Genetic basis of the neurovirulence of type 1 polioviruses isolated from vaccine-associated paralytic patients

Summary We examined four type 1 polioviruses isolated from the stools of patients with vaccine-associated paralytic poliomyelitis in China. All of these isolates were shown to be Sabin derived viruses by restriction fragment length polymorphism assay after polymerase chain reaction and by sequencing of the viral genome encoding the viral coat protein, VP1. However, the same analysis of the 3D coding region suggested that two of the four isolates had the sequence of wild type poliovirus in the tested region. Furthermore there were also point mutations in the 5 non-coding region. One was a single base change from U to C at nucleotide position 525, and the other three were from G to A at position 480. All the four strains were more neurovirulent than Sabin type 1 virus in transgenic mice with human poliovirus receptor gene. The data showed that the nucleotide positions of type 1 poliovirus which were identified to be in favor of the high neurovirulence were indeed changed during natural transmission, and suggested that the point mutation alone or a recombination of the vaccine type with wild type genome results in an acquisition of neurovirulence.     

Three cases of paralytic poliomyelitis associated with type 3 vaccine poliovirus strains in Bulgaria

Oral poliovirus vaccine (OPV) can cause, in extremely rare cases vaccine‐associated paralytic poliomyelitis in recipients, or contacts of vaccinees. Three cases of vaccine‐associated paralytic poliomyelitis (two contacts and one recipient) occurred in the Bourgas region of Bulgaria in the spring of 2006. The first two cases, notified as acute flaccid paralysis, were 55 days old unvaccinated twin brothers, having been in contact with vaccinees. The third case concerned a 4‐month‐old infant who had received the first OPV dose 37 days prior to the onset of illness. Complete clinical, epidemiological, virological, serological and molecular investigations of the children with paralysis and their contacts were undertaken. In all the three cases type 3 polioviruses were isolated from fecal samples and characterized as Sabin‐like poliovirus strains. Type 3 polioviruses isolated from the twin brothers demonstrated by sequence analysis U‐to‐C back mutation at nt 472 of the 5′ UTR, known to correlate with neurovirulence, and mutation in the VP1 region. Type 3 poliovirus isolated from the third child demonstrated in the 3D sequenced region a recombination with Sabin type 1 poliovirus. In the latter region, three silent mutations and one, resulting in amino acid substitution, were also observed. The clinical, epidemiological and virological data and the neurological sequelae observed 60 days following the onset of paralysis, confirmed the diagnosis of vaccine‐associated paralytic poliomyelitis in all the three patients. J. Med. Virol. 81:1661–1667, 2009. © 2009 Wiley‐Liss, Inc.     

Detection of unusual mutation within the VP1 region of different re-isolates of poliovirus Sabin vaccine

In the present study, a genomic analysis of full VP1 sequence region of 15 clinical re-isolates (14 healthy vaccinees and one bone marrow tumor patient) was conducted, aiming to the identification of mutations and to the assessment of their impact on virus fitness, providing also insights relevant with the natural evolution of Sabin strains. Clinical re-isolates were analyzed by RT-PCR, sequencing and computational analysis. Some re-isolates were characterized by an unusual mutational pattern in which non-synonymous mutations outnumbered the synonymous ones. Furthermore, the majority of amino-acid substitutions were located in the capsid exterior, specifically in N-Ags, near N-Ags and in the north rim of the canyon. Also mutations, which are well-known determinants of attenuation, were identified. The results of this study propose that some re-isolates are characterized by an evolutionary pattern in which non-synonymous mutations with a direct phenotypic impact on viral fitness are fixed in viral genomes, in spite of synonymous ones with no phenotypic impact on viral fitness. Results of the present retrospective characterization of Sabin clinical re-isolates, based on the full VP1 sequence, suggest that vaccine-derived viruses may make their way through narrow breaches and may evolve into transmissible pathogens even in adequately immunized populations. For this reason increased poliovirus laboratory surveillance should be permanent and full VP1 sequence analysis should be conducted even in isolates originating from healthy vaccinees.     

