Molvir Flavi Toga 01 19 06

Today: 

Today Positive-strand RNA viruses We will begin with the families Flaviviridae and Togaviridae Representative members of these families and the diseases with which they are associated Epidemiology of representative viruses Strategy of replication

Important Families of Positive- Strand RNA Viruses: 

Important Families of Positive- Strand RNA Viruses Togaviridae Caliciviridae Coronaviridae Flaviviridae Picornaviridae

Common Features of Positive- Strand-RNA Viruses: 

Common Features of Positive- Strand-RNA Viruses They replicate in the cytoplasm The genomic RNA serves as a message and is translated The genomic RNA is infectious The virions do not contain any enzymes viral proteins are translated as polyproteins

Features common to the Flaviviridae and Togaviridae: 

Features common to the Flaviviridae and Togaviridae A non-segmented (+) strand RNA genome Viruses are enveloped Viruses have two envelope proteins and one envelope protein Most, but not all viruses in these two families are arthropod-borne

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Transmission of Arboviruses In Nature Vertebrate Host Arthropod Host man and other primates small rodents birds mosquitoes ticks

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Sequential steps necessary for a mosquito to transmit an Arbovirus A female mosquito ingests an infectious blood meal and virus enters the mesenteron. Virus infects and multiplies in mesenteronal epithelial cells. Virus is released across the basal membrane of the epithelial cells and replicates in other tissues. Virus infects salivary glands. Virus is released from the epithelial cells of the salivary glands and is transmitted in the saliva during feeding. [From Monath (1988, p 91).]

Classification of the Flaviviridae: 

Classification of the Flaviviridae Genus flavivirus: >60 viruses Genus pestivirus: diseases of farm animals Genus hepacivirus: Hepatitis C virus

Medically Important Viruses in the Genus Flavivirus: 

Medically Important Viruses in the Genus Flavivirus Dengue (4 serotypes) Yellow fever St. Louis encephalitis West Nile Japanese encephalitis Tick-borne encephalitis

Epidemiology of Selected Flaviviruses: 

Epidemiology of Selected Flaviviruses SLE and WN viruses are maintained in nature by a mandatory cycling between a mosquito vector and an avian host Man is an accidental host With dengue and YF viruses, man is part of the natural cycle, and the cycling of these viruses therefore is between man and mosquito

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Dengue Hemorrhagic Fever and Shock Syndrome (DHF/SS) An Hypothesis An infant is infected with one serotype of DV, e.g type 2, makes antibody to the virus, and recovers. Although there are group antigens common to all 4 serotypes of DV, the neutralizing activity is type-specific. When this infant is later infected with a second serotype of DV, e.g. type 4, the anti-type 2 antibodies can bind to, but do not neutralize the the type 4 virus. Macrophages, which are infected by dengue virus, display Fc receptors.

DHF/SS (cont’d): 

DHF/SS (cont’d) Virus bound to Ab, is able to enter macrophages via the Fc receptor. This is termed antibody-mediated enhancement This makes the infection of macrophages more efficient, and leads to the infection of a larger number of macrophages, leading in turn to a more severe clinical presentation According to this hypothesis, DHF/SS should occur only when there is more than one serotype of DV circulating in a given area. This appears to be the case.

Replication of flaviviruses: 

Replication of flaviviruses Genome replication is basically like that of poliovirus Genome is about 10-11,000 nt in length and contains a single open reading frame Nucleocapsid is composed of the capsid (C) protein and the viral RNA

Replication (cont’d): 

Replication (cont’d) The two envelope proteins are made and glycosylated on the cytoplasmic membranes (ER and Golgi) Virus acquires its envelope by budding through cytoplasmic membranes modified by insertion of the envelope proteins

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Togaviridae

Replication strategy of Alphaviruses: 

Replication strategy of Alphaviruses Genome is 11-12,000 nt in length There is a 5’ cap and a poly A tail However, genome organization very different from that of picorna and flaviviruses There are two open reading frames One ORF encodes the nonstructural, and the other the structural proteins

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Genome Organization Both positive strand genomic and subgenomic RNA have 5’ 7-methylguanosine cap and 3’ polyA tail Genomic RNA serves as the mRNA for nonstructural polyproteins subgenomic RNA is the mRNA for the structural polyproteins The sequence of the subgenomic promoter overlaps with the 3’ end of the nsP4-coding region

General Properties of Alphaviruses: 

General Properties of Alphaviruses 1. Enveloped, spherical particles with spikes - about 70nm in diameter 2. Icosahedral structure 3. Contain 3 major proteins - E1 and E2 are membrane or envelope proteins and from the spikes - C is the capsid protein which together with the viral RNA forms the viral nucleocapsid 4. Virus entry is by receptor-mediated endocytosis

Properties of Alphaviruses (con’t): 

