Negative (-) - Sense ssRNA Viruses

Dan Matthew E. Burias

BS Biology (Microbiology Track)
Mindanao State University – Iligan Institute of Technology
Definition
 Also called antisense RNA
 Does not exhibit polarity of mRNA
 Must synthesize first the sense or positive strand RNA using viral
RNA-dependent RNA Polymerase (RdRp) and the antisense or
negative strand as template to proceed viral protein translation.
 Viruses can be classified based on the segmentation of their genome:
Non-segmented genome and Segmented Genome
 Encompasses the viruses responsible for several medically important
diseases like Lassa Hemorrhagic Fever, Influenza, Rabies,
Measles and Ebola Hemorrhagic Fever.
 Some single-stranded RNA viruses belonging to families of
Bunyaviruses and Arenaviruses can utilize some parts of their
segmented genomes in both positive-sense and negative sense.
Viral Replication Mechanism (Simplified Schematic Diagram)
Viral Replication
 Replication events:
 The RNA dependent RNA polymerase complex binds to the
leader sequence on the encapsidated (-)RNA genome, and starts
replication.
 The antigenome is concomitantly encapsidated during replication.
 The RNA dependent RNA polymerase complex ignores all
transcription signals when in replication mode.
 The antigenome is then replicated under the same process, the viral
polymerase complex binding first to the trailer sequence.

Notes: The viral RdRP complex is presumably the same for replication
and transcription. It may be a transcriptase by default and turn into
the replicase mode depending on nucleocapsid concentration.
Viral Mechanism (Replication)
Viral Transcription
 The RNA dependent RNA polymerase (RdRp)complex
initiates transcription by binding to the leader sequence in 3’ of
the genomic negative strand RNA.
 The RdRp transcribes a 5’ triphosphate-leader RNA, then stop
and restart on a transcription initiation signal. The RNA initiated
on this signal is capped.
 At the end of viral genes is a transcription stop signal on which
the RdRp will produce a polyadenylation signal by
stuttering on a U stretch before releasing the mRNA.
 On polycistronic genomes, the RdRp can then scan to the
next transcription initiation and resume transcription on the next
gene.
Viral Mechanism (Transcription)
Polymerase Stuttering
 Many negative stranded RNA virus polyadenylate their mRNA
through a polymerase stuttering mechanism during transcription.
 The stop signal present at the end of each gene comprises a
stretch of U on which the viral polymerase acquires a stuttering
behavior: after each Adenine inserted, it moves back one
nucleotide along with the mRNA. It resumes transcription adding
a new A, then again moves back. In this way a U on the genomic
template is copied hundreds of times at the end of viral mRNA
thereby producing a polyA tail.
 Eventually the polymerase will release the polyAdenylated
mRNA and stop transcription or scan to restart on the next
gene.
Polymerase Stuttering (Borna-, Filo-, Paramyxo-, Rhabdo- and Orthomyxoviridae)
Viral Mechanism (Replication Cycle)
NSssRNA Families
 SEGMENTED GENOME
 Orthomyxoviridae (Replication in Host Cell nucleus)
 Arenaviridae
 Filoviridae
 NON-SEGMENTED GENOME (Order Mononegavirales;
Replication in Cytoplasm)
 Paramyxoviridae
 Bornaviridae
 Rhabdoviridae
 Filoviridae
NSssRNA Viral Diversity
FILOVIRIDAE(Overview)
FILOVIRIDAE (Overview)
 Classified within the Order Mononegavirales; two genera;
Ebolavirus and Marburgvirus
 Causes infections in vertebrate animals, especially primates
FILOVIRIDAE (History)
 The first Filovirus was recognized in 1967 in Germany and
Yugoslavia. The virus was named after Marburg, Germany, the
site of one of the outbreak.

 Reemerge in 1975, when a traveler, most likely exposed in

Zimbabwe, became ill in Johannesburg, South Africa. A few
sporadic cases and 2 large epidemics (Democratic Republic of
Congo in 1999 and Angola in 2005) of Marburg hemorrhagic
fever (Margurg HF) have been identified since that time.
FILOVIRIDAE (History)
 Ebolavirus was first identified in 1976 when two outbreaks of
Ebola hemorrhagic fever (Ebola HF) occurred in northern Zaire
(now the Democratic Republic of Congo) and southern Sudan.

