RNA VIRUSES

1
RNA VIRUSES
2
Picornaviridae
3

• The smallest RNA-containing viruses known.
• Comprise one of the largest (230 members) and
  most important families of human and agricultural
  pathogens.
• The family is currently divided into five genera
  Rhinoviruses, Enteroviruses, Aphthoviruses,
  Cardioviruses, and Hepatoviruses (REACH).

4

• Aphtovirus (foot-and-mouth disease virus)-
  Infects cloven-hoofed (footed) animals and
  occasionally humans.
• Cardiovirus- Infects rodents
• Three genera Rhinovirus, Enterovirus, and
  Hepatovirus include primary human pathogens with
  numerous serotypes.

5
Genus Virus Serotypes
Rhinoviruses gt 100
Enteroviruses
Polio viruses 3
Coxsackie viruses A B 23 6 1-22, 24 (23 echorviruses 9) 1-6
Echoviruses 28 1-7,9,11-21, 24-27,29-33 Echo 8 Echo 1 Echo 10 Reovirus 1 Echo 28 Rhino 1A Echo 34 Coxsackie A 24 Echo 22,23 Parechovirus
Parechoviruses 2 Previously Echo 22 and 23
Enteroviruses 68-71 4 New naming system since 1967
Hepatovirus Hepatitis A virus 1 Previously enteroviruses 72
Total 67
6

• Enteroviruses and Hepatovirus differ from
  Rhinoviruses in
• - Stability at pH 3
• - Optimum T º of growth
• - Mode of transmission
• - Diseases caused

7
Enterovirus Particles
8

• The virion is roughly spherical, naked, and range
  in diameter from 24 to 30 nm.
• 60 subunits make up the icosahedral capsid each
  of which is composed of four polypeptide chains,
  VP1-VP4
• VP1, 2 and 3 are exposed at the virion surface,
  whereas VP4 lies buried in close association with
  the RNA core.

9

• Of the four proteins, VP1 exhibits the greatest
  sequence variability and VP4 the least.
• VP1 is also the dominant protein, playing key
  roles in surface topography, antigenicity,
  receptor binding, and probably viral uncoating.
• Genome is a ss RNA ( 7500 nt) of positive
  polarity, polyadenylated at 3- and has a protein
  of 22 to 24 amino acids (VPg) at the 5- end.

10
Receptors

• The receptors for polioviruses, Coxsackie
  viruses, echoviruses, and the major serogroup of
  rhinoviruses have all been mapped to human
  chromosome 19.
• The receptors for polioviruses and human
  rhinoviruses have been identified as members of
  the Ig superfamily, whereas the receptor for
  echoviruses has been identified as a member of
  the integrin family.

11

• Virus Receptor
• - Rhinovirus (major) ICAM-1
• - Rhinovirus (minor) LDL-R
• - Polioviruses PVR (similar
  to
• 
  ICAM-1) CD155
• - Coxsackie A ICAM-1
• - Coxsackie B Unknown
• - Echovirus DAF, VLA-2

12
Enteroviruses

• Arildone (pleconaril) contains a 3-methyl-
  isoxazole group that binds to the floor of the
  VP1 canyon and alters its conformation to prevent
  the uncoating of the virus.
• Poliovirus produces a protease that degrades the
  200.000 Dalton cap-binding protein of eukaryotic
  ribosomes, thereby blocking the translation of
  cellular mRNA.

13
Orthomyxoviridae
14
INFLUENZA VIRUS
15
ORTHOMYXOVIRUSES
type A, B, C NP, M1 protein sub-types HA or
NA protein
16

• Among the RNA viruses, influenza is very special
  in that all of its RNA synthesis take place in
  the nucleus.
• Short capped primers are generated from host cell
  RNAs by an influenza virus-encoded cap-dependent
  endonuclease.
• Influenza virus mRNAs undergo splicing in the
  nucleus.

17
Classification

• The family contains two genera Influenza A and B
  viruses, and influenza C virus.
• Genera are distinguished on the basis of
  antigenicity of nucleoprotein (NP) and matrix (M)
  proteins.
• Influenza A viruses are divided into subtypes
  based on the antigenicity of HA and NA
  glycoproteins.

