1. Replication of Negative-Sense RNA Viruses (Mutipartite)

2. (-)RNA Virus Mutipartite Genome
Orthomyxoviridae
8 gene segments
Bunyaviridae
3 gene segments (L, M, S; some S gene are ambisense)
Arenaviridae
2 gene segments (L, S; both ambisense)

3. Family Orthomyxoviridae
“normal” “mucus”
(-)RNA
Envelope , large peplomers, 120 nm
Helical nucleocapsid, 15 nm; ribonucleoprotein (RNP)

4. Genus: Influenza Virus
“influence” malign, supernatural
Envelope glycoproteins:
HA (1-16), NA (1-9)
Human groups (identify by capsid NP):
Type A infect humans and animals; epidemics
Type B infects humans; epidemics
Type C infects humans; mild disease

5. Classification of Human Influenza Virus
HA: H1, H2, H3 (H5, H7, H9 rare, does not spread well human-human)
NA: N1, N2
Type A or B
Geographic source
Isolate number
Year of isolation

6. Hemagglutinin subtype Neuraminidase subtype
World Health Organization Influenza Nomenclature (One of three strains in Vaccine)
Hemagglutinin subtype
Influenza type
Year of isolation
(H3N2)A/Brisbane/10/2007
World Health Organization (WHO) Influenza Nomenclature
The WHO has devised the nomenclature system for tracking influenza virus antigenic variants and new strains of influenza.
Subtypes of influenza type A are determined by surface antigens hemagglutinin (HA) and neuraminidase (NA); influenza type B has only one subtype; influenza type B surface HA and NA do not demonstrate the major variability that occurs suddenly in influenza type A (the basis for new subtypes).
Influenza type A (H1N1) designates HA subtype 1 and NA subtype 1, whereas influenza type A (H3N2) designates HA subtype 3 and NA subtype 2.
Geographic source
Isolate number
Neuraminidase subtype
Influenza type B does not occur as subtypes.

7. Influenza Virus: (-)RNA Genome
Eight gene segments (2.3 – 0.9 kb)
Total genome = 13.6 kb
Ten mRNAs translate for ten viral proteins (two smallest mRNAs are spliced)
Replication occurs in cell nucleus & cytoplasm

9. Influenza Virus: Entry / Uncoating
Entry by receptor-mediated endocytosis
Release of eight separate RNP into cytoplasm
RNP transported into nucleus
Viral transcription occurs in nucleus

10. Influenza Virus: mRNA Transcription complex: Viral (-)RNA genome
Three viral polymerase-associated proteins (PB1, PB2, PA)
“Cap snatching” viral endonuclease cleaves cell 5’ cap mRNA (10-13 bases)
Cell 5’ cap mRNA12-13 serves as “primer” for viral mRNA transcription

11. Influenza Virus: mRNAs
Eight mRNAs transcribed
Two smallest mRNAs (Segment 7, 8) spliced
Matrix: M1, M2
Nonstructual: NS1, NS2

12. Influenza Virus: mRNA Translation
Ten mRNAs (5’cap, 3’ polyA tail)
Transport from nucleus to cytoplasm
Translation on cell ribosome for ten viral proteins

13. Influenza Virus: Antigenome (RI-1)
(-)RNA genome serves as template
Synthesis of viral proteins in cytoplasm (NP, PB1, PB2, PA) and transport into nucleus
Increase levels of NP switch transcription to uncapped (+)RNA antigenome

14. Influenza Virus: Genome (RI-2)
(+)RNA antigenome serves as template
(-)RNA genome copied from antigenome:
Template for viral mRNA
For progeny virus
Assembly of RNP: genome (-)RNA, NP, PB1, PB2, PA in nucleus
Transported out to cytoplasm by viral M1 and NS2

15. Influenza Virus: Assembly & Release
HA, NA, M2 proteins glycosylated in ER / Golgi and inserted into plasma membrane
Viral RNP associates with matrix (M1) protein, guided to virus modified plasma membrane
Virus exits by budding

16. Virus Respiratory Infections
Primary site – oral & respiratory mucosa, ±eye
Migrate to lymphatic tissue
Enters blood (fever, malaise)
Secondary site - reticuloendothelial system organs (liver, spleen, bone marrow)
Re-enters blood and infects other target organs (extremities & skin, RT, GI tract, CNS, heart)

17. Influenza Infection/Disease
Virus replication in RT
Host defense compromised:
Destroys ciliated cells
MØ, T cells impaired
Viral or 2° bacterial pneumonia (Staphylococcus, Streptococcus, Haemophilus)

19. Influenza Epidemiology
Endemic - Winter, peaks Dec - Jan
Epidemics every ~5 years
Pandemics every ~10 years
1918 Spanish (H1N1) >20 M deaths
1957 Asian (H2N2) 80 M infected, USA 88,000 deaths
1968 Hong Kong (H3N2) USA 34,000 deaths
1977 Russian (H1N1)
USA estimates each year
10-20% get flu
>10,000 hospitalizations for flu-related complications
~36,000 deaths from complications of flu

20. Influenza Virus Epidemics
Ability of virus to change
Antigenic “drift” – gradual variation in HA, NA due to high RNA mutation rate
Antigenic “shift” – major variation due to dual infection and gene reassortment
Origin of new influenza A virus strains by exchange between different animal species i.e. avian » pigs » humans

21. Antigenic Drift & Shift

22. 1997 - Who’s Afraid Of The Big Bad Bird Flu (H5N1)?
Who’s Afraid Of The Big Bad Swine Flu (H1N1)?

