الفهرس 
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Abstract
Rotavirus group A infections are still the primary cause of acute gastroenteritis in infants and children under the age of five in both developed and developing countries, with symptoms ranging from asymptomatic infection to severe dehydration or death. According to epidemiological studies, this RNA virus is the top leading cause of non-bacterial gastroenteritis, accounting for more than 50% of all occurrences of intestinal viral infection in children. This study aimed to identify and isolate RVA genotypes circulating in a cohort of Egyptian children ranging from birth to 5 years of age admitted to two major university pediatric hospitals (Abo El-Reesh and El Demerdash).
A total of 230 fecal samples [128 males and 102 females] were obtained over two consecutive winters (December 2018 to April 2020) from children under the age of five who were hospitalized in the aforementioned public hospitals in the Great Cairo area. Diarrheal samples (n=230) were screened for RVA RNA using monoplex one-step RT-PCR for detection of VP6 and NSP4 fragment. Rotaviruses group A accounted for over one sixth (n=34) of all cases with acute gastroenteritis, with an incidence of 14.8%. It was obviously notable that the number of RVA cases among males (18%) was much higher than those detected in females (10.8%) with no significant difference statistically. The odds of infection was 1.8 times among males compared to females. Most of positive RVA cases were under the age of two years. By stratification of age into age groups, the highest rate of infection was among children 6-12 months (29.2%) followed by infants 0-6 months (17.6%) and the least affected group were those above 12 months 6.9% and the difference was highly significant statistically p<0.01. The odds of infection was 5.5 times higher in children 6-12 months compared to those above 12 months. Also the odds of infection of infants 0-5 months was 2.9 times higher compared to older age group >12 months. Rotavirus group A was discovered throughout the year except for the summer season; nevertheless, there was an evident seasonality of rotavirus diarrhea, with a larger prevalence in winter (18.1%), followed by spring (15.1%), and autumn (7.3%). The present cross-sectional study included more children from metropolitan regions (Cairo and Giza) rather than the rural areas in either the RVA (34 cases) or the non-RVA (196 cases) groups. All positive samples were subjected to G/P typing using a multiplex two step RT-PCR technique for detection of VP4 and VP7 fragments. Only 40% of the samples were typeable for G types and 56% were typeable for P types. G1P[8] was the only detected genotype in the present study.
All PCR positive RVA samples were subjected to isolation and propagation using both MA104 and Caco-2 cell lines. An initial three isolation passages were performed on MA104 cells for selective RV isolation from clarified stool samples, followed by Caco-2 propagation passages. In Caco-2 cells, 5 out of the 34 (14.7%) RVA strains were isolated on the first passage, and all five strains passed the second and third passage well. On the other hand, MA104 cells did not support the growth of the virus on the first passage. However, 3 out of the 34 (8.82%) strains identified on MA104 cells grew on the second passage, while only 2 (5.9%) grew on the third one. It should be noted that the strains (n=5) propagated successfully on Caco-2 cells were the same as the strains propagated on MA104 cells and those strains were used to complete the present study. Caco-2 cultures supported rotavirus proliferation of a substantially higher number of isolates than MA104 cells in the first and second passages, but the difference was not statistically significant. It should be emphasized that the isolated RVA strains exhibited different degree of virus-induced CPE, where some strains showed complete destruction of the mammalian cell monolayer, others produces partial destruction.
The identity of all isolated RVA strains was confirmed by multiplex RT-PCR using VP4 specific primers. Sequence analyses of some RVA genes (VP6, VP4, VP7, NSP3, NSP4, NSP5) were carried out. The nucleotide sequences of these fragments were submitted to the GenBank database. They were accepted and reported under the accession codes: OL963766, OL963767, ON152314, OL963768, ON152315, ON152316. The amino acids’ multiple alignments of RVA proteins obtained from the final consensus of the above mentioned genes against their homologous proteins of human rotaviruses, found in the GenBank database, were carried out using Clustal Omega alignment tool. The alignments showed similarity ranging from 99.49% to 99.90%. Phylogenetic analyses for each gene were generated by Maximum Likelihood (ML) approach using MEGA X software v.10.1.7.
