Download Negative sense RNA viruses – Hantavirus, influenza

NEGATIVE STRAND RNA VIRUSES
(-) sense RNA genome: Genomic RNA is not translatable
Viral RNA is transcribed into (+)sense mRNA
RNA alone is not infectious
Virions contain RNA dependent RNA polymerase
Most, if not all viruses are enveloped
A diverse array of negative-strand RNA viruses infect vertebrate hosts
Plant
Negative-strand RNA viruses are less diverse, but very successful in plant and
invertebrate hosts
Rhabdoviruses and bunyaviruses infect and are successful in many different
vertebrate, invertebrate, and plant hosts
All plant-infecting negative strand RNA viruses also infect and replicate in their
invertebrate vectors
Negative strand RNA viruses with segmented genomes most likely evolved from
nonsegmented negative strand RNA viruses
Invertebrate
Vertebrate
FAMILIES of NEGATIVE STRAND VIRUSES
NON-SEGMENTED (-)STRAND VIRUSES
RHABDOVIRIDAE Rabies, VSV, & Plant viruses
FILOVIRIDAE - Marburg & Ebola viruses
PARAMYXOVIRIDAE - Measles, Mumps, RSV, & Distemper
BORNAVIRIDAE – Neurological diseases of humans and many animals
SEGMENTED (-)STRAND VIRUSES
ORTHOMYXOVIRIDAE - Influenza virus
SEGMENTED AMBISENSE VIRUSES
BUNYAVIRIDAE - Hantavirus, plant Tospovirus and Tenuivirus
ARENAVIRIDAE - Lassa fever
Segmented Negative Strand RNA Viruses
• Orthomyxoviridae
– Three types of flu virus
• Genus Influenzavirus A – 8 genome segments
• Genus Influenzavirus B - 8 genome segments
• Genus Influenzavirus C - 7 genome segments, no
neuraminidase
– Several insect-transmitted viruses
• Genus Thogotovirus - 6 genome segments
– Thogoto virus – tick-transmitted
– Dhori virus – tick-transmitted
– Batken virus – mosquito-transmitted
– Influenza A is by far the most important
Segmented Negative Strand RNA Viruses
• Arenaviridae
– Relatively small group
– Unique characteristic of encapsidating host
ribosomes
– Often associated with persistent infections of rodents
– Several viruses associated with hemorrhagic fevers
– Ambisense genomes
• Bunyaviridae
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Large group of 200+ viruses
Infect vertebrates, invertebrates, plants
Major component of classic “arbovirus group”
Biology usually involves vectors
Many have ambisense genomes
Influenza etiology
• Spread person-to-person by aerosol, direct or
indirect contact, in water – no vectors
• Incubation period 1-3 days
• Causes myalgia, sore throat, fever, headache,
cough which may be protracted
• Symptoms typically last 2-7 days
• Intensity of symptoms differs greatly depending
on virus strain
Viruses from different
families are associated
with infections of the
respiratory tract
• Negative-strand RNA
viruses constitute a
significant portion of
bronchial and
influenza viruses
– Respiratory syncytial
virus – RSV
– Influenza A and B
– Parainfluenza
Influenza A virus
• Orthomyxoviridae
– Influenza virus most important; among the most important diseases
worldwide
– 100 nm spheres
– 8 nucleocapsids per virion
– 13 kb genome divided into 8 segments
– Highly variable natural biology well-studied
– Replication in nucleus, assembly in cytoplasm
Influenza A virus components
• Envelope proteins
– HA – hemagglutinin: trimer of
glycoproteins involved in attachment
– NA – neuraminidase: tetramer
involved in release from endosome
– M1 – membrane matrix protein
– M2 – membrane ion channel protein –
absent in type B and C
• Ribonucleoproteins (RNPs)
– 8 RNP segments per genome
– Each of 8 (-) strand segments wrapped in NP
– One copy of polymerase complex, (PB1, PB2, PA, products of segs
1-3) at 3’-end of each segment
• Nonstructural proteins
– NS1 and NS2 localized to nucleus, encoded from overlapping ORFs
– Overlapping ORFs from spliced RNAs; NS2 is fusion from NS1
Influenza A virus structure and genome organization
•
A. Micrograph and drawing
of Influenza A virus.
– 9 viral proteins are
associated with virion
– All 8 negative-sense
genome segments are
contained in a single
particle
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B. Genome organization.