23. Influenza Treatment Antivirals:
Rimantadine for Flu A
Tamiflu and Relenza for Flu A & B)
Inactivated killed whole virus or subunit vaccine (HA, NA) for:
Elderly, nursing home residents
Patients with chronic diseases
Health care workers
Anyone desiring protection
Live cold adapted (25ºC) virus vaccine:
Given as nasal spray
Ages 5-50 years
Use of aspirin to treat fever due to virus infection of children contraindicated; associated with Reye’s Syndrome (injury to liver, encephalopathy)

24. Flu Vaccine

25. Flu Vaccine: Risks vs. Benefits
> Million flu infections/year in USA
>100,000 hospitalizations/year due to flu
>20,000 – 40,000 deaths/year due to flu or its complications
Vaccine Side Effects (What to Expect Flu Shot)
Kill inactivated, cannot get flu
Soreness, redness, swelling
Fever (low grade)
Aches
Rare serious problem – allergic reaction toegg protein

26. “Don’t Blame Flu Shots for All Ills, Officials Say”
N. Y. Times, Sept. 28, 2009
Dr. Harvey V. Fineberg, President, Institute of Medicine
Every year:
1.1 million heart attacks
795,000 strokes
876, 000 miscarriages
200,000 have first seizure

27. Similar Genomes: (-) RNA Viruses

28. Reading & Questions
Chapter 15: Replication Strategies of RNA Viruses Requiring RNA-directed mRNA Transcription as the First Step in Viral Expression.

29. QUESTIONS???

30. Class Discussion – Lecture 7a
1. Why can’t influenza virus replicate in a cell where the nucleus has been removed?
2. You lab is researching the Spring fever virus (SpFV) and the debilitating variant SpFV-4 that causes senioritis. Others have identified SpFV as an Influenza virus but your team’s research results show it may be a new genus tenatively called Procrastinovirus. The following table list properties of SpFV strains studied in your lab:

31. (a) Which features of SpFV are similar to Influenza virus?
(b) Which features are different from Influenza virus?
(c) Which viral proteins do you predict will be different between SpFV and SpFV-4?
(d) What might account for the ability of SpFV-4 strain to produce senioritis?

32. Family Bunyaviridae (-)RNA Envelope, 90-120 nm
Three helical, circular, nucleocapsids, 2.5 nm
Most are arboviruses
Infect arthropods, birds, mammals

33. Bunyaviridae: (-)RNA Genome
Three segments of (-)RNA:
L = polymerase (RNA pol)
M = G1, G2 (envelope gp), NSM
S = RNP (nucleocapsid), ± NSS
Total: kb

34. Genus: Bunyavirus Mosquito vector
Bunyamwera virus – Africa; fever, rash, encephalitis
California encephalitis virus – endemic in USA
La Crosse encephalitis virus - endemic in USA

35. Genus: Phlebovirus “vein” Sandfly vector
Rift valley fever virus – Africa
Often fatal hemorrhagic fever

36. Genus: Hantavirus Transmission by contact with rodent excreta
Hantaan virus – Korea; hemorrhagic fever + renal syndrome
Sin Nombre virus – S.W. USA; hantavirus adult respiratory distress syndrome (HARDS)

37. Various Coding Strategy for Bunyaviridae S Gene
Virus replication occurs in cytoplasm
Transcribe mRNA for N, ±NSS protein
mRNA has 5’ cap, 3’ no polyA tail

38. Coding Strategy for S Gene Hantavirus: No NS
Transcribe single mRNA for N protein
Does not code for NSS protein

39. Coding Strategy for S Gene Bunyavirus: Overlapping ORF
Two partially overlapping ORFs
NSS ORF within N ORF
Transcription of a single mRNA
Translation for both N and NSS proteins using alternate reading frame of mRNA

40. Coding Strategy for S Gene Phlebovirus: Ambisense Genome
S genome RNA, two ORF:
(+)NSs gene
(-)N gene
Transcribes for two subgenomic mRNAs:
N mRNA from genome
NSS from antigenome

41. Similar Genomes: (-) RNA Viruses

42. Family Arenaviridae “sandy” – ribsomes in virions (-)RNA
Envelope, nm
Two helical, circular nucleocapsids, 9-15 nm
Natural hosts are rodents
Virus transmission by excreta

43. Genus: Arenavirus
Lymphocytic choriomeningitis virus (LCM) – mild “flu” in mice, humans
Lassa fever virus – Africa; highly fatal hemorrhagic fever, Biosafety Level 4 pathogen
Junin virus – Argentine hemorrhagic fever
Machupo virus – Bolivian hemorrhagic fever

44. Arenavirus: (-)RNA Genome
Two RNA segments
Total genome = 10 kb
Both are ambisense genomes

45. LCM: Persistent Infections
Infection of host early in life
Persistent chronic infection
Viremia
Virus shedding in saliva and urine
Little or no neutralizing antibody
Model to study virus/host factors for chronic infections

46. Similar Genomes: (-) RNA Viruses

47. Reading
Chapter 15: Replication Strategies of RNA Viruses Requiring RNA-directed mRNA Transcription as the First Step in Viral Expression.

48. QUESTIONS???

49. Class Discussion – Lecture 7b
1. How are two different ways Bunyavirus makes more than one protein from a “monocistronic” mRNA?
2. Why are the (-)RNA viruses thought to have appeared fairly recently?

50. Group Case Study Tuesday, Oct. 30:
Group 6 – Influenza Virus
Group 7 – Bunyavirus
Group 8 - Prions
Ten minute oral presentation on patient case history and questions using PowerPoint
Written report due in class (also for Group #1-5)
PowerPoint and Word file of report to Instructor to post on Instructional1 for class study or save to computer in classroom