1. RNA Polymerase = enzyme that makes mRNA from the DNA gene template
The DNA -> RNA -> Protein Pathway
RNA Polymerase = enzyme that makes mRNA from the DNA gene template

2. Plus-strand RNA Viruses
Plus-strand RNA viruses have genomes comprised of RNA. They have no DNA in their replication cycles.
In most cases, the replication cycle takes place in the cytoplasm of the cells.
No involvement of the transcription machinery in the nucleus; so, the plus-strand RNA viruses produce their own enzymes for RNA transcription and replication, which recognize RNA as the template.
The plus-strand RNA viruses do use the host cell’s translation machinery to generate viral proteins.
Many plus-strand RNA viruses produce numerous viral proteins from a single “gene.”

3. Types of Plus-Strand RNA Viruses with single-stranded, non-segmented plus-strand genomes
Genome Polarity
Polymerase in Virions?
RNA by itself infectious?
Types of mRNAs
Types of protein products
Example virus family I +
(mRNA) No
Yes
One
Long polyprotein; subsequently cleaved
Picornaviruses
(ex. Rhinovirus)
Flaviviruses
(ex. West Nile Virus) II
Multiple
One for each mRNA
Coronaviruses
Class

4. Picornavirus Diseases
ENTEROVIRUSES (stable in GI tract)
Poliomyelitis
Hepatitis A Virus
Coxsackieviruses
Enteroviruses Types 68-72
Echoviruses
RHINOVIRUSES (not stable in GI tract)
Common Cold
Numerous subtypes

5. Fecal-Oral Transmission

6. Enterovirus Pathogenesis: Target Tissues

7. Poliovirus Pathogenesis
Only a fraction of patients develop paralytic disease
Asymptomatic infection – 90%
Abortive/minor illness – 5%
Non-paralytic progression to the CNS – 1%-2%
Paralytic poliovirus – 0.1%-2%
3-4 days after minor illness
Virus infects motor neurons in anterior horn of spinal cord and the motor cortex

8. Poliovirus Infection: Progression to CNS Disease

9. Protection by Antibodies
Secretory antibodies can prevent primary infection
Serum antibodies prevent viremic spread to target tissues

10. Poliovirus Vaccination
Salk Vaccine
Killed Virus (formalin inactivated)
IPV
Sabin Vaccine
Live, attenuated viruses
OPV
Attenuated strains can revert to virulent forms
Vaccinees shed the attenuated viruses in feces
Plusses
Minuses

11. WHO Eradication of Poliovirus
Eradication campaign started in 1988
Current target date for eradication: Beyond 2009

12. Is Eradication Possible?
Oral Poliovirus Vaccine
Preferred for Third World Vaccination
Cheaper, No Sterile Needles Required
Campaign of usage has drastically reduced wild poliovirus transmission
Major issue is REVERSION of OPV strains to virulent forms
Laboratory Stocks
Poliovirus present in many laboratories around the World
Poliovirus contaminants in stocks of other viruses, i.e. Coxsackievirus, Rhinovirus
Bioterrorism
cDNA copies of poliovirus genome exist in many laboratories; transfection into tissue culture cells results in virus production
The poliovirus genome has been generated on a gene synthesizer (Plus-strand RNA viral genomes alone are infectious!)

13. Picornavirus Diseases
ENTEROVIRUSES (stable in GI tract)
Poliomyelitis
Hepatitis A Virus
Coxsackieviruses
Enteroviruses Types 68-72
Echoviruses
RHINOVIRUSES (not stable in GI tract)
Common Cold
Numerous subtypes

14. Human Rhinovirus Structure
5’-NTR
Open Reading Frame Encoding Polyprotein
AAAAAAAA
~630 nts
VPg
~7200 bases

15. Human Rhinovirus Structure
Virus exterior = protein shell
Icosahedral shape
Cross-section
Viral RNA genome inside
Protein capsid layer outside

