Detection of MEF-1 laboratory reference strain of poliovirus type 2 in children with poliomyelitis in India in 2002 & 2003.

BACKGROUND & OBJECTIVES: Significant progress has been made towards eradication of poliomyelitis in India. Surveillance for acute flaccid paralysis (AFP) has reached high standards. Among the 3 types of polioviruses, type 2 had been eliminated in India and eradicated globally as of October 1999. However, we isolated wild poliovirus type 2 from a small number of polio cases in northern India in 2000 and again during December 2002 to February 2003. Using molecular tools the origin, of the wild type 2 poliovirus was investigated. METHODS: Polioviruses isolated from stool samples collected from patients with AFP were differentiated as wild virus or Sabin vaccine-like by ELISA and probe hybridization assays. Complete VP1 gene nucleotide sequences of the wild type 2 poliovirus isolates were determined by reverse transcriptase polymerase chain reaction (RT-PCR), followed by cycle sequencing. VP1 nucleotide sequences were compared with those of wild type 2 polioviruses that were indigenous in India in the past as well as prototype/laboratory strains and the GenBank database. RESULTS: Wild poliovirus type 2 was detected in stool samples from 6 patients with AFP in western Uttar Pradesh and 1 in Gujarat. In addition, the virus was isolated from one healthy contact child and from environmental sewage sample in Moradabad where three of these patients were reported. These isolates were identified as genetically closely related to laboratory reference strain MEF-1. Molecular characterization of the isolates confirmed that there was no evidence of extensive person-to-person transmission of the virus in the community. INTERPRETATION & CONCLUSION: Laboratory reference strain (MEF-1) of poliovirus type 2 caused paralytic poliomyelitis in 10 patients in September 2000 and November 2002 to February 2003. The origin of the virus was some laboratory as yet not identified. This episode highlights the urgent need for stringent containment of wild poliovirus containing materials in the laboratories across the country in order to prevent recurrence of such incidents.

Preimplantation genetic diagnosis of chromosomal abnormalities by multicolour fluorescence in situ hybridisation.

Preimplantation genetic diagnosis (PGD) is an early diagnosis of genetic disorders, prior to the onset of pregnancy. PGD incorporates the latest techniques in assisted reproduction and molecular genetics. Embryos or oocytes are biopsied during culture in vitro and genetic analysis is carried out on the blastomeres or polar bodies. Embryos shown to be free of the genetic disease under investigation are transferred to the uterus. Multicolour fluorescence in situ hybridisation (FISH) is used to diagnose numerical and certain structural abnormalities of chromosomes in the embryo. The common probes used are for chromosomes 13, 18, 21, X and Y. FISH can also be used for PGD of translocations, when one of the parents is a carrier. PGD was carried out recently in 4 cases using multicolour FISH. In one of the embryos, trisomy 18 was detected. Tetraploidy was seen in another embryo. Only chromosomally normal embryos were transferred back to the uterus. Care has to be taken while interpreting FISH signals as the signal may be split, diffused, superimposed or in a different focus.

Effect of passively transferred anti-poliovirus antibodies on seroconversion.

A prospective study enrolling 50 mother-infant pairs was undertaken to determine the effect of maternal antibodies on poliovirus antibody titres and seroconversion rates in infants and to determine the difference in titres and seroconversion rates following three and five doses of oral poliovaccine (OPV). Cord blood samples and samples collected 4 weeks after 3rd and 5th doses of trivalent oral poliovaccine were processed for estimation of anti-poliovirus antibody titres. These were expressed as geometric mean titres (GMT). Significance was analyzed using unpaired 't' test. The relationship between maternal antibody titres and seroconversion was determined by correlation coefficient test. Post OPV5 titres were significantly higher than post OPV3 titres for type 1 and type 2 polioviruses. Seroconversion rates against type 1, 2 and 3 polioviruses were 92.9%, 100.0% and 92.9% following OPV3 and 100.0%, 100.0% and 93.2% following OPV5. The cord blood titres did not have any relation to post-OPV3 or post-OPV5 titres. Although there is significant passive transfer of poliovirus antibodies across the placenta, this does not affect titres achieved after immunization. Post-OPV5 titres against type 1 and type 2 viruses are significantly higher than post-OPV3 titres. The seroconversion rates following OPV5 are higher than those obtained post-OPV3 but this difference is not statistically significant.

Absence of wild poliovirus circulation among healthy children in a rural area with high oral poliovirus vaccination coverage.