Characterization of echovirus 25 (ECV 25) in the VP1/2A gene junction region

Summary.  Genetic relationships among echovirus type 25 (ECV 25) isolates associated with aseptic meningitis in Germany 1997/98 and a 40-year-old ECV 25 prototype strain were investigated using RT-PCR and direct sequencing the VP1/2A gene junction region. Sequences were compared to each other and to nonpolio enterovirus representatives (phylogenetic analysis). The analysis indicated that the sequences of recent isolates have drifted over time distinctly away from the prototype strain sequence. Genetic drift may change biological features of isolates possibly leading to new antigenic variants. Received April 6, 1999 Accepted June 8, 1999     

Isolation of vaccine-derived type 1 polioviruses displaying similar properties to virulent wild strain Mahoney from sewage in Japan

Type 1, 2, and 3 vaccine-derived polioviruses were isolated from a sewage disposal plant located downstream of the Oyabe River in Toyama Prefecture, Japan, between October 1993 and September 1995. Neurovirulence was analyzed in 13 type 1 vaccine-derived strains, using mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC). Nine strains (69%) were estimated to have marked neurovirulence. Some of the neutralizing antigenic sites, temperature sensitivity, and plaque-forming ability of two virulent vaccine-derived poliovirus strains were similar to Mahoney strain. The neutralizing activity of human sera obtained after oral poliomyelitis vaccine (OPV) administration against one of the virulent vaccine-derived polioviruses was examined. Although all human sera showed sufficient neutralizing activity for the prevention of poliomyelitis by vaccine-derived poliovirus strains, a lower titer than that against Sabin type 1 strain was observed. Vaccination against virulent vaccine-derived poliovirus will be effective. However, the environmental presence of viruses that have properties similar to those Mahoney strain is a threat. The introduction of inactivated poliovirus vaccine (IPV), and well-maintained herd immunity, together with reinforced environmental surveillance is important for the final phase of the polio eradication program by the World Health Organization (WHO).     

The results of genetic marker estimations of type 1 polioviruses isolated from paralytic patients in Czech regions in the period after the start of the first field trial with Sabin's oral vaccine in Czechoslovakia

Summary The results of the investigation of 32 type 1 viruses isolated in paralytic patients in the course of 15 months after first feeding type 1Sabin's vaccine are presented.The results of the rct marker determination indicate that 20 of these strains possessed the rct+character, 9 rct±Character and 3 rct—character.In the intratypic serodifferentiation test all strains tested were distinguished from the LSc 2 ab virus and also from the Mahoney virus, the parental strain of LSc 2 ab virus (type 1Sabin's vaccine).On the basis of these data the suggestion is made that the type 1 viruses investigated are not related to the vaccine strain.     

Characterisation of germline mutations in the neurofibromatosis type 1 (NF1) gene.

Neurofibromatosis type 1 is one of the most common inherited disorders with an incidence of 1 in 3000. The search for NF1 mutations has been hampered by the overall size of the gene, the large number of exons, and the high mutation rate. To date, fewer than 90 mutations have been reported to the NF1 mutation analysis consortium and the details on 76 mutations have been published. We have identified five new mutations using single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) and three intragenic deletions with the microsatellite markers. Of the five new mutations, two were in exon 27a, two in exon 45, and one in exon 49 and these include 4630delA, 4572delC, R7846X, T7828A, and one in the 3' untranslated region (3' UTR). The two nucleotide alterations in exon 27a and the one in exon 45 are predicted to produce a truncated protein.     

