Analysis of antigenic profiles of inactivated poliovirus vaccine and vaccine-derived polioviruses by block-ELISA method.

A new block-ELISA test for quantitative evaluation of relative reactivity of antigenic sites was developed and used to reveal the detailed epitope structure of inactivated poliovirus vaccines (IPV) and live poliovirus strains. Poliovirus was captured on ELISA plates coated with rabbit anti-poliovirus IgG and blocked by monoclonal antibodies (Mabs) specific to individual epitopes before the remaining reactive antigenic sites were quantified by polyclonal anti-poliovirus IgG conjugate. The decrease of conjugate binding by the pre-treatment with a Mab reflects its contribution to the overall reactivity of poliovirus antigen. The level of block activity of Mabs for a given antigen can be expressed as a percent of reduction of antigenic reactivity as determined by ELISA test. It can be normalized by expressing this value as a ratio to the block activity of a reference sample. The data on the blocking-activity of a panel of monoclonal antibodies specific to different antigenic sites represents the epitope composition (antigenic profile) of a sample. Quantitative differences in epitope composition were determined for nine samples of inactivated poliovirus vaccine (IPV) and compared with the International Reference Reagent. This method could be used for monitoring consistency of IPV production, comparison of vaccines made by different manufacturers, and for the analysis of antigenically modified strains of attenuated poliovirus. Antigenic structures of two isolates of type 1 vaccine-derived poliovirus (VDPV) were compared with the structures of parental Sabin 1 and wild-type Mahoney strains using 17 monoclonal antibodies and revealed significant differences, suggesting that the method can be used for screening of field isolates and rapid identification of antigenically divergent VDPV strains.     

Antigenic evolution of vaccine-derived polioviruses: changes in individual epitopes and relative stability of the overall immunological properties

The Sabin oral poliovirus vaccine (OPV) readily undergoes changes in antigenic sites upon replication in humans. Here, a set of antigenically altered descendants of the three OPV serotypes (76 isolates) was characterized to determine the driving forces behind these changes and their biological implications. The amino acid residues of OPV derivatives that lie within or close to the known antigenic sites exhibited a marked tendency to be replaced by residues characteristic of homotypic wild polioviruses, and these changes may occur very early in OPV evolution. The specific amino acid alterations nicely correlated with serotype-specific changes in the reactivity of certain individual antigenic sites, as revealed by the recently devised monoclonal antibody-based enzyme-linked immunosorbent assay. In comparison to the original vaccine, small changes, if any, in the neutralizing capacity of human or rabbit sera were observed in highly diverged vaccine polioviruses of three serotypes, in spite of strong alterations of certain epitopes. We propose that the common antigenic alterations in evolving OPV strains largely reflect attempts to eliminate fitness-decreasing mutations acquired either during the original selection of the vaccine or already present in the parental strains. Variability of individual epitopes does not appear to be primarily caused by, or lead to, a significant immune evasion, enhancing only slightly, if at all, the capacity of OPV derivatives to overcome immunity in human populations. This study reveals some important patterns of poliovirus evolution and has obvious implications for the rational design of live viral vaccines.     

Circulation of endemic type 2 vaccine-derived poliovirus in Egypt from 1983 to 1993

From 1988 to 1993, 30 cases of poliomyelitis associated with poliovirus type 2 were found in seven governorates of Egypt. Because many of the cases were geographically and temporally clustered and because the case isolates differed antigenically from the vaccine strain, it was initially assumed that the cases signaled the continued circulation of wild type 2 poliovirus. However, comparison of sequences encoding the major capsid protein, VP1 (903 nucleotides), revealed that the isolates were related (93 to 97% nucleotide sequence identity) to the Sabin type 2 oral poliovirus vaccine (OPV) strain and unrelated (<82% nucleotide sequence identity) to the wild type 2 polioviruses previously indigenous to Egypt (last known isolate: 1979) or to any contemporary wild type 2 polioviruses found elsewhere. The rate and pattern of VP1 divergence among the circulating vaccine-derived poliovirus (cVDPV) isolates suggested that all lineages were derived from a single OPV infection that occurred around 1983 and that progeny from the initiating infection circulated for approximately a decade within Egypt along several independent chains of transmission. Complete genomic sequences of an early (1988) and a late (1993) cVDPV isolate revealed that their 5' untranslated region (5' UTR) and noncapsid- 3' UTR sequences were derived from other species C enteroviruses. Circulation of type 2 cVDPVs occurred at a time of low OPV coverage in the affected communities and ceased when OPV coverage rates increased. The potential for cVDPVs to circulate in populations with low immunity to poliovirus has important implications for current and future strategies to eradicate polio worldwide.     