Poliovirus circulation in a rural community was studied by a stool sample survey. No acute paralytic poliomyelitis case had been reported from the study area during the previous 5 months. Immunization coverage in age groups 7 to 12 months and 12 to 60 months was 95.8 and 94 per cent, respectively. Of the 257 children from whom stool samples were collected (about 6% of the child population), 161 (62.6%) were positive for virus isolation. Poliovirus was isolated from 60 (23.3%) children. All three poliovirus types were detected (41 type 1, 16 type 2 and 3 type 3). Intratypic differentiation tests classified these isolates as vaccine-like. Among the children excreting poliovirus, the proportion of those who did not receive polio vaccine within 30 days prior to the sample collection was 46.3, and 68.7 per cent for poliovirus type 1 and 2, respectively. It was concluded that these poliovirus excreting children were infected by the vaccine strains circulating in the environment. The survey showed that wild poliovirus was not detectable within five months after the last case of acute poliomyelitis. Displacement of the wild virus from the environment and circulation of vaccine virus was achieved by high vaccination coverage in this area.

An evaluation of cold chain in Maharashtra & Karnataka states by potency testing of field samples of oral poliovirus vaccine.

The cold chain for oral poliovirus vaccine was monitored in Maharashtra and Karnataka by potency testing of vaccine vials collected from various stages of the delivery system. Results showed that cold chain maintenance improved in the state of Maharashtra within a period of three years as the monitoring began in 1987. Of the 6289 samples of trivalent OPV collected from all 30 districts of the state during 1990 to 1992, 5834 (92.8%) had retained virus titre of at least 10(5.81) TCID50/dose. In comparison, 72 per cent of the 1660 samples collected from the state of Karnataka during the same period were found to contain this minimum required virus titre. Defects in cold chain maintenance in Karnataka could be demonstrated by plotting virus titre of samples of individual batches collected from different outlets. It was concluded that potency retesting of OPV samples for cold chain monitoring will ensure proper storage, transport and use of potent vaccine in the field.

Prevalence of antibodies to polioviruses & enteroviruses excreted by healthy children in Bombay.

A total of 132 healthy children between the ages one month and 12 yr were surveyed to determine the prevalence of antibodies to the three poliovirus serotypes. Among infants up to six months of age, 73.2, 85.4 and 56.1 per cent had antibodies to poliovirus types 1, 2 and 3, respectively. In children of age groups 7 months to 3 yr and above 3 yr, antibody prevalence to the three poliovirus serotypes was 90.2, 86.9 and 57.4, and 83.3, 96.7 and 76.7 per cent, respectively. Immunization coverage with three doses of OPV exceeded 85 per cent in children above 7 months of age. Low seroprevalence to type 3 poliovirus in the children was conspicuous. Of the 80 faecal samples studied from these children, 24 (30%) were positive for virus. Among these isolates, 16 were poliovirus type 1 and three type 2. Intratypic differentiation revealed that 15 of the 16 poliovirus type 1 isolates were of wild origin. Two out of the three poliovirus type 2 isolates were of oral poliovaccine origin. Our data indicate that in spite of good vaccination coverage wild poliovirus type 1 circulation was endemic in Bombay and; that a large number of children were susceptible to poliovirus type 3 infections.

Regulation of sialyltransferase activity in intestinal segments of rats.

A differential distribution of sialyltransferase (ST) in different regions of intestine has been shown. Jejunum and ileum homogenates from rats showed almost exclusive presence of alpha-2-3 ST (to Gal in Gal beta-1-4GlcNAc and/or to Gal in Gal beta-1-3GalNAc). In contrast, colon homogenates showed the presence of both alpha-2-3 ST (as above) and alpha-2-6 ST. Incubation of intestinal slices in presence of heat-inactivated horse serum (HHS) showed a time- and temperature-dependent secretion of soluble ST into the medium. Both jejunum and ileum slices showed high rates of secretion of alpha-2-3 ST. Colon slices, though rich in alpha-2-6 ST, secreted only alpha-2-3 ST. Colchicine, an anti-mitotic drug, injected into rats caused about 10-fold increase of the serum ST level. Jejunum slices from colchicine-treated rats showed an increased secretion of alpha-2-6 ST, suggesting that intestine undergoes a change in the expression of normal secretion of alpha-2-3 ST to a secretion of alpha-2-6 ST. The secretion of ST from incubated intestinal slices was inhibited by heparin. Certain protein factors (anti-proteases) in HHS bind to heparin-sepharose column and these protein factors are responsible for causing the secretion of ST into the medium. It has also been found that a supernatant fraction of the colon homogenate activated ST. Gel chromatography on HPLC produced 3-4 protein fractions from the colon cytosol and one of this fraction bearing high molecular weight proteins produced the maximum activation of ST.(ABSTRACT TRUNCATED AT 250 WORDS)