16. Rhinovirus Life Cycle

17. Rhinovirus Binding to Receptor and Antibodies
ICAM-1 RECEPTOR
BOUND TO RHINOVIRUS
ANTIBODIES BOUND
TO RHINOVIRUS

18. ICAM-1 Receptor Binds to Canyon on Rhinovirus Surface

19. Rhinovirus Life Cycle

20. Rhinovirus Polyprotein Processing: Method for Generation of Individual Viral Proteins

21. Rhinovirus Life Cycle

22. Rhinovirus Pathogenesis
Entry of virus into upper respiratory tract
Hands and fomites
Inhalation of droplets that contain virus
Unable to replicate in the GI tract (not stable to acid)
Receptor is ICAM-1 (Intercellular Adhesion Molecule 1)
Preferentially replicates at 33oC
Over 100 serotypes
Reason for repeat infections throughout lifetime
Antigenic sites change, but receptor-binding
site is protected

23. Rhinovirus Pathogenesis

24. Targets for Rhinovirus Therapy

25. New Rhinovirus Therapies
62 million cases of Rhinovirus infection in U.S. each year
Cause more than 50% of respiratory tract infections
Traditional vaccine approaches as with poliovirus not possible (too many serotypes), so antiviral therapies must exploit unique properties of the virus
Soluble ICAM-1: Blocks virus binding to its receptor
AG7088: 3C protease inhibitor (being formulated for intranasal delivery)
Pleconaril: Binds a pocket in the capsid; interferes with attachment and uncoating
Promising early clinical studies – reduction in disease length
Not approved due to possible drug-drug interactions
Reformulations being developed

26. West Nile Virus

27. Types of Plus-Strand RNA Viruses with single-stranded, non-segmented plus-strand genomes
Genome Polarity
Polymerase in Virions?
RNA by itself infectious?
Types of mRNAs
Types of protein products
Example virus family I +
(mRNA) No
Yes
One
Long polyprotein; subsequently cleaved
Picornaviruses
(ex. Rhinovirus)
Flaviviruses
(ex. West Nile Virus) II
Multiple
One for each mRNA
Coronaviruses
Class

28. Flavivirus Gene Structure
(West Nile Virus = member of Flavivirus family)
Figure 1. Genomic structure of flaviviruses. The flavivirus genome is 11,000 to 12,000 nucleotides long. Both the 5'- and 3'- ends contain noncoding (NC) regions. The genome encodes 10 proteins, 3 of which are structural proteins (C, M, and E), and 7 of which are nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). The M protein is synthesized as a precursor (prM) protein. The prM protein is processed to pr + M protein late in the virus maturation by a convertase enzyme (furin).

29. West Nile Virus Genetic Structure and Protein Expression
virol/flavi1.jpg

30. West Nile Virus Structure

31. West Nile Virus Replication Cycle
*The WNV receptor in vertebrate cells is V3 integrin, which is highly conserved in vertebrates
Figure 2 The WNV replication cycle. A. Attachment and entry of the virion. B. Uncoating and translation of the virion RNA. C. Proteolytic processing of the polyprotein. D. Synthesis of the minus-strand RNA from the virion RNA. E. Synthesis of nascent genome RNA from the minus-strand RNA. F. Transport of structural proteins to cytoplasmic vesicle membranes. G. Encapsidation of nascent genome RNA and budding of nascent virions. H. Movement of nascent virions to the cell surface. I. Release of nascent virions. SHA, slowly sedimenting hemagglutinin, a subviral particle that is also sometimes released.

32. Coronaviruses
Another virus group with members responsible for the “common cold”
Coronavirus member in the news = SARS Virus

33. Types of Plus-Strand RNA Viruses with single-stranded, non-segmented plus-strand genomes
Genome Polarity
Polymerase in Virions?
RNA by itself infectious?
Types of mRNAs
Types of protein products
Example virus family I +
(mRNA) No
Yes
One
Long polyprotein; subsequently cleaved
Picornaviruses
(ex. Rhinovirus)
Flaviviruses
(ex. West Nile Virus) II
Multiple
One for each mRNA
Coronaviruses
Class

34. Coronaviruses

35. Coronaviruses

36. Coronaviruses

37. Coronavirus Receptor = ACE-2
ACE-2 = angiotensin-converting enzyme 2
Expressed in heart, lung, kidney, GI tract

38. SARS Virus Budding

39. Targets for SARS Therapy