1. Rotavirus Presented by: Niaj MD. Tanvir (ID-13326010) Sarah Shawly (ID-13126015)

2. Rotavirus Family: Reoviridae  spherical virions with structures resembling the spokes of a wheel (rota = wheel)  isolated from respiratory tract, enteric tract of human  viral gastroenteritis:the second most common viral illness after upper respiratory tract infection  a major killer of undernourished infants in developing countries  responsible for half a million deaths a year

4. Rotavirus Epidemiology Reservoir: Human-GI tract Transmission : Fecal-oral, fomites Temporal Fall and winter pattern: (temperate areas) Communicability: 2 days before to 10 days after onset

5. Structure

6. spherical nonenveloped structures icosahedral viruses with double stranded RNA diameter around 70 nm made up of 3 concentric protein layers outer layer having 2 surface viral proteins:VP7 and VP4(VP4 forms 60 spikes.) middle capsid being made of VP6 protein core being composed of VP1, VP2 and VP3 proteins

8. FIG:ROTA VIRUS UNDER ELECTRON MICROSCOPE

9. Genome

10. total genome size=18,550 bp. 11 segments of dsRNA transcripts representing genome-length mRNAs monocistronic gene segment (only segment 11 is bicistronic.) reassortment of gene segments between related strains viral mRNAs containing 5’-methylated cap structures but lacking polyA tail short 5’ and 3’ non-translated regions

13. Viral encoded structural proteins

14. VP1: located in core of virus particle; act as RNA dep RNA pol; produces mRNA transcripts for synthesis of viral proteins and produces copies of genomic RNA segments VP2: forms core layer of virion and binds to RNA genome VP3: part of inner core; act as enzyme guanylyl & methyl transferase (capping enzyme that catalyses the formation of the 5' cap in post-transcriptional modification of mRNA; cap stabilises viral mRNA by protecting it from host NA degrading enzymes)

15. VP4 : on surface as spike; binds to cell surface receptors & drives entry of virus into cell; modified by protease (trypsin) (found in the gut) into VP5* & VP8* before virus is infectious VP6 : forms bulk of capsid; highly antigenic & can be used to identify rotavirus species VP7: glycoprotein forms outer surface ; highly antigenic

16. Viral encoded nonstructural proteins

17. NSP1- product of gene 5; RNA-binding protein NSP2- RNA-binding protein; accumulates in cytoplasmic inclusions (viroplasms) & required for genome replication NSP3- bound to viral mRNAs in infected cells & responsible for shutdown of cellular protein synthesis NSP4- viral enterotoxin to induce diarrhoea & first viral enterotoxin discovered NSP5- encoded by segment 11 & accumulates in viroplasm NSP6- NA binding protein, encoded by gene 11

18. Replication

19. Entry Virion first attaches to the target cell. Binding of a virion to a cell is initially via sites on the spike proteins (VP4 and after cleaving VP8*, VP5*) and then via sites on the capsid surface (the glycoprotein VP7). Proteolytic enzymes like trypsin cleaves VP4 which results in much more rapid entry into the cell. 2 possible mechanisms for entry- *direct penetration of the virion across the plasma membrane *endocytosis Receptor mediated endocytosis delivers the virion to the early endosome.

20. FIG:Entry of Rotavirus

21. Reduced calcium concentration in endosome triggers uncoating (breakdown of VP7) of the triple-layered particle (TLP) leaving behind VP2 and VP6 protein coats around viral dsRNA known as the double-layered particle (DLP).

22. Synthesis There are three phases of replication: (1) translation and synthesis of viral proteins (2) replication of the dsRNA, genome packaging, and DLP assembly (3) budding of DLP into the ER for the acquisition of the outer layer Releasing DLP into the cytosol activates the internal polymerase complex (VP1 and VP3) to transcribe capped positive-sense RNA ((+)RNAs) from each of the eleven double-stranded RNA (dsRNA) genome segments. Early transcription of the dsRNA genome by viral polymerase occurs inside DLP so that dsRNA is never exposed to the cytoplasm.

23. (+)RNAs serve either as - *mRNAs for synthesis of viral proteins by cellular ribosomes or, *as templates for synthesis of negative-sense RNA/(–)RNA during genome replication NSP1 and NSP3 are produced first. NSP1 acts as an IFN antagonist while NSP3 shuts off host mRNA translation. NSP2,NSP5,NSP6,VP1,VP2,VP3,VP6 are then produced by early capped mRNA translation. NSP2 & NSP5 interact to form viroplasm.

24. Figure: Ultrastructure of rotavirus-infected cell.

25. Figure: Schematic representation of the steps of rotavirus replication

26. Assembly and Release  Genome packaging is initiated when newly synthesized VP1 (and, presumably, VP3) bind the 3′ end of viral (+)RNA segments.  It is currently thought that interactions among the 11 segments of (+)RNAs cause the formation of `assortment complex’.  Condensation of the inner capsid protein, VP2, around the assortment complex triggers dsRNA synthesis by VP1.  The intermediate capsid protein, VP6, then assembles onto the nascent core forming DLP.

27.  The current model proposes that interaction with the rotavirus viroplasm protein, NSP4, recruits DLPs and the outer capsid protein VP4 to the cytosolic face of the endoplasmic reticulum (ER) membrane to form the DLP.  The DLP–VP4–NSP4 complex buds into the ER.  Subsequent removal of the ER membrane and NSP4 permits assembly of the ER-resident outer capsid protein, VP7, and formation of the TLP.  Mature virions are released following cell death and associated breakdown of host plasma membrane.

28. Pathogenesis Virus enters through mouth. Virus then adhere to GI tract epithelium (jejuna mucosa). Replication occurs in epithelium of small intestine. Infection leads to isotonic diarrhea because of NSP4 enterotoxin.

29. Trivia First infection usually does not lead to permanent immunity. Reinfection can occur at any age. The first rotavirus vaccine, RotaTeq, is made from a strain of rotavirus that was originally isolated from a calf.

30. Reference Materials Fields. Virology (6th Ed) J. Carter & Saunders. Virology:Principles and applications Ruiz, M.C., Leon, T., Díaz, Y., and Michelangeli, F. (2009) Molecular biology of rotavirus entry and replication. The Scientific World JOURNAL 9, 1476–1497. DOI 10.1100/tsw.2009.158.