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 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 Plant

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 –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 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

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 Replication-associated and virus core proteins are translated on free ribosomes and imported to nucleus RNA replication in nucleus 14.M1 and NS1 proteins bind to nascent – strand RNAs and shut down viral RNA synthesis; nucleocapsids exported to cytoplasm Viral structural proteins and nucleocapsids assemble at plasma membrane 19. Virions complete assembly and bud through plasma membrane Influenza infection cycle

Influenza A virus RNA synthesis All termini of genomic and mRNAs from individual genomic segments are different 1.Genomic 3’ end unmodified; 5’-end ppp (no cap) 2.Viral mRNA transcription primed with capped nt RNA fragments stolen from cellular mRNA 3.Before the 5’-end, viral polymerase pauses and reiteratively copies internal U 7 tract to add ~ 150 A residues to 3’-end of mRNA transcript 4.Genomic RNA is copied to full-length antigenome (+ strand RNA)

Cap snatching by influenza virus Proceeds in nucleus First, cleavage of capped 5’- terminal fragments from newly synthesized cellular messages (pre-mRNA) 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 multiply- infected 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 (=2 8 ) 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 –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; million killed More died in a year than in the four year peak of the Black Plaque in the 14 th 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 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 culture- based 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 PNAS Online Nov 2, 2005)

- Construction of infectious cDNA clones of nonsegmented negative- strand 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, 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

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

Three RNAs, designated L (large) M (middle) and S (short) are contained in bunyavirus particles. No matrix protein is present. Bunyavirus particle composition

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 Bunyavirus genome organizations

For expression of ambisense segments, transcripts representing both senses of the ambisense segment must be made and capped. Ambisense genome expression

Replication of bunyaviruses is cytoplasmic; there is no nuclear component.

Relationships among Bunyaviruses and phylogenetic comparison to other negative-sense RNA viruses (plant hosts)