Genomic sequence of an avian paramyxovirus type 1 strain isolated from Muscovy duck (Cairina moschata) in China

The complete sequence of an avian paramyxovirus type 1 (APMV-1) strain, FP1/02, isolated from Muscovy duck in China, was determined. Sequence analysis indicated that the complete genome of strain FP1/02 contained 15,192 nucleotides (nt), following the rule of six. The genome contained an extra 6-nt insertion in the non-coding region of the NP gene when compared with other APMV-1 strains, such as strains La Sota and Beaudette C. The cleavage site of the F protein was (112)R-R-Q-K-R↓F(117), indicating that the FP1/02 strain was virulent, but the morbidity and mortality varied with the species of duck. Genotypic analysis based on the F gene revealed that APMV-1 FP1/02 was a member of genotype VII. Phylogenetic analysis showed that the FP1/02 strain shared high identity with other APMV-1 strains such as ZJ1, SF02 and NA-1 isolated from geese.     

Alteration in oligonucleotide fingerprint patterns of the viral genome in poliovirus type 2 isolated from paralytic patients.

A close relationship was demonstrated by oligonucleotide fingerprinting between genomes of the poliovirus type 2 Sabin vaccine strain and recent isolates from paralytic cases associated with vaccination in Japan. The oligonucleotide maps of isolates from an agammaglobulinemic patient, who continued to excrete poliovirus type 2 for 3.5 years after the administration of oral vaccine, showed that the genomic alteration proceeded gradually, retaining the majority of the oligonucleotides characteristic of the vaccine strain for a long period, indicating vaccine origin for the isolates. The final isolate at month 41, however, lost the majority of these oligonucleotides. The heterologous antigenic relationship between the final isolate and the previous isolates was also observed. The serial alteration in electrophoretic mobility of the major structural proteins (VP1, VP2, and VP3) was observed throughout the excreting period. These results indicate that the population of the virus in this individual changed markedly during the last short period (about 3 months), in which the treatment with secretory immunoglobulin A was carried out. Genome comparisons in oligonucleotide maps show that some oligonucleotides in the genome of the vaccine strain are highly mutable after passage in humans.     

Characterization of four mutations in the neurofibromatosis type 1 gene by denaturing gradient gel electrophoresis (DGGE)

Neurofibromatosis type 1 (NF1) Is one of the most common Inheriteddisorders. The gene responsible for the disease has a very highmutation rate, approximately fifty per cent of NF1 patientsappear to have a de novo mutation. The search for mutationsis hampered by the large size of the NF1 gene and up to date,relatively few mutations have been characterized. In the presentwork, we report the results of screening seventy unrelated NF1patients for mutations in NF1 exons 29 and 31 by using an experimentalapproach that combines the polymerase chain reaction (PCR) withdenaturing gradient gel electrophoresis (DGGE). Four mutationswere Identified and characterized. Three of them consist ofC-T transitions resulting in nonsense mutations, two In exon29, C5242T and C5260T, and one In exon 31, C5839T. The fourthmutation consists of a two-base pair deletion In exon 31, 5843delAA,also resulting in a premature stop codon. The finding In ourpatients of mutation C5839T, previously reported In three Independentstudies, supoports that thisposition is a hotspot within theNF1 gene.     

Characterization of a neurotoxigenic Clostridium butyricum strain isolated from the food implicated in an outbreak of food-borne type E botulism.

Neurotoxigenic Clostridium butyricum was isolated from the food implicated in an outbreak of clinically diagnosed type E botulism in China. PCR assay showed that the isolate (LCL 155) contained the type E botulinum toxin gene. This appears to be the first report of neurotoxigenic C. butyricum causing food-borne botulism.     

Genetic variability within the VP1 coding region of echovirus type 30 isolates

Summary.  Genetic relationships of the prototype Bastianni strain of 1958 and of 13 echovirus type 30 (ECV30) isolates associated with meningitis cases in Germany during a period from 1966 to 1997 were investigated using direct sequencing of amplicons derived from a part of the capsid protein VP1 gene. Sequences were aligned both with each other and with known sequences of other type 30 echovirus strains. Phylogenetic analysis indicated that isolates investigated in this work fell into at least three genetic clusters apart from the prototype Bastianni strain. This suggests that genetically distinct groups of ECV30 variants have developed over time. Received January 17, 2000 Accepted February 22, 2000     