Genomic characterization of human and environmental polioviruses isolated in Albania.

Between April and December 1996, a serious outbreak of poliomyelitis occurred in Albania; almost 140 subjects were involved, and the episode presented an unusually high mortality rate (12%). During the outbreak, water samples from the Lana River in Tirana, Albania, and stool samples from two cases of paralytic poliomyelitis were collected and analyzed for the presence of polioviruses. Six polioviruses were isolated from the environmental and human samples, according to standard methods. All the samples were characterized by partial genomic sequencing of 330 bases across the 5' untranslated region (5'-UTR) (nucleotide positions 200 to 530) and of 300 bases across the VP1 region (nucleotide positions 2474 to 2774). Comparison of these sequences with those present in data banks permitted the identification of environmental isolates Lana A and Lana B as, respectively, a Sabin-like type 2 poliovirus and an intertypic recombinant poliovirus (Sabin-like type 2/wild type 1), both bearing a G instead of an A at nucleotide position 481. The two other environmental polioviruses were similar to the isolates from the paralytic cases. They were characterized by a peculiar 5'-UTR and by a VP1 region showing 98% homology with the Albanian epidemic type 1 isolates reported by other authors. This study confirms the environmental circulation in Albania of recombinant poliovirus strains, likely sustained by a massive vaccination effort and by the presence in the environment of a type 1 poliovirus, as isolated from the Lana River in Tirana about 2 months before the first case of symptomatic acute flaccid paralysis was reported in this town.     

The wild boar of Egypt]

The wild boar, Sus scrofa, is not a typical member of the Egyptian wild fauna, although it appears to have lived in the Nile Delta and other suitable regions in the north of the country. However, historic populations were probably of mixed origin, including feral domestic pigs. It is incorrect, as is sometimes still done, to include the wild boar in the iconographic bestiary of Ancient Egypt and assume that the domestic pigs of Ancient Egypt derive from local wild boars.     

Evolution of circulating wild poliovirus and of vaccine-derived poliovirus in an immunodeficient patient: a unifying model

We determined nucleotide sequences of the VP1 and 2AB genes and portions of the 2C and 3D genes of two evolving poliovirus lineages: circulating wild viruses of T geotype and Sabin vaccine-derived isolates from an immunodeficient patient. Different regions of the viral RNA were found to evolve nonsynchronously, and the rate of evolution of the 2AB region in the vaccine-derived population was not constant throughout its history. Synonymous replacements occurred not completely randomly, suggesting the need for conservation of certain rare codons (possibly to control translation elongation) and the existence of unidentified constraints in the viral RNA structure. Nevertheless the major contribution to the evolution of the two lineages came from linear accumulation of synonymous substitutions. Therefore, in agreement with current theories of viral evolution, we suggest that the majority of the mutations in both lineages were fixed as a result of successive sampling, from the heterogeneous populations, of random portions containing predominantly neutral and possibly adverse mutations. As a result of such a mode of evolution, the virus fitness may be maintained at a more or less constant level or may decrease unless more-fit variants are stochastically generated. The proposed unifying model of natural poliovirus evolution has important implications for the epidemiology of poliomyelitis.     

