Transcript NEGATIVE STRAND RNA VIRUSES (-) sense RNA genome: Genomic RNA is not translatable Viral RNA is transcribed into (+)sense mRNA RNA alone is.

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
NONSEGMENTED NEGATIVE STRAND RNA VIRUSES
ORDER MONONEGAVIRALES
The family RHABDOVIRIDAE
-
45-100 X 100-430 nm bacilliform or bullet-shaped particles
membrane spikes composed of only G protein
helical nucleocapsids unwind to 20 X 700 nm
1 segment, I0-14kb genome
5-10 genes encode 5-10 proteins; most have 5-6 genes
200+ virus species
infect vertebrates, invertebrates and plants
Vesicular stomatitis & Rabies virus; many plant & fish viruses
replicate in both host & insect vectors
Rhabdovirus structure
All 5 proteins encoded by most animal rhabdovirus genomes
are part of the virion.
G protein
M Protein
P Protein
N Protein
L Protein
Relationships among rhabdoviruses
Fish hosts
Animal hosts
Plant hosts
FAMILY RHABDOVIRIDAE
Genus Vesiculovirus
- Vesicular stomatitis virus, type species, is best studied rhabdovirus
- causes epizootics in cattle, swine, & horses; insect & tick hosts also
- rapid replication leads to many defective-interfering (DI) particles
Genus Lyssavirus - Rabies virus
- continuous health problem worldwide - thousands of cases per year
- in dogs and cattle in Central & South America; skunks, racoons, foxes in North America
- vampire bats associated with endemic spread in Central America
- prevention by animal immunization; rarely used for humans
- treatment shortly after transmission required; natural mortality in untreated humans is 15-25%
- particularly unpleasant death makes this one of the most feared viruses
Genus Ephemerovirus
- Bovine ephemeral virus is most important
- low mortality, but fever, lameness, anorexia of livestock make it a serous problem
- more complex genome – 10 genes
Genus Novirhabdovirus
- cause diseases of fish of significant economic importance
- similar to VSV in genome structure, but contain 6th gene for nonstructural protein
Genus Cytorhabdovirus
- Lettuce necrotic yellows virus is type member
- different species replicate in and transmitted by different vectors
- functional transcriptase can be isolated
- bud through membrane in cytoplasm
Genus Nucleorhabdovirus
- Potato yellow dwarf virus is type member; Sonchus yellow net virus is best studied
- different species replicate in and are transmitted by different vectors
- functional transcriptase has not been isolated
- replicate in nucleus and bud through inner nuclear membrane
Entry of Vesicular Stomatitis Virus into Cells
Virus moves to clathrin
coated pits and is taken
into the cell by receptor
mediated endcytosis.
G protein binds to Sialic
Acid receptors on Cell
surface. VSV has broad
host range and has
many potential receptor
proteins.
Endosome moves
into the cytoplasm to
a moderately low pH.
The G protein has a
conformational
change and the
nucleocapsid is
released into the
cytoplasm.
The M protein is
released to activate
mRNA
transcription.
Animal rhabdovirus infection cycle
1.