– 8 negative-sense RNAs
encode 10 proteins
– Segments 7 and 8 each
encode 2 proteins: one by
translation stop/start (7)
and one by splicing (8)
– 3’-ends of mRNAs are
polyadenylated by cellular
machinery; caps are
added to 5’-ends by
“snatching” from cellular
mRNAs
Influenza infection cycle
1-3. Virus HA binds sialic acid receptor,
enters by endocytosis, and releases
core NPs which migrate to nucleus
4-6. Transcription of mRNAs is primed
by capped termini snatched from
cellular mRNAs; transcripts
processed and/or exported to
cytoplasm
7. Translation of membrane proteins in
ER
8-10. Replication-associated and virus
core proteins are translated on free
ribosomes and imported to nucleus
11-13. RNA replication in nucleus
14. M1 and NS1 proteins bind to
nascent – strand RNAs and shut
down viral RNA synthesis;
nucleocapsids exported to cytoplasm
15-18. Viral structural proteins and
nucleocapsids assemble at plasma
membrane
19. Virions complete assembly and bud
through plasma membrane
Influenza A virus RNA synthesis
•
All termini of genomic and mRNAs from individual genomic
segments are different
Genomic 3’ end unmodified; 5’-end ppp (no cap)
Viral mRNA transcription primed with capped 10-13 nt RNA
fragments stolen from cellular mRNA
Before the 5’-end, viral polymerase pauses and reiteratively copies
internal U7 tract to add ~ 150 A residues to 3’-end of mRNA transcript
Genomic RNA is copied to full-length antigenome (+ strand RNA)
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4
Cap snatching by influenza virus
• Proceeds in nucleus
• First, cleavage of capped 5’terminal fragments from
newly synthesized cellular
messages (pre-mRNA)
• 10-13 nt fragments serve as
primers for mRNA synthesis
• Elongation proceeds as
usual, resulting in RNA that
cannot serve as a replication
template
Polycistronic segments
of orthomyxoviruses
Most orthomyxovirus segments
are monocistronic, but several are
polycistronic. These express
downstream genes by alternative
splicing (A, B, D), translational
stop-start (C), and leaky
scanning to alternative start
codon (E). All of these
mechanisms lead to synthesis of
second and/or third protein at
reduced levels compared to the
first.
Influenza A virus genome packaging
• Is one copy of each of the 8 segments of Influenza A virus
RNA packaged, or are 8 random segments packaged?
• Answer: one copy of each segment per particle. How?
– Mechanism is not known; likely RNA/RNA interactions in trans
– 3’ and 5’ ends of each segment contribute to incorporation.
– 3’-terminal 183 and 5’-terminal 157 nt of neuraminidase coding
sequence are important for packaging.
Mutant of
neuraminidase
coding segment
Influenza A virus genome packaging
• Mutants containing fewer than 8 segments, lacking hemagglutinin (HA),
neuraminidase (NA), or both, are viable for replication and virion
production
• Substitution of VSV glycoprotein gene (G) for HA and the green
fluorescent protein (GFP) for NA resulted in viable recombinant virus
expressing both ; the recombinant VSV G protein was mutated during
passage resulting in a shorter-than-full-length protein
• Recombinant virus was stable on passage
Influenza virus variability
• Antigenic drift
– RNA encoding HA or NA
mutates resulting in a new
codon
– Minor coding alteration
results in variation in
folding of antigenic protein
– Mutated protein may have
minor selective advantage
– Continued
mutation/selection results
in gradual buildup of novel
antigenic variants
Influenza virus variability
• Antigenic shift
– Pseudorecombination, or
reassortment
– May occur in any multiplyinfected host
– Results in immediate
change in antigenic
properties
– 256 possible new strains;
one may be more fit than
either parent and rapidly
selected
Influenza epidemiology
• Experiments in Italy demonstrated
flu virus recombination in swine
• Pigs were infected with H3N2
(Hong Kong 68) and HswN1 (now
called H1N1)
• 256 possible reassortants could
result (=28)
• All reassortants of HA and NA
identified following double
infection
Depiction of same
experiments from your
text
Influenza epidemiology
• Influenza A has wide host range
– Birds (natural), sea mammals, horses, pigs, humans
• Strains are described by antigenicity of HA and NA, which
are designated by numbers
• Currently 15 HA (1-15) and 9 NA (1-9) described
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1918 “Spanish flu” pandemic – H1N1
1957 “Asian flu” epidemic – H2N2
1968 “Hong Kong flu” pandemic – H1N2
1977 “swine flu” epidemic – H1N1
1999 – current threat is H5N1, similar to 1918 strain
• Epidemiology involves close contact of humans, farm
animals, and birds – this especially in Asia
• Kills >20,000 per year in the US normally
Influenza epidemiology
• Flu is spread worldwide by migratory birds
– Birds usually affected little by virus
– Virus is shed into cold water, and is stable there
• Spread by shearwaters (migratory birds) from Australia to California turkeys
in the same season
• Arctic Terns spread flu to chickens in Scotland, Alaska and Russia
• 1980 seal flu epidemic linked to duck/tern recombinant; also found in humans
Spanish flu pandemic, 1918
• One of the greatest pandemics in the history of mankind
• Infected >200 million people; 20-40 million killed
• More died in a year than in the four year peak of the Black
Plaque in the 14th century
• People between 15 and 34 years old most differentially affected
– death rate 20 times higher than previous years
• Approximately half of soldiers who died in WWI died of flu
• Some evidence strain first appeared in France in 1916
• Some evidence recombinant HA led to this strain
• Infectious cDNA clones of complete 1918 strain recently
finished – many similarities to current H5N1 avian flu
• Could this happen again? Vaccine/theraputic availability and
knowledge of epidemiology makes another flu pandemic of the
same proportions unlikely, but possible
Influenza vaccine
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Live attenuated and inactivated vaccine can be used
Inactivated vaccine commonly used now
70-80% effective
May be made against: 1) whole virus; 2) subviral particles; 3)
surface antigen
• Multiple virus strains used in production of a given vaccine
• Prediction of strain or strains most likely to be epidemic in a
given year is critical to vaccine production
– WHO meets twice a year, makes recommendations
– Recommendations given to vaccine manufacturers, which make
vaccines for northern and southern hemispheres
• 250 million doses made per year
• Effective H5N1 vaccine made recently, now being increased
Steps in current egg-based flu vaccine production
Requires: 1) advance knowledge of most important strain to
fight and 2) billions of eggs and a lot of money. Cell culturebased vaccine is only a few years from common use.