18
Distinguishing Characteristics

• Influenza A viruses naturally infect humans,
  several other mammalian species and a wide
  variety of avian species whereas Influenza B and
  C are human pathogens.
• The surface glycoproteins of influenza A virus
  exhibit much greater amino acid sequence
  variability than their counterparts in influenza
  B virus. Influenza C has a single multifunctional
  glycoprotein.
• Influenza A and B viruses contain 8 RNA segments,
  whereas influenza C contains 7 segments

19
Virion Structure

• Influenza A and B are morphologically
  indistinguishable but there are morphological
  features that distinguish influenza A and B
  viruses from influenza C virus.
• The RNPs consist of four protein species and the
  RNA genome which occurs in eight separate
  segments containing 10 genes.
• The segments are complexed with nucleoprotein to
  from a nucleocapsid with helical symmetry.
• Genomic segments range from 890 to 2340 bases.

20
Replication

• Unlike replication of other RNA viruses,
  replication of orthomyxovirus depends on the
  presence of active host cell DNA synthesis.
• Replication in the nucleus is necessary because
  the virus lacks capping and methylating enzymes
  activities.
• The virus scavenges cap sequences from the
  nascent mRNA generated in the nucleus and
  attaches it to its own mRNA.

21
Genome Organization

• RNA segment 1 codes for PB2
• RNA segment 2 for codes PB1
• RNA segment 3 for codes PA
• RNA segment 4 for codes HA
• RNA segment 5 for codes NP
• RNA segment 6 for codes NA
• RNA segment 7 for codes M1 and M2
• RNA segment 8 for codes NS1, and NS2

22
Virion Proteins

• PB2, PB1 (Basic), PA (acidic)
• NP
• HA (16 subtypes)
• HEF (Influenza C)
• NA (9 subtypes)
• M1 and M2
• NS1 and NS2

23
Genetics

• RNA segment Reassortment (Antigenic shifts)
• RNA Mutations (antigenic drifts)
• RNA Recombination
• Nomenclature
• - A/Swine / lowa/15/30/H1N1
• - A/ Bangkok/1/79/H3N2

24
Paramyxoviridae
25

• Enveloped viruses with a negative single stranded
  nonsegmented RNA genome.
• They have special relationships with
  orthomyxoviruses and rhabdoviruses.
• They encode and package their own RNA
  transcriptase.
• They range in size from 150 350 nm

26
Classification

• The Paramyxovirinae
• Paramyxovirus Parainfluenza virus
  types
• 1 and 3.
• Rubulavirus mumps virus,
  
• 
  parainfluenza virus types
• 2, 4a and
  4b.
• Morbillivirus measles virus.
• The pneumovirinae
• Pneumovirus Respiratory Syncytial
• Virus (RSV)

27

• Nucleoprotein (NP)
• Phosphoprotein (P Protein)
• large (L) protein
• The matrix (M) protein
• Envelope Glycoproteins
• - Attachment protein (HN,H, G)
• - Fusion Protein (F)
• Other proteins
• - SH, C, V, W, I, D, NS1 and NS2.

28
Parainfluenza Virus

• ssRNA virus
• enveloped, pleomorphic morphology
• 5 serotypes 1, 2, 3, 4a and 4b
• No common group antigen
• Closely related to Mumps virus

29
Parainfluenza Viruses

• Important respiratory tract pathogens of infants
  and children causing 30-40 of such infections.
• They are second only to RSV as a cause of serious
  respiratory tract disease in infants and children
  (HPIV 1-3).
• Pleomorphic, 150-200 nm in diameter, enveloped
  with HN and F envelope glycproteins.

30
Respiratory Syncytial Virus (RSV)

• RSV is the most important cause of viral lower
  respiratory tract disease in infants and children
  worlwide.
• RSV infection is an important agent of disease in
  immunosuppressed adults and the elderly.
• Ranges in diameter from 150-300 nm

31

• Research on RSV has been impeded because
• - It grows poorly in tissue culture and most
• exprerimental animals
• - it does not shut off host macromolecular
• synthesis
• - The virion is unstable.

