REOVIRUS Neira- Una Hrapović Kanita Šabanović
Contents: Introduction to Reovirus Rotavirus Virion Rotavirus Replication Attachment and Entry Early and Late Events Overview Diseases Other dsRNA viruses
Introduction to reoviruses Icosahedral viruses with dsRNA genomes isolated from the respiratory tracts and enteric tracts of humans and animals, and with which no disease could be associated, became known as reoviruses. A large number of similar viruses have been found in mammals, birds, fish, insects, plants and fungi. Many of these viruses are causative agents of disease. Original name - Reoviridae
The original reoviruses are incorporated into the genus Orthoreovirus. Most orthoreovirus infections of mammals are asymptomatic. The majority of humans become infected with orthoreoviruses early in life and have specific serum antibodies by early adulthood.
Family Reoviridae
Rotavirus virion Rotaviruses were first described in 1963, when they were observed during electron microscopy of faecal samples from monkeys and mice. Spherical virions at 75 nm in diameter, with structures similar to wheel. The virion, has triple-layered particle as the capsid: three layers, each constructed from a distinct virus protein (VP) The inner and middle layers, VP2 and VP6, are perforated by channels The middle layer contains the ‘spokes’ of the ‘wheel ’ The outer layer VP7
Rotavirus replication Rotaviruses infect cells called enterocytes at the ends of the villi (finger -like extensions) in the small intestine
Attachment and entry The mechanisms by which rotaviruses attach to and enter their host cells are complex There are two possible ways in which a virion can enter the cell: I. direct penetration of the virion across the plasma membrane and II. endocytosis
Entry
Early transcription, translation, and assembly of double-layered particles
Late events
Overview
Rotavirus diseases Destroyed enterocytes on vili Reduced absorption of water, salts and sugars from the gut Tight junctions between cells damaged by NSP4 Leakage of fluid into the gut Diarrhea Treatment not everywhere available: half a million deaths of infants and young children Infect other tissues
Other dsRNA viruses Icosahedral symmetry, most of them naked Exception: Cystoviridae Problem: dsRNA viruses - potent inducer of a number of cell defense mechanisms (apoptosis, interferon production, RNA silencing) Overcome: enclosed within virus protein structures, never free in the cytoplasm to trigger these defenses
PAPER DISCUSSION “Molecular characteirisation of rotavirus strains detected during clinical trial of the human neonatal rotavirus vaccine in Indonesia”
Contents: Introduction Methods and Materials Results Discussion
INTRODUCTION The RV3-BB human neonatal rotavirus vaccine aims to provide protection from severe rotavirus disease from birth. The aim of the study was to characterise the rotavirus strains causing gastroenterits during Indonesian Phase IIb efficiency trial.
Rotavirus is the most common cause of gastroenteritis in the children under the age of five. Rotavirus vaccines have been introduced into a national immunisation programs of 92 countries globally. RV3-BB vaccine is based on naturally attenuated asymptomatic neonatal G3P rotavirus strain, first identified in Melbourne. A randomized, placebo-controlled trial to evaluate the efficiency of an oral human-strain neonatal rotavirus vaccine was recently completed in Indonesia.
Vaccine efficiency against severe rotavirus gastroenteritis from 2 weeks after dose 3 and 18 months of age was 63% in the combined vaccine group, 75% in the neonatal vaccine group and 51% in the infant vaccine group. The rotavirus strains demonstrate significant genetic diversity. The rotavirus genome is comprised of 11 segments of double stranded RNA, encoding six structural and six non structural proteins.
Method and materials 1) Study design and participants Double-blind placebo-controlled trial inolved 1649 participants was conducted (January 2013-July 2016) in Indonesia. Healthy full term babies 0-5 days of age, birth weight of 2.5-4.0 kg) were randomized into one of three groups: Neonatal vaccine groups Infant vaccine groups Placebo group The investigation product (IP) consisted of RV3-BB vaccine or Placebo 2) Sample collection and processing Samples were collected from at least two faecal samples from skin or nappy, stored in specimen container. 3) Rotavirus antigen testing Tested using commercial rotavirus enzyme immunoassay (EIA) ProSpecT
4) Rotavirus genotyping Viral RNA was extracted from 10% and 20% faecal extracts of each specimen using Viral Nucleic Acid Extraction Kit II 5) Polyacrilamide gel electrophoresis 11 segments of of rotavirus dsRNA were separated on 10% polyacrylamide gel with 3% polyacrylamide stacking gel at 25mA for 16h. 6) Amplification of complete rotavirus genomes The 11 gene segments were reverse transcribed and amplified by PCR. 7) Nucelotide sequencing 8) Phylogenic analysis 9) Accession numbers
Results 1649 participants, 1640 received at least one dose of IP and 1588 were followed to 18 months There were 1110 unique episodes of gastroenteritis Rotavirus enzyme immunoassay (EIA) antigen testing was performed on 1246 stool samples 9.5% - rotavirus positive 21.9% - episodes in the neonatal vaccine 27.6% - infant vaccine and 50.4% - in the placebo schedule
Results Most common genotype identified was G3P[8], this genotype represented 85.7% rotavirus strains Majority of severe rotavirus gastroenteritis cases score - due to a G3P[8] strain Both Indonesian strains clustered with contemporary human equine-like G3P[8] strains from Australia, Brazil, Japan, Spain, Thailand sharing > 99.1% nucleotide identity The equine strain - 90.6% nucleotide identity
Discussion RV3-BB provided protection against severe gastroenteritis in a Phase IIb efficacy trial, caused by G3P[8] strain G3P[8] inter-genogroup reassortant, containing equine-like G3 VP7, a P[8] VP4 gene and a genogroup 2 backbone Not been previously reported in strain surveillance conducted in Indonesia Most similar to strains detected in Spain and Hungry Share a similar genogroup 2 backbone with G1P[8] and G3P[4] strains and similarity to an equine-like G3P[8] inter- genogroup reassortant strain emerged in Australia, Asia and South America
Discussion Strains demonstrate considerable genetic variation To be effective rotavirus vaccines must provide heterotypic protection against a diverse population of strains Following infection antibody responses to the capsid proteins and non-structural proteins have been reported VP4 (VP5* and VP8*), VP7 and VP6 - cross reactive epitopes Contribute to heterotypic protection Protection provided by RV3-BB in the Indonesian trial was cross protective and likely not solely dependent on homotypic responses Strain was genetically similar to the equine-like G3P[8] inter- genogroup reassortant strain which has emerged globally since 2013
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