Parasite identification in the surveillance of imported leishmaniasis cases in Italy

An accurate Leishmania classification was defined since 1980s by the use of isoenzyme analysis. To date, this procedure still represents the reference identification technique, despite the increasing use of molecular approaches. Studies and surveillance methods on leishmaniases are strongly conditioned by the knowledge and mapping of all the parameters characterizing each nosogeographical entity. On this respect, the identification of parasites from all the actors of the natural life cycle plays a key role. With the increasing population movements and climate changes, novel risk factors could be identified associated to Leishmania geographical distribution and spreading: a) the introduction into Italy of new populations of L. infantum from other countries; b) the introduction of new Leishmania species that may find a suitable milieau to support their life cycle in our country. The objective of this report is to present the surveillance activity on imported leishmaniases by the Leishmania Identification Reference Centre, ISS. Two different methodologies were routinely applied: a) isoenzyme electrophoretic analysis, which requires parasite culture, and b) a number of molecular techniques, used for both diagnosis and parasite identification, differently applied according to the geographical origin of the suspected leishmaniasis case. When possible, both types of methodologies were applied. From 1986 to June 2002, 38 imported cases of leishmaniases were identified: 9 visceral (VL) and 29 cutaneous (CL) cases, of which 22 from the Old and 7 from the New World. Pathology, Leishmania species/zymodeme and geographical origin features are reported in the paper. Seven out of 9 VL patients were HIV positive, of whom 5 detected in the period 1993-1995. This high importation rate can be associated to the general increase in Mediterranean Leishmania/HIV coinfections in that period. Following HAART treatment, VL imported cases became occasional; no introduction of new L. infantum populations has been detected. On the other hand, our findings show an increase of CL imported cases from different areas of the Old and New Worlds. This phenomenon, however, is so far limited to new Leishmania species that could hardly be introduced in our country, because of their strict biological requirements (i.e. vectors and/or natural reservoir hosts). Since June 2002, 10 further suspected imported cases were recorded. For these patients--whose Leishmania identification is still in course--the origin/visited geographical areas were only slight different from the previous. However, the characteristics of the patients are changing: there are more immigrants that occasionally visited their place of origin, and Italian military staff.     

Immunity to poliomyelitis in adult population of Moscow region. Role of vaccine and wild polioviruses in its formation

Assessment of immunity to poliomyelitis in adults from 8 towns of Moscow region was conducted. Low levels of population immunity against some serotypes of poliovirus in several towns have been found. At the same time, these levels were high and very high in other towns. Measurement of levels of strain-specific antibodies to vaccine and wild polioviruses demonstrated wide circulation of wild polioviruses during past decades which had significant influence on formation of immunity. Substantial number of non-immune adults represents favorable conditions for circulation of vaccine polioviruses after cessation of vaccination, which, in its turn, could result in reestablishment of their neurovirulent properties and subsequent reemergence of poliomyelitis.     

Antibodies to lymphocytic choriomeningitis virus in wild rodent sera in Egypt

Specific antibodies to lymphocytic choriomeningitis (LCM) virus were detected in sera of wild rodents trapped in different regions of Egypt. The highest proportion of positive sera (11.5%) was found in Sinaa Governorate of the Asian Continent, followed by Sharkia Governorate (5.6%) adjacent to Sinaa and Giza Governorate (1.8%). In Upper Egypt, however all 610 rodent sera collected in Aswan, Red sea Pernees and New Valley adjacent to Sudan were negative. The highest percentage of positive sera was in Rattus rattus frugivorus and Mus musculus species.     

Advanced environmental surveillance and molecular analyses indicate separate importations rather than endemic circulation of wild type 1 poliovirus in Gaza district in 2002

An improved sewage surveillance algorithm (sample acquisition, processing, and molecular analysis) for wild and vaccine-derived polioviruses was developed and validated. It was based on plaque isolation with sensitive and high-throughput methods. The molecular analysis included sequencing; a comparison of the type, rate, and distribution of nucleotide substitutions with a profile for outbreaks evolving from a single progenitor; and phylogenetic analysis for relative similarity. The analyses revealed that two environmental wild type 1 isolates from the Gaza district in 2002 were imported separately, most likely from Egyptian southern governorates, and were not linked by endemic circulation. These findings illustrate the continuous spreading potential of wild-type poliovirus and underscore the value of extensive environmental surveillance employing advanced molecular analysis to monitor wild poliovirus in poliomyelitis-free regions.     