Properties of Alphaviruses (con’t) 5. The viral genome - a positive-strand RNA molecule about 11,700 nt long 6. Replication and Transcription of viral RNA - genome RNA is template for genome length minus strand RNA genome length minus-strand RNA serves as template not only for genome length plus-strand RNA, but also for a subgenomic (SG) plus-strand Like the genome plus-strand RNA, the SG RNA is capped at its 5’ end and polyadenylated at its 3’ end - type O cap at 5’ end - poly A at 3’ end

Properties of Alphaviruses (con’t): 

Properties of Alphaviruses (con’t) - both mRNAs are initially translated into polyproteins which must be cleaved or processed 8. Viral nucleocapsid is attracted to sites on the plasma membrane which have been modified by the insertion of viral membrane proteins - nucleocapsid becomes enveloped, budding from the modified plasma membrane and thus becoming a mature virus particle 7. The genome size plus-strand RNA serves as the message for the ns proteins. The subgenomic plus-strand RNA serves as message for the structural proteins. 9. Replication is entirely cytoplasmic

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Figure 1 Negatively stained electron micrographs of (A) SF virus particles and (B) isolated Sindbis virus nucleocapsids. Bar 100 nm (courtesy of Dr. C.H. von Bonsdorff)

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Sindbis Virus (SV) Classification: Virion Structure: Prototype virus of genus alphavirus, family Togaviridae Nearly all alphaviruses are maintained in nature by alternate replication in a mosquito host and a vertebrate host. Genome Organization: Icosahedral nucleocapsid core enveloped by host derived membrane with E1-E2 heterodimer spikes Positive strand genomic RNA (11703 nt) with 5’ 7- methylguanosine cap and 3’ poly A tail Genome encodes 3 structural proteins (Capsid, E2, and E1) and 4 nonstructural proteins (nsP1,2,3,4) From Fields Virology, 3rd ed.

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Fig. 5 Production of glycoproteins E2 and E1. The left side illustrates signal sequences (dark-blue cylinders) that lead to the insertion of the glycoproteins into the endoplasmic reticulum (ER). The right side illustrates an E1-E2 heterodimer after heterodimerization of PE2 and E1, fatty acid acylation, reorientation of the tail of E2 to the cytoplasm and cleaveage of PE2 to E2. Three cysteines in this region (light-blue circles) are palmitoylated and the fatty acid side chains are probably inserted into the bilayer. A possible site for phosphorylation is shown (red star). The model is not to scale: the conformations of E2 and E1 are unknown and the illustration is schematic.

Functions of the Alphavirus ns Proteins: 

Functions of the Alphavirus ns Proteins nsP1: - capping and methylation of the viral plus- strand RNAs - regulation of minus strand RNA synthesis nsP2: - the protease which is responsible for the processing of the ns proteins - has an RNA helicase domain nsP3: - ?? nsP4: - the RNA-dependent RNA polymerase

Three of the Sindbis virus ns proteins show homology to ns proteins encoded by a number of plant viruses representing different virus families: 

Three of the Sindbis virus ns proteins show homology to ns proteins encoded by a number of plant viruses representing different virus families Haseloff, Goelet, Zimmern, Ahlquist, Dasgupta and Kaesberg (1984) PNAS 81, 4358 Ahlquist, Strauss, Rice, Strauss, Haseloff, and Zimmerman (1984) J. Virol. 53,536 These observations gave rise to the concept that families can be grouped into superfamilies. Thus viruses such as TMV, BMV, and CMV are now included in what is called the alphavirus superfamily

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Sindbis Virus with Double Sub-genomic Promoters

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The SARS virus 1. Family Coronaviridae, genus coronavirus 2. These are spherical large RNA viruses about 100-120 nm in diameter. 3. They have a positive-strand RNA genome which is 29- 32 kb in length. This is the longest RNA genome so far found in any virus. 4. The RNA is capped and polyadenylated, and is infectious.

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SARS virus (cont) 5. The virus is enveloped and has a characteristic morphology, due to the very prominent glycoprotein spikes. These give the virus a crown like appearance, hence the name. 6. The viruses bud, not at the plasma membrane, but at a membrane compartment intermediate between the ER and the Golgi system.

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SARS virus (cont) 7. The previously known coronaviruses fell into 3 antigenic groups, and included viruses that infected domestic animals as well as humans. 8. The human viruses were known only as causes of the common cold syndrome or gastroenteritis. In general they were not considered very important human pathogens. Some coronaviruses did cause serious disease in animals.

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Identification of the SARS virus 1. Inoculation of appropriate clinical material onto various cell lines. 2. Observation of CPE on 2 cell lines (including Vero cells )after several days. 3. Passage of culture medium onto fresh cultures of the same cell line. 4. Examination of infected cells by EM and visualization of coronavirus-like particles.

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Sequence analysis of 405 nt sequence of polymerase gene was done on 12/19 patients. The sequence was identical in all of them. Therefore the clinical syndromes in all was caused by the same virus. The above findings were described in the NEJM of April 10, 2003. In MMWR of April 11, 2003 brief clinical descriptions are given of 5 cases, all of whom survived. None had an elevated white blood cell count.