32

• RSV survives on surfaces for up to 6 hours and on
  gloves for less than 2 hours.
• The virus loses viability with freeze-thaw
  cycles, in acidic conditions and with treatment
  by disinfectants.
• It encodes a larger number of mRNAs than do the
  paramyxoviruses (10 compared with 6 or 7)
• Additional genes are SH, M2, NS1, and NS2

33

• Although six proteins appear to correspond (N, P,
  M, G/H/HN F and L) only F and L exhibit
  unambiguous sequence relatedness between the two
  subfamilies.
• Variation in the G glycoprotein (RSV-A and B)
• RSV utilizes ICAM-1 as its receptor.

34
Mumps Virus

• to mump means to grimace or grin.
• The virion is 120 200 nm in diameter
• Contains in addition to the six major proteins V
  (viral) protein and S (soluble) protein.
• One serotype.

35

• MEASLES (RUBEOLA)
• Measles is a relatively new disease of humans.
• Probably it has evolved from an animal
  morbillivirus (rinderpest).
• It is related to canine distemper virus.
• Abu- Becr Al- Razi of 10th century is credited
  with distinguishing smallpox from measles.
• He referred to measles as hasbah eruption in
  Arabic and regarded it as a modification of
  smallpox.

36

• It is highly infectious and almost always
  produces clinical disease in those infected.
• Virion is similar to other members of the
  paramyxoviridae but it lacks the neuraminidase.
• Membrane cofactor protein (MCP) or CD46 is the
  receptor for the virus.
• Measles virus is a stable monotypic virus with
  some degree of variability (strains).

37
Human Metapneumovirus

• In 2001, van den Hoogen and colleagues reported
  
• that they had isolated a paramyxovirus from
  28
• young children in the Netherlands identified
  as a new
• member of the metapneumovirus genus by
• - Virological data
• - Sequence homology
• - Gene constellation
• Previously, avian pneumovirus was the sole member
  
• of this recently assigned genus, hence the
  provisional
• name for the newly discovered virus human
• metapneumovirus.

38
hMPV Features

• Negative stranded RNA virus
• Paramyxoviridae family
• Related to avian pneumovirus and turkey
  rhinotracheitis virus
• Causative agent of respiratory tract disease in
  humans
• Most children are seropositive by the age of 5
  years
• 2 genetic clusters of hMPV that may represent
  different serotypes

39
Rubella virus

• Rubella virus is a member of the togaviridae but
  unlike most other togaviruses, rubella virus has
  no known invertebrate host, and the only known
  natural reservoir for rubella virus is man.
• Rubella virus is a spherical, icosahedral,
  enveloped particle that measures 60-70 nm in
  diameter.
• It has a ss RNA genome of about 10.000 nt that
  is encased by multiple copies of the capsid
  protein (C). Two glycoproteins, E1 and E2, are
  embedded in the envelope

40
Rhabdoviridae
41

• A large number of member viruses that are
  serologically unrelated.
• Rabies belongs to the genus lyssa virus (rabies
  in Greek means mad or frenzy).
• It is bullet shaped, enveloped and has a diameter
  of 75X180 nm.

42
Rabies Virus
G, M, L, N, and NS Proteins
43

• The genome is helical and is associated with N
  protein.
• Virions bud from the endoplasmic reticulum
• Replication of rhabdoviruses is followed by cell
  death except for rabies virus which is nonlytic
  causing no discernable damage

44
CORONAVIRUSES
45
HISTORY AND CLASSIFICATION

• Avian Infectious Bronchitis (IBV)
• (Schalk and Hawn, 1931).
• Recovery of virus in the Laboratory (Beaudette
  and Hudson 1937).
• Discovery of human coronaviruses
• (Tyrrell and Bynoe, 1965).
• Distinctive morphology.

46
(No Transcript)
47
(No Transcript)
48
(No Transcript)
49
Genome Structure
50
Human Coronaviruses

• Genus Coronavirus
• Species HCoV-229E
• HCoV-OC43
• SARS- CoV
• HCoV-NL63
• HCoV-HKU1
• HCoV-EMC
• Responsible for about 10-20 of common colds
• re-infection is common
• infections year-round, most prevalent in fall and
  spring
• incubation period about 2 to 5 days

51
VIRION STRUCTURE

• There is considerable diversity in both the
  lengths and nucleotide sequences of the S1
  glycoproteins of different coronaviruses and even
  of different strains of a single coronavirus.
• This diversity in S1 probably results from
  mutation and recombination between coronaviruses
  and strong positive selection in vivo.