Infectious cDNA clones of negative-strand RNA viruses
Two systems for the generation of negative-sense RNA viruses from cloned cDNA. (A)
Generation of nonsegmented negative-sense RNA viruses. Cells are cotransfected with protein
expression plasmids for the N, P and L proteins and with a plasmid containing a full-length viral
cDNA, all under the control of the T7 RNA polymerase promoter. Following infection with
recombinant Vaccinia virus encoding T7 RNA polymerase, vRNA is synthesized and the virus
replication cycle is initiated. (B) Generation of influenza A virus. Cells are cotransfected with
plasmids that encode all eight vRNAs under the control of the RNA polymerase I promoter.
Cellular RNA polymerase I synthesizes vRNAs that are replicated and transcribed by the viral
polymerase and NP proteins, all provided by protein expression plasmids.
More efficient method for infectious clones of flu virus recently published (Neumann et al. 2005 PNAS Online Nov 2, 2005)
Infectious cDNA clones of negative-strand RNA viruses
- Construction of infectious cDNA clones of nonsegmented negativestrand RNA viruses is much more difficult than positive-strand RNA viruses
-First done for nonsegmented virus with Rabies virus
(Schnell et al., 1994, EMBO J. 13:4195)
Review: Neunmann et al., 2002, J. Gen. Virol., 83, 2635-2662
Requires:
1. Plasmids encoding nucleoprotein (N) and polymerase proteins
(L and P), all under control of bacteriophage T7 promoter
2. Plasmid containing complete viral sequence (+)strand (the antigenome)
under control of phage T7 promoter
3. Recombinant Vaccinia virus encoding phage T7 RNA polymerase
These three are co-transfected to susceptible cells; once replication cycle
begins, it is no longer dependent on initially added components
T7 polymerase is required because these are cytoplasmic – there is no
cellular DNA-dependent RNA polymerase available
Infectious cDNA clones of negative strand
RNA viruses have many uses
• Examination of:
- 3' and 5' sequences required for replication
- minimal sequences for amplification of DI RNAs
- sequences responsible for DI RNA interference
- effect of changing intergenic dinucleotide sequence
- effect of changing virus gene order
- packaging signals
- effects of non-structural genes
- host range and virulence
• Expression of:
- various reporter genes
- structural genes of other (-) strand RNA viruses
Family Bunyaviridae
• Largest family of RNA viruses, more than 200
members
• All members replicative but not pathogenic in
invertebrate vectors
• Maintained in insect vectors and wild animals in
nature; rarely transmitted to humans and
domestic animals as dead end hosts
• May cause encephalitis and hemorrhagic fevers
• Replication and genome organization somewhat
similar to orthomyxoviruses, but not nuclear
Five genera of Family Bunyaviridae
• Genus Bunyavirus
– Prototype Bunyawera virus; mosquito vectored
• Genus Hantavirus
– Rodent alternate hosts; occasionally major outbreaks
• Genus Nairovirus
– Cause hemorrhagic fevers; tick-borne
• Genus Phlebovirus
– Cause Rift Valley Fever and similar disease; sandfly
vectored
• Genus Tospovirus
– Important plant pathogen with the widest host range of
any plant virus; thrips vectored
Bunyavirus particle composition
Three RNAs, designated L (large) M (middle) and S
(short) are contained in bunyavirus particles. No matrix
protein is present.
Bunyavirus genome organizations
Segments of two genera of the Bunyaviridae are ambisense.
That is, transcripts from both polarities are used as mRNAs. All
transcripts are capped with 5’-terminal fragments snatched from
cellular, cytoplasmic mRNAs, and are not polyadenylated
Ambisense genome expression
For expression of ambisense segments, transcripts
representing both senses of the ambisense segment must
be made and capped.
Replication of bunyaviruses is cytoplasmic;
there is no nuclear component.
Relationships among Bunyaviruses and phylogenetic
comparison to other negative-sense RNA viruses
(plant hosts)