1. March 06 2008 Notes: The Panama Puzzle http://www-micro.msb.le.ac.uk/Tutorials/Panama/Panama.html Walter Reed: Major, US Army Medical Corps.

2. Hemorrhagic fevers

3. Hemorrhagic fevers (often infection is mild or asymptomatic) -initial fever, then leaky capillaries (other organs involved) bleeding into skin and mucous membranes Yellow fever virus - jaundice + "black vomit" - imported to "New World" by traders in 17th C. - spread by mosquitoes (partic strains, not in Indian subcontinent) - animal reservoirs, get sporadic cases, - urban epidemics: if enough people infected, mosquitoes can transmit between people - halted building of Panama Canal, 1000's of deaths. Control of YFV - elimination of vectors - immunization against yellow fever virus (attenuated strain) - good immunity - not heat stable - needs refrigeration

4. YFV Diameter: 37 - 50 nm ~ 11 kb genome; translated as single polyprotein - structural proteins at 5' end of genome Single capsid protein + membrane protein (M) - E glycoprotein spikes attached to M - synthesized as single precursor

5. YFV Virus buds into ER / Golgi and is released by exocytosis Ab-modified enhancement of replication. - non-neut. Abs bind virus - compex taken up by cells with Fc receptor (eg macrophage) - more serious infection (x 20 - 1000 fold more virus)

6. Attenuation of YFV Extensive passaging of virus in non-host tissue - change in virulence between 89th and 114th passage. Stable changes. 68 nucleotides = 32 aa changes. (capsid protein unchanged) E protein had most changes ?? change in tissue tropism, efficiency of infection Single nucleotide change in 5' UTR also associated with reduced virulence. There are also host resistance factors - single dominant gene in mice - general resistance to flaviviruses (x 100 - 1000 fold) - get lower titres of virus in resistant mice - thought not to be linked to IFN or immune response - ? Associated with reduced replication of viral genome

7. Japanese Encephalitis virus - major cause of human encephalitis in Asia - ~ 35,000-50,000 symptomatic cases/year. - Local incidence rates range from 1-10 cases per 100,000 - > 100 cases / 100,000 during outbreaks. Mortality/Morbidity: 1 per 250 infections results in symptomatic disease. Mortality rates in places with intensive care capabilities are 5-10%. In less developed areas, mortality rates may exceed 35%. Worldwide, more than 10,000 reported deaths occur per year. Approximately 33-50% of patients with symptomatic disease who survive have major neurologic sequelae at 1 year, including seizure disorders, motor or cranial nerve paresis, or movement disorders.

8. Current JEV vaccines - Formalin-inactivated, virus from mouse brain need multiple doses, high cost, adverse reactions - Live-attenuated vaccine produced in 1 o hamster kidney cells Only licensed in China (cells not allowed for vaccine elsewhere) ChimeriVax-JE Replace M and E genes of YFV with attenuated JEV counterparts Recombinant virus can be grown in same system as YFV vaccine

9. Multiple changes in JEV proteins (wt vs attenuated) Single reversions don’t restore neurovirulence Most sets of 3 reversions are only sublethal

10. Coronaviruses SARS Many human coronaviruses are very difficult to grow in TC; - murine hepatitis virus was used a model Bovine coronavirus

11. Coronaviruses The envelope carries three glycoproteins: * S - Spike protein: receptor binding, cell fusion, major antigen * E - Envelope protein: small, envelope-associated protein * M - Membrane protein: transmembrane - budding & env formation In a few types, there is a third glycoprotein: * HE - Haemagglutinin-esterase The genome is associated with a basic phosphoprotein, N.

12. Subgenomic RNAs **Notice the same 5’ end on all of the subgenomic mRNAs**

18. Subgenomic RNAs

19. intergenic sequences

21. Coronavirus genome and replication replicase polyprotein pol Genome + RNA 32 kb SEMN genes 2-7 gene 1 (replicase) R C Leader Borrowed from web file, Mark R. Denison, M.D., Vanderbilt

22. Coronavirus Transcription and Replication 7 RNA 5' 3' Genomic RNA Leader RNA (+) (+) (-) 3' Spike (S) M N E NS NS Gene 1 Replicase Proteins 1 23456 Genes 2-7 RNA 3 4 5 6 7 2 5' 3' AnAnAnAn AnAnAnAn AnAnAnAn AnAnAnAn AnAnAnAn AnAnAnAn AnAnAnAn AnAnAnAn 5' UnUnUnUn SubgenomicRNAs ( +/- ) Genomic RNA (+) Replicase Borrowed from web file, Mark R. Denison, M.D., Vanderbilt

23. Severe Acute Respiratory Syndrome (SARS) Virus

25. Live fruit bats hang in a Sumatran food stall. Having the bats in such close proximity to people and live animals for sale in the market could be the reason new viruses have emerged to infect humans.