Polyphasic taxonomy of symbiotic rhizobia from wild leguminous plants growing in Egypt

About 20 strains of rhizobia from wild legumes were characterized based on numerical analysis of phenotypic characteristics, nodulating ability, fatty acid methyl esters (FAME) and SDS-PAGE profiles of whole cell proteins. FAME analysis revealed that palmitic (16:0), stearic (18:0) and arachidonic (20:0) were detected in most of wild-legume rhizobia, the latter being uncommon in fatty acid profiles of Rhizobium and Sinorhizobium. Numerical analysis of FAME classified strains of wild-legume rhizobia into 9 clusters and one heterogeneous group. There was both agreement and disagreement with the clustering data based on phenotypic analysis and FAME analysis. Four strains were grouped together in the same cluster based on both methods. However, 4 another strains, which were placed in one cluster of phenotypic analysis, were distributed in several clusters after FAME analysis. SDS-PAGE of whole-cell proteins revealed that the rhizobial strains exhibited protein profiles with peptide bands ranging from 5-19 band per profile and showed molar mass of 110-183 kDa. As in the case of FAME analysis, numerical analysis of protein bands was compared with clustering of phenotypic analysis. Agreement of the two methods was obvious when clustering some strains but conflicted in the classification of some other strains. However, integration of the three methods could be the basis of a polyphasic taxonomy. The twenty strains of wild-legume rhizobia were finally classified as follows: 12 strains related to Rhizobium leguminosarum, 5 strains related to Sinorhizobium meliloti and 3 strains to Rhizobium spp. Rhizobia nodulating wild herb legumes are among indigenous strains nodulating crop legumes in cultivated as well as noncultivated lands.     

Floristics and environmental relations in two extreme desert zones of western Egypt

• 1 The present study provides quantitative estimates of soil, vegetation structure and species distribution in 144 stands along two transects in two extreme desert zones of western Egypt. This study had two aims: (1) to describe the floristic composition of the accidental type of vegetation growing in parts of the Western Desert of Egypt; and (2) to analyse the distribution of species in relation to certain environmental factors by multivariate data analysis.
• 2 Eight environmental variables were recorded: salinity, pH, calcium carbonate, soil moisture, organic matter content, and percentages of sand, silt and clay. A total of 60 species belonging to 19 families of the angiosperms and Ephedra alata was recorded. Chamaephytes and therophytes were the most frequent, denoting a typical desert life‐form spectrum, mainly of Saharo‐Arabian distribution.
• 3 The samples were classified into eight twinspan groups. Detrended correspondence analysis (DCA) showed that these groups could be distinguished by the first two DCA axes. They could also be effectively interpreted and explained with canonical correspondence analysis (CCA). Based on forward regression, salinity, fine sediments, organic matter and soil moisture content related closely to the first three canonical axes, and accounted for 84.1% of the species–environment relationship among the stands.
• 4 Both classification and ordination resulted in a clear demonstration of the vegetation pattern in the study area. Other twinspan groups were characterized in addition to those identified previously. In conclusion, the study area has a transitional character where the Nubian Desert and the Northern Mediterranean vegetation meet.

     

Long-term excretion of vaccine-derived poliovirus by a healthy child

A child was found to be excreting type 1 vaccine-derived poliovirus (VDPV) with a 1.1% sequence drift from Sabin type 1 vaccine strain in the VP1 coding region 6 months after he was immunized with oral live polio vaccine. Seventeen type 1 poliovirus isolates were recovered from stools taken from this child during the following 4 months. Contrary to expectation, the child was not deficient in humoral immunity and showed high levels of serum neutralization against poliovirus. Selected virus isolates were characterized in terms of their antigenic properties, virulence in transgenic mice, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 1 strain. The VDPV isolates showed mutations at key nucleotide positions that correlated with the observed reversion to biological properties typical of wild polioviruses. A number of capsid mutations mapped at known antigenic sites leading to changes in the viral antigenic structure. Estimates of sequence evolution based on the accumulation of nucleotide changes in the VP1 coding region detected a "defective" molecular clock running at an apparent faster speed of 2.05% nucleotide changes per year versus 1% shown in previous studies. Remarkably, when compared to several type 1 VDPV strains of different origins, isolates from this child showed a much higher proportion of nonsynonymous versus synonymous nucleotide changes in the capsid coding region. This anomaly could explain the high VP1 sequence drift found and the ability of these virus strains to replicate in the gut for a longer period than expected.