Virus binds receptor and enters
by endocytosis
2-3. Membrane fusion and core
release
4-6. Sequential transcription and
translation of mRNAs
7-8. After nucleocapsid protein (N)
accumulates, switch from
transcription to replication occurs
5,9,10. Glycoprotein (G) enters
secretory pathway and is
embedded in plasma membrane
11,12. Nucleocapsid with associated
phosphoprotein (P) and RdRp (L)
are organized into bullet-shaped
core particles by matrix protein
(M)
13. Cores bud through membrane at
sites of G-protein accumulation
Rhabdovirus transcription and replication
VSV is the model rhabdovirus
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Rhabdovirus genes are
transcribed sequentially, in
order of greatest requirement
and time of requirement
Regulatory leader sequence is
transcribed first and binds N
protein early and often,
resulting in maintenance of
transcription mode
Sufficient N-protein
accumulation and
phosphorylation state of P
results in switch from
transcription to replication
Full-length antigenome (+
sense RNA) is template for sense genome replication
SEE AND UNDERSTAND
FIGURE ON P. 200-201
Details of genome structure of VSV. Coding regions for the nucleoprotein (N), phosphoprotein (P),
matrix protein (M), glycoprotein (G) and polymerase protein (L) are separated by regulatory
sequences that contain a transcription stop (gene end) signal, a polyadenylation signal, a
nontranscribed intergenic region and a transcription start (gene start) signal. Transcription units are
flanked by a leader (Le) and trailer (Tr) region that contain the genomic and antigenomic viral
promoters, respectively. Note that the genome sense strand is shown, so by convention, the 3’-end
is on the left. From Neumann et al., J. Gen Virol. DOI 10.1099/vir.0.18400-0
Rabies Virus
FIG. 1. (A) Organization of the 12-kb genome of RV. Filled boxes represent the open reading frames of the N, P, M
(matrixprotein), G, and L genes. c represents the nontranslated 39 sequence of the G cistron (pseudogene). (B)
Organization of chimeric viruses. Open reading frames are drawn as shaded boxes, and transcriptional
stopypolyadenylylation signals are represented by flags. In SAD VCAT, a DNA fragment composed of the NyP
cistron border and the CAT open reading frame was used to replace the major part of the pseudogene sequence
of standard RV SAD L16; for construction of SAD XCAT, the fragment was inserted into the StyI site of the
pseudogene. The former coding part of the N gene is shown as an open box.
Transcription and replication initiate at separate sites on the
vesicular stomatitis virus genome
Fig. 6. Model for RNA synthesis: Schematic of the VSV genome depicting the leader region (Le) and the N and P
genes. During transcription the RdRP, a complex of L (large oval) and a trimer of P (small oval), binds to specific
sequences and initiates synthesis at the N gene start. The products of this reaction are the VSV mRNAs, of which
the N and P mRNAs are shown. The 5'-terminal cap is depicted by a black diamond, and the 3' polyadenylate tail
by A(n). During replication, the RdRP initiates at the 3' end of the genome. Initiation at the 3' end provides leader
RNA (not shown) and the full-length antigenome. Replication requires protein synthesis to supply N protein for
encapsidation of nascent RNA. N protein (hatched oval) is kept soluble by interaction with P, in a 2:1 complex.
Positions of initiation are indicated by the black triangles.
From Whelan and Wertz, 2002, PNAS 99: 9178-9183
Particle structure, genome organization, and expression of
Vesicular stomatitis virus, a cytoplasmic rhabdovirus
Nucleorhabdovirus
Cytorhabdovirus
Two genera of plant-infecting rhabdoviruses: one replicates in cytoplasm, the
other replicates in nucleus
RABIES
Rabies
• Serious and highly contagious
• Mostly in wild animals; domestic animals now less
than 10% of cases
• Symptoms include insomnia, anxiety, confusion,
slight or partial paralysis, excitation,
hallucinations, agitation, hypersalivation, difficulty
swallowing, and hydrophobia, then death
• Transmitted by bites of infected animals
• Bats are commonly associated with rabies
www.lmb.uni-muenchen.de/groups/
Conzelmann/default.htm
Rabies is a virus that attacks the central nervous system of
warm-blooded animals. It can be transmitted by the bite of a
rabid animal or via the introduction of the saliva of a rabid animal
into a fresh (less than 24 hours old) wound. Transmission via
other routes is rare.
History of Rabies Virus
Causes the most lethal of all infectious
diseases. Even the most extreme modern
medical interventions are usually not
successful.
The disease was recognized in Egypt before
2300 B.C. and in ancient Greece, where it
was described by Aristotle.
The method of transmission of rabies was not
recognized until 1804.
Rabies is thought to be responsible for the
origin of vampire legends (bats, biting,
hypersexuality are associated with rabies).
Infects all Mammals. In North America,
rabies is most common in skunks, foxes,
raccoons, bats, dogs, and cats.
Occurs worldwide, with the exception of
Hawaii, Japan, Great Britain, & smaller
islands
Racoons, Skunks, Foxes and Bats Are Major Rabies Reserviors
Racoon
Red Fox
Striped Skunk
Silvertailed Bat
Bat Bites Account for About Half of USA Rabies Cases
CDC
Rabies Disease Syndrome
 Incubation normally 1-2 months
occasionally up to two years.