 Two different species of Ebola virus; both were named after the
nations in which they were discovered.
 90% of the Zairian cases and 50% of the Sudanese cases resulted in
death.
FILOVIRIDAE (History)
 Past outbreaks have been determined to have origins in Africa
and in one instance, the Philippines.
 Subsequent outbreaks of EBOV, has led to the identification of
three additional distinct species - EBOV-S (Sudan), EBOV-IC
(Ivory Coast), EBOV-R (Reston).
FILOVIRIDAE (History)
FILOVIRIDAE (Epidemiology)
FILOVIRIDAE (Genome Structure)
 Non-segmented and is comprised of a linear, negative-sense,
single-stranded RNA molecule. The complete genome is
approximately 18,900-19,000 bases in length.
FILOVIRIDAE (Genome Structure)
FILOVIRIDAE (Phylogeny)
FILOVIRIDAE (Virion Structure)
 Non-uniform, pleomorphous shape
 More similar to Rhabdoviruses, but considerably longer and filamentous (Latin filus
meaning “thread”).The filaments can be branched, U-shaped or spirally coiled.
 The particles have a constant diameter of 80 nm; however, their length is highly
variable (up to 14,000 nm).
 Marburg virus is about 665 nm long, whereas Ebola virus is 805 nm long.
 The filaments consist of a helical nucleocapsid, which is composed of the
 RNA genome and the viral proteins NP (nucleoprotein),
 P (alsoVP35),
 VP30 and;
 L (RNA-dependent RNA polymerase).
 The nucleocapsid is surrounded by an envelope.
 The matrix proteins VP24 (minor) and VP40 (major) are associated with both the inner
side of the envelope and the protein components of the nucleocapsid.
 The trimeric glycoproteins (GP) are embedded in the envelope and project
approximately 7 nm from the surface of the virus
FILOVIRIDAE (Virion Structure)
FILOVIRIDAE (Virion Structure)
FILOVIRIDAE (Virion Structure)
FILOVIRIDAE (Replication)
 CYTOPLASMIC
 Attachment to host receptors through GP glycoprotein mediates
is endocytosed into vesicles in the host cell by apoptotic mimicry.
 Fusion of virus membrane with the vesicle membrane;
ribonucleocapsid is released into the cytoplasm.
 Sequential transcription, viral mRNAs are capped and
polyadenylated by polymerase stuttering in the cytoplasm.
 Replication presumably starts when enough nucleoprotein is present
to encapsidate neo-synthetized antigenomes and genomes.
 The ribonucleocapsid interacts with the matrix protein under the
plasma membrane, buds via the host ESCRT complexes from the
plasma membrane, releasing the virion.
FILOVIRIDAE (Replication)
FILOVIRIDAE (Receptors)
FILOVIRIDAE (Fusion)
FILOVIRIDAE (Budding)
Many enveloped viruses recruit components of the
cellular ESCRT (endosomal sorting complex required for transport)
system to mediate host-assisted viral budding.
FILOVIRIDAE (Diseases)

Suspected Natural
Virus Disease Signs/ Symptoms
Host

Sudden onset of fever,

malaise, muscle pain,
Marburg Hemorrhagic
Bats headache, inflammation
Marburg virus (MARV) Fever
of the pharynx, vomiting,
bloody diarrhea

Sudden onset of fever,

Ebola Hemorrhagic malaise, muscle pain,
Bats
Ebola virus (EBOV) Fever headache, inflammation
of the pharynx, vomiting,
bloody diarrhea
FILOVIRIDAE (Host)
 Filoviruses are zoonotic
 Despite numerous attempts to locate the natural reservoir or
reservoirs of Ebolavirus and Marburgvirus species, their origins
were undetermined until recently when Marburgvirus and
Ebolavirus were detected in fruit bats in Africa.
 Marburgvirus has been isolated in several occasions from
Rousettus bats in Uganda.
FILOVIRIDAE
 Pathogenesis
 Clinical and biochemical findings support anatomical observations of:
 extensive liver involvement,
 renal damage,
 changes in vascular permeability, and
 activation of the clotting cascade.
 Visceral organ necrosis is the consequence of virus replication in
parenchymal cells. However, no organ is sufficiently damaged to cause death.
 Fluid distribution problems and platelet abnormalities indicate
dysfunction of endothelial cells and platelets.
 The shock syndrome in severe and fatal cases seems to be mediated by
virus-induced release of humoral factors such as cytokines.
 Filovirus glycoproteins carry a presumably immunosuppressive domain, and
immunosuppression has been observed in infected monkeys.
FILOVIRIDAE (Transmission, Treatment and
Prevention)
 Transmission
 person-to-person by way of contact with infected bodily fluids.
 route of transmission from animals to humans remains unknown.
 Treatment
There is no standard treatment for filovirus infections. Infections are
generally treated through:
 pain management, and
 supportive therapy for maintaining blood volume and electrolyte balance.
 Prevention
 The best means of preventing the spread of filovirus infections is through universal
precaution measures.
 Vaccines have been proven effective in protecting non-human primates. Currently
there are no approved vaccines available for humans.
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
 The term “Ortho” distiguishes this group from paramyxoviruses
 Greek word myxo means “mucus”.
 Structure
 Influenza viruses are spherical or filamentous enveloped particles 80 to
120 nm in diameter.
 The helically symmetric nucleocapsid consists of a nucleoprotein and a
multipartite genome of single-stranded antisense RNA in seven or eight
segments.
 The envelope carries a hemagglutinin attachment protein and a
neuraminidase.
 Classification and Antigenic Types
 Influenza viruses are divided into types A, B, and C on the basis of variation
in the nucleoprotein antigen. In types A and B the hemagglutinin and
neuraminidase antigens undergo genetic variation, which is the basis for the
emergence of new strains; type C is antigenically stable.
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE (Molecular Structures)
ORTHOMYXOVIRIDAE (Molecular Structures)
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
NUCLEUS

