PHYSICAL METHODS FOR STUDYING VIRAL STRUCTURE:-
It has been known for many years that viruses are smaller than micro organisms.The first unequivocal demonstration for an animal virus occurred in 1898,where Loeffler and Frosch demonstrated that foot and mouth disease , an important infectious disease of cattle could be transferred by material which could pass through a filter of average pore diameter too small to allow passage of bacteria . The new group of organisms became known as filterable viruses.For some time they were called as ultra microscopic since c most viruses are beyond the limit of resolution of light microscope [200 nanometers (nm) = 2000 angstroms (A)].Only with the advent of the electron microscope was it possible to study the morphology if viruses properly .It then became apparent that they range in size from about the size of smallest micro organisms down to little bigger than the largest protien molecules.
Early electron microscopic studies of viruses by Ruska in 1939-1941 were expanded during the 1950s to include thin sectioning of infected cells and metal shadowing of purified virus particles.Then in 1959 our knowledge of viral ultra structure was transformed when negative staining was applied to the electron microscopy of the viruses.
(Coronavirinae), bacilliform, 170–200 to75–88 nm (Bafinivirus) or found as a mixture of both, with bacilliform particles characteristically bent into crescents (Torovirus). The particles are typically decorated with large, club- or petal-shaped surface projections (the “peplomers” or “spikes”), which in electron micrographs of spherical particles create an image reminiscent of the solar corona. This inspired the name of the “true” coronaviruses (now grouped in the subfamily Coronavirinae), which was later adopted for the whole family.
Nucleocapsids are helical and can be released from the virion by treatment with detergents. Whereas the coronavirus nucleocapsid appears to be looselywound, those of the Torovirinae are distinctively tubular.
In terms of genome size and genetic complexity, the Coronaviridae are the largest RNA viruses identified so far, rivaled only by the okaviruses, large nidoviruses of invertebrates assigned to the family Roniviridae. Replication has been studied in detail only for coronaviruses, but the limited data available for toro- and bafiniviruses suggest that the latter viruses use essentially similar strategies.
Virions attach to dedicated host cell surface receptors via their spikes and release their genome into the target cell via fusion of the viral envelope with the plasma membrane and/or the limiting membrane of an endocytic vesicle. The entire replication cycle takes place in the cytoplasm and involves the production of full-length and subgenome-sized (sg) minus-strand RNA intermediates with the viral genome serving both as mRNA for the replicase polyproteins and as a template for minus-strand synthesis. RNA synthesis is catalyzed by an as yet poorly characterized replication–transcription complex, composed of viral and host proteins and associated (at least in coronaviruses) with an interconnected network of modified intracellular membranes and double membrane vesicles that are presumably endoplasmic reticulum (ER)-derived.
The genome contains multiple ORFs. Its 5-most two-thirds are occupied by the replicase gene, which is comprised of two overlapping ORFs called 1a and 1b (Figure 1). The replicase gene istranslated to produce polyprotein pp1a and, subject to programmed 1 ribosomal frameshifting,a C-terminally extended product, pp1ab. The polyproteins are co- and post-translationally processed by a set of virus-encoded proteinases and, thus, are not detectable as full-length proteins in virus-infected cells. The N-termini of pp1a and pp1ab are processed by one or two papain-like proteinases, whereas the C-terminal half of coronavirus pp1a and the ORF1b-encoded part of pp1ab are cleaved at 11 well-conserved sites by the main proteinase (Mpro or 3CLpro), a nidovirus-wide conserved enzyme with a chymotrypsin-like fold, a poliovirus 3C proteinase-like substrate specificity and either a serine (torovirus, bafinivirus) or a cysteine (coronavirus) as active site nucleophile.
In coronaviruses, proteolytic processing results in the production of 15 (in viruses belonging to the species Avian coronavirus) or 16 mature products, commonly referred to as non-structural proteins (nsp’s) and numbered according to their position – from N- to C-terminus – in the viral polyproteins.
Many nsp’s are unique enzymes involved in one or more essential step(s) in viral replication. Others appear to be exclusively involved in virus–host interactions (including immune evasion) and are dispensable for virus propagation in vitro . Polyprotein processing in toro and bafini viruses has not been studied in detail.
