Molecular characterization of intestinal viruses causing acute flaccid paralysis in children isolated directly from stool samples without the use of tissue transplantation technique

 

Inventors: Assis. Prof. Dr. Hala Younis Fadhil/Department of biology, College of science, University of Baghdad

Abstract

The Enteroviruses (PV+ NPEVs) presently by a large number of different serotypes are currently the most common cause of acute flaccid paralysis in children. Currently, the Iraqi health institutions depended on their isolation for these viruses on the tissue culture technique from stool samples as an indicator for the viral infection diagnosis. But, this method represented with disability of detection for the false negative isolates, and weak ability for the weak positive isolates detection in the tissue culture. Beside for the long time periods and the high cost demanded for this technique and the unavailability of cells along the year. From other hand, the uses of serological methods in the previous researches do not provide inclusive and accurate detection for the all serotypes of this viral family, next to its easily affected with the genetic mutations in the viral Ribonucleic acid. So that, this study aimed to modify the way of enteroviruses detection from the tissue culture technique to the molecular detection technique, by isolation of the viral particles directly from the stool samples without culturing it to compensate for multiplication of the viruses by the tissue culturing method by concentrating the viral particles load with filtration and getting the concentrated filtrate that contain the enteroviral particles only, that in turn leads for exploitation of the founded laboratory materials in the Central Public Health Laboratory to extract the viral RNA from the viral particles suspension then detect the viral particles by RT- PCR technique by using a pair of primers specific for the non coding region (NCR) of the viral nucleic acid. Also, its aimed to performing the nucleotide sequence analysis for the viral nucleic acid of the non polio enteroviruses group only by using pair of primers for the fourth viral capsid protein gene (VP4 primers) to genotyping the most important strains, that coordinated with residual paralysis cases and death cases. Then classifying it by the phylogenetic tree and documenting it for the first time in Iraq. The samples were collected from the infected children stool and grouped according to its viral particles isolation method into two groups, the first group (the viral particles suspension) prepared directly from stool samples and the second group (the cell suspension group) prepared from the harvesting of the suspension from the cell line after its inoculation with the viruses. That in turn classified into strong positive isolates, weak positive isolates and false negative isolates to be compared with the viral particle suspension molecular detection results. The results of the RT- PCR technique for the viral particle suspension showed 100% diagnosis to all the positive isolates, the strongly and weakly growing in the cell culture isolates with high quickness and accuracy. From other hand, 35% of the false negative isolated of the cell culture technique diagnosed as positive results, in which some of their cases show residual acute flaccid paralysis in the infected children. As well, RT- PCR method of the viral particle suspensions overcame the problems of the cell culture technique from the time, cost, contamination and the demanded efforts sights. The nucleotide sequence analysis technique detect repeated nine of seventeen genotype genotypes of the non- polio enterovirus genus from type B, which Involved echovirus genotype that include (E- 4, E- 9, E- 14, E- 7, E- 20) and Coxsackie virus genotype that include Cox- B1 only, and new genotypes included (EV-B “unknown type” ,EVB- 74 and EVB- 11). This study documented two death cases for NPEVs group that harmonized with Echovirus- 4 and EVB- 74 strains which distinguished with high appearance of mutations in their RNA during its comparison with their matched international strains of the NCBI. Also, EVB- 111 strain was documented in this study which presented as a rare strain coordinated with residual acute flaccid paralysis case. In the false negative isolates, Echovirus- 20 and Cox- B1 strains were noticed repeatedly and this pointed on the hidden danger of these viruses that is not detected clearly by the traditional cell culture technique in compassion with the molecular ways that coordinated with directly isolated viral particle.