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.
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(-) 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
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
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
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
All 5 proteins encoded by most animal rhabdovirus genomes are part of the virion.
- 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
- Bovine ephemeral virus is most important
- low mortality, but fever, lameness, anorexia of livestock make it a serous problem
- more complex genome – 10 genes
- cause diseases of fish of significant economic importance
- similar to VSV in genome structure, but contain 6th gene for nonstructural protein
- 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
- 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
Virus moves to clathrin coated pits and is taken into the cell by receptor mediated endcytosis.
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.
G protein binds to Sialic Acid receptors on Cell surface. VSV has broad host range and has many potential receptor proteins.
The M protein is released to activate mRNA transcription.
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
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
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
Two genera of plant-infecting rhabdoviruses: one replicates in cytoplasm, the other replicates in nucleus
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 rabiesRabies
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.
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
Brain tissue showing Negri bodies.
Rabies Disease Syndrome
Inactivated whole virus vaccines are available for humans.
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
- 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.
- 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)
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 disulphide-linked 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
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).
Images from Linda M Stannard
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
Rash that starts on the face and neck, then spreads
Red, watery eyes
Average 10 days from exposure to onset of fever
Average 14 days from exposure to onset of rash
Other symptoms and complications
CNS/ brain infection (as SSPE, subacute sclerosing panencephalitis)
Complications may be lethal
More serious in infants and adults, less in children and teens
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 shownMeasles
Defective form of virus that is more strongly cell-associated 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
Recovery often associated with permanent neurological or brain damageSubacute-sclerosing panencephalitis (SSPE)
RNA viruses have many uses:
- 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
- various reporter genes
- structural genes of other (-) strand RNA viruses
Will be especially useful for vaccine production
- Construction of infectious cDNA clones of nonsegmented negative-
strand RNA viruses is much more difficult than positive-strand RNA viruses
- First done with Rabies virus (Schnell et al., 1994, EMBO J. 13:4195)
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
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