Afterwards, all isolated RVA strains were subjected to qPCR to confirm RVA isolation step and relatively quantify the number of copies of two genes (VP6 and VP4) in each isolate. RVA144 strain had the lowest Ct when using VP6 fragment. End point dilution assay (TCID50) was carried out to measure the infectivity of the isolated RVA strains and to assess viral titer before vaccine preparation. RVA144 titer was 104 TCID50/ml, however, the titer of the other strains did not exceed 102 TCID50/ml. Therefore, RVA144 strain was selected as candidate for IRVV preparations. For efficient vaccine production, RVA144 titer was raised by multiple passages on Caco-2 cell monolayers to reach 105 TCID50/ml. For bulk production, the selected strain was progressively propagated at MOI of 0.1 on Caco-2 cells. The virus bulk was harvested, clarified and chemically inactivated using BPL 1:3000 (v/v). Alum hydroxide AL(OH)3 was added as an adjuvant to the parenterally administered IRVV.
The immunogenicity of the IRVV trial preparations was experimentally tested in mice, which is considered as well-grounded model for studying viral vaccines immunization. Six tested groups, 10 mice in each, were included in the experiment. Groups (GPs) I and II; included mice vaccinated subcutaneously with 0.5 and 1 ml IRVV, respectively. GPs III and IV; included mice vaccinated subcutaneously with 0.5 and 1 ml of Alum-adjuvanted IRVV formula, respectively. GPs V and VI; included mice vaccinated orally with 1 and 1.5 ml IRVV, respectively. Three control groups (10 mice each) were included. GP VII (reference vaccine); included mice vaccinated orally with 1.5 ml Rotarix®, to compare the immunogenicity of the prepared IRVV to the mentioned reference vaccine available in the market. GP VIII (Placebo 1); included mice vaccinated with vaccine preparation medium GP IX (Placebo 2); included mice vaccinated with sole Alum in vaccine production.
The same vaccine preparation and dose regimen was used for the two booster vaccinations. Each GP was boosted with the second and third doses of the same initial preparations used at 4-weeks intervals (on 0, 28th, 56th day). Blood was collected at 2-weeks intervals after the prime immunization and continued up to 3 weeks post the final booster shot (on 0, 14th, 28th, 42th, 56th, 70th, 84th day) for total IgG evaluation studies. The total serum IgG-antibodies were evaluated using homemade ELISA where the plates were coated overnight with the inactivated RVA144 suspension. In the present study, the inactivated RV candidate elicited substantial levels of serum antibody responses in mice. The seroconversion of the immunized mice was deduced from ELISA readings. Comparisons between the different studied groups regarding the seroconversion at different time intervals were carried out. The seroconversion post-vaccination with non-adjuvanted IRVVs trials given subcutaneously (GPs I and II) was maximized at the middle and late immune stage. GP II seroconversion was maximized earlier than the lower one at the middle immune stage, however, both reached the maximum titer at the same time. The seroconversion post-vaccination with adjuvanted low dose IRVV trial given subcutaneously (GP III) exceeded the corresponding group of higher dose (GP IV). Moreover, there was a great difference in the seroconversion between GP III and the reference one (GP VII) and the difference was highly significant statistically. This denoted the higher immune response to the low dose adjuvanted IRVV given subcutaneously (GP III) compared to the high dose one as well as the commercially available vaccines oral effect. The seroconversion of adjuvanted low dose IRVV trial given subcutaneously was higher than the non-adjuvated one which highlights the importance of Alum as an adjuvant in IRVV preparation. Regarding the oral IRVVs (GPs V and VI), the maximum seroconversion was observed after the first booster dose, however the second booster one did not affect the immune response obviously and the titer began to DROP after nearly two weeks from the first booster vaccination. GP VI had a higher mean response compared to the reference (GP VII, Rotarix®) group with no statistically significant difference. The mean endpoint IgG dilution titers of the studied groups were compared to give an overview on the overall effect of each studied group. A significant mean seroconversion was noticed post-vaccination with adjuvanted low dose IRVV trial given subcutaneously (GP III) when compared to other groups. The oral IRVV (GP VI) had higher mean seroconversion when compared to other low dose and reference Rotarix ® GPs with no statistically significant difference.
Finally, statistical analysis was carried out using the SPSS program version 23. Quantitative data was summarized by mean, standard deviation while qualitative data was summarized by frequencies and percentages. The statistical analyses used included Student t test, Chi square test, one way analysis of variance (ANOVA) and Bonferoni test.