, comprises the C-terminal part of the P2C protein and the N-terminal half of the P3A protein. The fourth domain, located at position 1500-1519 aa, includes almost the entire P3B, and the last domain, located at position 1719-1764 aa, contains the C-terminal region of the P3C protein and the N-terminal region of the P3D protein. It is interesting to note that four of the five most immunoreactive domains are derived from small HAV proteins and/or encompass protein cleavage sites separating different HAV proteins. The HAV-specific immunoreactivity of each antigenically reactive peptide was confirmed by using seven HAV seroconversion panels. Collectively, these data demonstrate that HAV structural and nonstructural proteins contain antigenic epitopes that can be efficiently modeled with short synthetic peptides.

Sequence heterogeneity within three different regions of the hepatitis G virus genome.

Two sets of primers derived from the 5'-terminal region and the NS5 region of the hepatitis G virus (HGV) genome were used to amplify PCR fragments from serum specimens obtained from different parts of the world. All PCR fragments from the 5'-terminal region (5'-PCR, n = 56) and from the NS5 region (NS5-PCR, n = 85) were sequenced and compared to corresponding published HGV sequences. The range of nucleotide sequence similarity varied from 74 and 78% to 100% for 5'-PCR and NS5-PCR fragments, respectively. Additionally, five overlapping PCR fragments comprising an approximately 2.0-kb structural region of the HGV genome were sequenced from each of five sera obtained from three United States residents. These sequences were compared to 20 published sequences comprising the same region of the HGV genome. Nucleotide and deduced amino acid sequences obtained from different individuals were homologous from 82.9 to 93. 6% and from 90.4 to 99.0%, respectively. Sequences obtained from follow-up specimens were almost identical. Comparative analysis of deduced amino acid sequences of the HGV structural proteins and hepatitis C virus (HCV) structural proteins combined with an analysis of predicted secondary structures and hydrophobic profiles allowed prediction of processing sites within the HGV structural proteins. A phylogenetic sequence analysis performed on the 2.0-kb structural region supports the existence of three previously identified HGV genetic groups. However, phylogenetic analysis performed on only small DNA fragments yielded inconsistent genetic grouping and failed to confirm the existence of genetic groups. Thus, in contrast to HCV where almost any region can be used for genotyping, only large or carefully selected genome fragments can be used to identify consistent HGV genetic groups.

[Modifying methylase SsoI from Shigella sonnei 47 cells]. / Modifitsiruiushchaia metilaza SsoI iz kletok Shigella sonnei 47.

A simple and fast method for isolation and purification of SsoI methylase from the bacterial strain Shigella sonnei 47 has been proposed. The enzyme is a modifying component of host cell specificity system and protects the acceptor DNA from hydrolysis by restriction endonucleases SsoI and EcoRI. The method is based on the hydrophobic chromatography of ammonium sulphate fraction on the phenylsepharose. The enzyme preparation obtained is devoid of specific and nonspecific endonucleases traces and is stable at storage in 30% glycerol during a year. The conditions of manifestation of "stroke" activity by the enzyme were studied.

[Cytosine DNA-methylase of a SsoII-type from Shigella sonnei 47 cells]. / Tsitozinovaia DNK-metilaza SsoII-tipa iz kletok Shigella sonnei 47.

Shigella sonnei 47 cells were found to contain DNA-methylase SsoII which is a modifying component of the system of host specificity of SsoII. The recognition sequence (RS) of methylase SsoII is represented by a five-member palyndromic structure--5'...CCNGG...3'--with a degenerated central nucleotide. Modification of SsoII affords protection of acceptor DNA not only from SsoII type restriction, but also from other restrictases, e. g., Eco RII having an analogous RS but with a less degenerated central nucleotide pair. A simple and rapid procedure for isolation and purification of DNA-methylase ScoII, which employs hydrophobic chromatography on phenyl-Sepharose, has been developed. The enzyme preparation does not contain trace amounts of specific and nonspecific endonucleases and keeps stable on storage in 30% glycerol over a period of one year.

[Restriction endonucleases from Shigella sonnei 47]. / Restriktiruiushchie éndonukleazy iz Shigella sonnei 47.

Two restrictases SsoI and SsoII, belonging to the enzymes of restriction of the class II, were isolated from a strain of dysenteric bacteria. The structure of the site sensitive to SsoI and SsoII was studied after fragmentation of testor DNA as well as by means of direct determination of nucleotide sequence. SsoI was shown to be an isoschizomer of the EcoRI restrictase from E. coli. Restrictase SsoII proved to be a new enzyme, which hydrolyzed the sequence 5' ...CCNGG.. 3' and was distinct from the known restrictases as shown by studies of the type of DNA hydrolyzed. A three-step procedure is developed for isolation of SsoII restrictase involving the consecutive chromatography on Blue Sepharose, phosphocellulose PII and phenyl-Sepharose. Restrictase SsoI and EcoRI were isolated by means of isoelectrofocusing using ampholines.

[Recognition site in the restriction endonuclease Eco CK]. / Izuchenie uchastka uznavaniia restriktiruiushchei éndonukleazy Eco CK.

5'-Terminal nucleotide was degenerated in the fragments produced by means of hydrolysis with vestrictase Eco CK. Analysis of the nucleotide sequence, carried out in four DNA fragments after the enzymatic hydrolysis' enabled to detect the only one common 13 residues nucleotide sequence, which is apparently involved in the recognizing site for vestrictase Eco CK: (A/T) (A/T) N (A/T) CGCNCNNNG. This sequence was not found in several DNA molecules with well-known primary structure, stable to the action of this enzyme.