 Multiplies in muscle and
connective tissue.
 Enters peripheral nerves to CNS
 First symptoms of malaise, sore
throat, fever.
 Increased sweat, hydrophobia,
difficulty swallowing, muscle
spasms, convulsions
 Levels of virus in the blood is not
very high.
 Invariably fatal.
 Death by respiratory paralysis.
 Virus can be diagnosed in brain
tissue by staining for Negri bodies
and by fluorescent antibody tests.
Positive Fluorescent Antibody
Test
Brain tissue showing Negri bodies.
Rabies Vaccines for Humans
Inactivated whole virus vaccines are available for humans.
– Nervous Tissue Preparations - Associated with the rare
complication of demyelinating allergic encephalitis.
– Duck Embryo Vaccine is grown in embryonated duck
eggs The vaccine has a lower risk of allergic
encephalitis but is considerably less immunogenic.
– Human Diploid Cell Vaccine (HDCV) - this is currently
the best vaccine available with an efficacy rate of nearly
100% and rarely any severe reactions. However it is very
expensive.
– Other Cell culture Vaccines - because of the expense of
HDCV, other cell culture vaccines are being developed
for developing countries. However recent data suggests
that a much reduced dose of HDCV given intradermally
may be just be effective.
World-Wide Control of Rabies
Urban - Canine rabies accounts for more than 99% of all
worldwide human rabies. Control measures against
canine rabies include;

stray dog control.
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vaccination of dogs.
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quarantine of imported animals.
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Wildlife - Rabies in wild animals is much more difficult
to control than canine rabies. However, on-going trials in
in the USA and Europe using bait containing rabies
vaccine given to foxes and raccoons reduces rabies
levels.

Rabies Control
• Eliminate rabies from hosts/victims
– Oral baits w/antivirus.
• Has been effective (Europe, Canada).
• Slowed potential outbreak in Ohio raccoons.
• Used in Texas for coyotes & foxes.
– Trap/vaccinate/release (TVR)
• Effective in Canada raccoons.
• Often combined with oral baits.
Several negative sense RNA viruses infect the central nervous system
Neurotropic – infect neural cells
Neuroinvasive – can enter CNS (spinal cord or brain)
Neurovirulent – can cause disease of nervous tissue
Mumps – highly neuroinvasive, but low neurovirulence; most infection leads to
infection of CNS, but neurological disease is mild
Rabies – highly neuroinvasive, high neurovirulence – readily infects periferal
NS and spreads to CNS, with 100% lethality if no antiviral therapy
(Herpes) – low neuroinvasiveness, high neurovirulence – always infects
periferal NS rarely spreads to CNS, severe consequences when it does
From Flint et al., 2004 – your text
FAMILY
T FILOVIRIDAE
- 80 x 800-1000nm or filaments sometimes longer in EMs
- very long branched, 6-shaped or U shaped particles
- helical nucleocapsids
- genome is single negative sense ssRNA, 19Kb
-only 2 antigenically unrelated member viruses - Marburg virus & Ebola virus,
several strains of the latter
- Marburg epidemic of lethal hemorrhagic fever reported in Germany & Bulgaria
(1967)
- epidemiology linked to green monkey kidney handling
- Ebola epidemic in the Sudan area in 1976 up to 88% lethal in the 550 cases
identified; most lower (<35% lethal)
These viruses appear to be among the least successful of negative strand RNA
viruses in that they have a restricted host range and limited distribution. They
are among the most feared by humans because of their spectacular
symptomatology and often fatal outcome of infection.