Natural variants of the Sabin type 1 vaccine strain of poliovirus and correlation with a poliovirus neutralization site

Independent substitution mutations have been detected in capsid polypeptide VP1 of the type 1 oral poliovirus vaccine isolated from normal infant vaccine recipients. These mutations map at amino acid residues 142 and 147 of VP1, a region only minimally hydrophilic. A synthetic peptide, corresponding to residues 141 to 147 of VP1 was synthesized, conjugated to a carrier polypeptide of bovine serum albumin. The conjugate was found to elicit a weak poliovirus neutralizing antibody response. It was also capable of priming the immune system for the production of IgG-type antibodies able to neutralize greater than 99.999% of infectious type 1 virus. It is suggested that region 141 to 147 of VP1 may be involved in neutralization of the virus and that the mutants may have accumulated by antibody selection.     

Characterization of the neurotoxin isolated from a Clostridium baratii strain implicated in infant botulism.

Botulism is widely known to result from ingestion of food containing botulinum neurotoxin produced in situ by certain strains of Clostridium botulinum. Infant botulism caused by C. botulinum, unlike the food-borne intoxication, is the toxicoinfectious form of botulism (S. S. Arnon, p. 331-345, in G. E. Lewis, ed., Biomedical Aspects of Botulism, 1981). The strain of Clostridium baratii implicated in infant botulism produced a neurotoxin that was neutralized with antiserum for botulinum neurotoxin serotype F (J. D. Hall, L. M. McCroskey, B. J. Pincomb, and C. L. Hatheway, J. Clin. Microbiol. 21:654-655, 1985). We developed a procedure to culture the toxigenic C. baratii (strain 6341) in dialysis bags and a simple purification scheme (precipitation of 900-ml culture supernatant with ammonium sulfate and two anion-exchange chromatographic steps at pH 5.5 and 8.0) that yielded up to 150 micrograms of purified neurotoxin. It is an approximately 140-kDa single-chain protein and has the following sequence of amino acid residues at the N terminus: Pro-Val-Asn-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asn-Asn-Thr-Thr-Ile- Leu. Comparison of this amino acid sequence with those of the botulinum neurotoxin serotypes A, B, and E showed 40 to 50% identical residues in comparable positions. The specific toxicity of the neurotoxin, approximately 2 x 10(6) 50% lethal doses for mice per mg of protein injected, was not enhanced significantly by mild trypsinization, although the protease cleaved the neurotoxin within a disulfide loop that generated at least two primary fragments, approximately 47 and approximately 86 kDa, that remained linked by an interchain disulfide. These two fragments resembled the light and heavy chains of the well-characterized neurotoxin serotypes A, B, C, D, E, and F produced by C. botulinum.     

Characterization of four herpesviruses isolated from owl monkeys and their comparison with Herpesvirus saimiri type 1 (Herpesvirus tamarinus) and herpes simplex virus type 1

Four herpesviruses were previously isolated from four outbreaks of lethal disease in owl monkeys. All four isolates have been shown to be antigenically closely related to each other and to Herpesvirus saimiri 1 (HVS-1) by kinetic neutralizations. The owl monkey strains also share similarities to HVS-1 and to each other with respect to host range, growth cycles and molecular weights of peptides and of DNA fragments generated by restriction endonuclease (R.E.) digestion. R.E. analysis, however, can differentiate strains by the use of certain enzymes. All four isolates share a common G-C ratio percentage with HVS-1 of 67 per cent. On the basis of these findings, we believe that these owl monkey virus isolates are strains of HVS-1.     

Characterization of a Legionella anisa strain isolated from a patient with pneumonia.

Legionella anisa, previously found only in environmental specimens, was isolated from a bronchial lavage specimen of an immunocompromised patient with pneumonia. Growth, physiologic, gas-liquid chromatographic, serologic, and DNA characteristics were consistent with those of the type strain of L. anisa, WA-316-C3 (ATCC 35292).