Virus attaches to sialic acid receptor though HA protein and

is endocytosed by clathrins in the host cell.
endosome acidification induces fusion of virus membrane with the
vesicle membrane; encapsidated RNA segments migrate to the nucleus.
Transcription of genomic segments by the viral polymerase produces
mRNAs that are capped and polyadenylated by the viral polymerase.
Replication of genomic segments.
High level of M1 protein induces genomes segments export from
nucleus by NEP protein.
Virus assembly and ESCRT-independent budding occurs at the plasma
membrane.
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
 Multiplication
ORTHOMYXOVIRIDAE
 Pathogenesis
 The virus is transmitted in aerosols of respiratory secretions. It multiplies in
the respiratory mucosa, causing cellular destruction and inflammation.
 Clinical Manifestations
 Classic influenza is a febrile illness of the upper and lower respiratory tract,
characterized by:
 sudden onset of fever,
 cough,
 myalgia,
 malaise,
 and other symptoms.
 Pneumonia is the most common serious complication.
 Host Defenses
 Both a cell-mediated response and antibody develop after infection.
Antibody provides long-lasting immunity against the infecting strain.
ORTHOMYXOVIRIDAE
 Gene Reassortment
 Because the influenza virus genome is segmented, genetic
reassortment can occur when a host cell is infected
simultaneously with viruses of two different parent strains. If a
cell is infected with two strains of type A virus, for example,
some of the progeny virions will contain a mixture of genome
segments from the two strains. This process of genetic
reassortment probably accounts for the periodic appearance of
the novel type A strains that cause influenza pandemics.
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
ORTHOMYXOVIRIDAE
 Epidemiology
 Influenza epidemics involving all age groups occur each winter;
worldwide pandemics appear irregularly.
 Changes in the hemagglutinin and neuraminidase surface antigens are
responsible for the appearance of antigenically novel strains that evade host
immunity and cause reinfections.
 Diagnosis
 The diagnosis is suggested by the symptoms, particularly if an
influenza epidemic is under way. Definitive diagnosis depends on
detecting the virus or a rise in antibody titer.
 Control
 An inactivated virus vaccine is developed each year against the strains
most likely to cause disease the next winter. The drugs amantadine
and rimantadine can be used for prophylaxis and treatment of
influenza A infections.
REFERENCES
 MicrobiologyBytes (2008). Negative Sense RNA Viruses.
https://microbiologybytes.wordpress.com/2008/02/04/negative-sense-rna-viruses/
 Viral Zone (N.D.) Negative-stranded RNA virus Transcription.
http://viralzone.expasy.org/all_by_species/1917.html
 Viral Zone (N.D.) Negative-stranded RNA virus Replication.
http://viralzone.expasy.org/all_by_species/1096.html
 Viral Zone (N.D.) Negative-stranded RNA virus Polymerase Stuttering.
http://viralzone.expasy.org/all_by_species/1916.html
 https://www.google.com.ph/imgres?imgurl=http://elifesciences.org/content/elife/4/e05
378/F8.large.jpg&imgrefurl=http://elifesciences.org/content/4/e05378&h=2010&w=299
7&tbnid=1IlcD0Pza3OhBM:&docid=DBJ5_aBqmoJOBM&itg=1&ei=vGFfVpPyN8m30QSo
-6DACw&tbm=isch&ved=0ahUKEwiTwPPIjr7JAhXJW5QKHag9CLgQMwgcKAIwAg
 ViPR (2015). Filoviridae. http://www.viprbrc.org/brc/aboutPathogen.spg?decorator=filo
 http://vignette3.wikia.nocookie.net/mmg-233-2014-genetics-
genomics/images/c/cd/Ebolavirus_cycle.jpg/revision/latest?cb=20141129194710
 http://viralzone.expasy.org/all_by_species/23.html