FAMILY PARAMYXOVIRIDAE
- pleomorphic, 150-250nm diameter; filamentous forms
common
- evident spike glycoproteins (1 or 2) and unglycosylated
proteins (1 or 2)
- filamentous helical nucleocapsids
- 15-16 kb ssRNA genome
- 7-8 functional genes encode 10-12 polypeptides
- found only in vertebrates; no vectors, most airborne;
horizontal transmission only
- large family of viruses, 3 genera contain serious human &
animal pathogens measles, mumps, distemper,
respiratory syncytial virus
- genome organization similar to rhabdovirus; structure and
biology more similar to orthomyxovirus (influenza)
Subfamily Paramyxovirinae
Genus Morbillivirus
- measles virus, canine distemper virus main pathogens
- Members contain hemagglutinin, not neuraminidase activity
Genus Paramyxovirus
- Sendai virus of mice is the model system
- Members contain both hemagglutinin and neuraminidase activity
- Members encode C protein
Genus Rubulavirus
- Mumps virus is major pathogen
- Members contain both hemagglutinin and neuraminidase activity
- Members do not encode C protein
-Subfamily Pneumovirinae
- Genus Pneumovirus
- Respiratory syncytial virus is major pathogen
- Members lack hemagglutinin or neuraminidase activity
Paramyxoviridae
Glycoproteins - do not form
such prominent spikes as on
influenza virus:
HN - haemagglutinin +
neuraminidase activities;
Measles - referred to as H
protein - no neuraminidase
activity;
RSV - G protein - neither
activity.
F - consists of 2 disulphidelinked subunits (F1 + F2) responsible for cell fusion +
haemolytic function.
Other proteins:
The M (matrix) protein lines the
inner surface of the envelope.
NP - nucleoprotein.
L and P - polymerase activity
From Alan Cann website
Measles
Family – Paramyxoviridae
Genus – Morbillivirus
Enveloped virions contain a helical nucleocapsid
composed of negative-sense ssRNA,
nucleocapsid protein, and replication-associated
proteins. Virions are roughly spherical ~ 200nm
in diameter but may appear larger and more
pleomorphic in negative stained EMs. The virus
envelope is a lipid bilayer, studded with virus
encoded glycoproteins which have properties of
haemagglutination and fusion (the F protein).
Stain penetrating
Images from Linda M Stannard
Measles virus
From Flint et al., ASM Press, 2004
Measles virus
From Flint et al.,
ASM Press, 2004
Measles
• Serious and highly contagious
– Usually found in non-immunized or partially-immunized
(single vaccine, no booster)
– Most born before 1957 have had measles
• Measles virus is spread easily
– Through air by coughs or sneezes
– By direct contact with nose or throat secretions
• Symptoms
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Rash that starts on the face and neck, then spreads
High fever
Runny nose
Red, watery eyes
Cough
Measles
• Symptoms start about 10 days after exposure
– Average 10 days from exposure to onset of fever
– Average 14 days from exposure to onset of rash
• Other symptoms and complications
– Ear infection
– Pneumonia
– CNS/ brain infection (as SSPE, subacute sclerosing
panencephalitis)
– Complications may be lethal
– More serious in infants and adults, less in children and teens
• Vaccine
– Measles (Paramyxoviridae), mumps (Paramyxoviridae), rubella
(Togaviridae, + sense) (MMR) vaccine is a live vaccine
– Has been very effective in limiting spread
– Links of vaccine to autism have been proposed but not shown
Subacute-sclerosing panencephalitis
(SSPE)
• Neural manifestation of measles virus
• Defective form of virus that is more strongly cellassociated found in SSPE brains
• Specific mutations result in virus that is more fit in
neural cells
• Usually occurs 6-12 years after measles infection
• More rarely associated with live measles vaccine
• Infection rate 1 per million; higher in males & rural
• Mortality high
• Recovery often associated with permanent neurological
or brain damage
Many lawsuits trying to link MMR vaccine to autism are still pending
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
Expression of:
- various reporter genes
- structural genes of other (-) strand RNA viruses
Will be especially useful for vaccine production
Infectious cDNA clones of nonsegmented 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 with Rabies virus (Schnell et al., 1994, EMBO J. 13:4195)
- 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
Neumann et al. 2002 JGV 83, 2635
Infectious cDNA clones of negative-strand RNA viruses
Fig. 2. Systems for the generation of negative-sense RNA viruses from cloned cDNA. (A)
Schematic diagram for the 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 VV encoding T7 RNA polymerase, vRNA is synthesized and
the virus replication cycle is initiated. (B) Schematic diagram for the 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.
Neumann et al. 2002 JGV 83, 2635
Infectious cDNA clones of a multipartite
negative sense RNA virus such as Influenza
virus are especially difficult to make
Figure from The Scientist, based on Neumann et al., PNAS 96: 9345