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

Thank you for your attention!