52
Functions of Coronavirus Proteins

•  Membrane (M) glycoprotein
• May determine budding site on intracellular
  membranes
• Essential for envelope formation
• May interact with viral nucleocapsid
• May induce alpha interferon

53
Functions of Coronavirus Proteins

• Spike (S) glycoprotein
• Binds to specific host cell receptor glycoprotein
• May induce fusion of viral envelope with cell
• membrane
• Induces cell fusion
• Binds immunoglobulin at Fc receptor site
• Binds to 9-O-acetylated neuraminic acid
• Induces neutralizing antibody
• Elicits cell-mediated immunity

54
Coronavirus Stability

• Stable
• In body fluids (e.g. urine and faeces) for up to
  4 days.
• For ? 21 days at cold temperature (4 and -80ºC).
• At a pH of 6.
• Inactivated rapidly
• At a mild alkaline pH.
• By disinfectants
• By heating to 56 ºC

55
REPLICATION OF CORONAVIRUSES

• Primary translation.
• Transcription of viral RNA.
• Replication of viral RNA.
• Processing and intracellular transport of viral
  proteins (S glycoprotein).
• Assembly and release of virions.

56
(No Transcript)
57
Coronavirus Genetics

• Variation is due to Mutation and
• Recombination
• Mutation
• High frequency (several point mutations during
  each round of replication).
• Analysis has shown extensive sequence
  variability in S and N genes especially due to
  deletion mutations.

58

• The most striking example of the biological
  importance of deletion mutations is the emergence
  of porcine respiratory coronavirus (PRCV) from
  transmissible gastroenteritis virus (TGEV) which
  causes epizootic enteric infection of pigs.
• In the early 1980s, PRCV emerged in Europe as a
  new virus that causes widespread, devastating
  epizootics of respiratory disease in pigs.

59

• Recombination
• High frequency (up to 25). Mechanism is by
  discontinuous transcription and polymerase
  jumping (Copy-choice). Example is acquisition of
  HE glycoprotein from influenza C
• The capacity of coronaviruses both to recombine
  and to mutate suggests that diversity will also
  be a feature of human coronaviruses and that
  changes in pathogenicity may occur over time
  (Kenneth McIntosh, 1996).

60
Propagation and Assay in Cell Culture

• In tissue culture, coronaviruses have a latent
  period of about 5 to 7 hours.
• Infectivity of virions is fairly stable at pH
  6.0, but rapidly inactivated at mildly alkaline
  pH.
• Coronaviruses can cause either cytocidal or
  persistent infections of cells in vitro and in
  vivo, depending on the virus strain and the host
  cell.

61

• None of the human coronaviruses, except HCoV-EMC,
  grows well in cell culture without extensive
  adaptation by passage.
• They have been propagated in human embryonic
  tracheal organ culture, in primary or secondary
  human embryonic kidney cell lines, in many
  diploid human fibroblast cell lines, and in few
  heteroploid lines.
• The most sensitive cell line for isolation of
  virus from clinical specimens appears to be the
  diploid intestinal cell line MA 177.
• The highest titers of both 229E and OC43 are
  obtained by growth in human rhabdomyosarcoma
  cells.

62
Reoviridae
63

• Respiratory Enteric Orphan viruses (Albert Sabin,
  1959)
• Non enveloped with double-layered protein capsid,
  containing 10-12 segments of the double-stranded
  RNA genomes (double double).
• Stable over wide PH and temperature ranges and in
  air-borne aerosols.

64

• Human Pathogens
• - Orthoreoviruses
• - Rotaviruses
• - Orbiviruses
• - Coltiviruses

65

• Rotaviruses cause human infantile
  gastroenteritis.
• They account for approximately 50 of all cases
  of diarrhea in children requiring hospitalization
  because of dehydration.
• In underdeveloped countries, rotaviruses may be
  responsible for causing as many as 1 million
  deaths each year from uncontrolled viral diarrhea.

66
Rotavirus Particle
67

• Proteolytic cleavage of the outer capsid
  activates the virus and produces an
  intermediate/infectious subviral particle (ISVP).
• Rotaviruses resemble enveloped viruses and they
  acquire an envelope and loose it during
  replication.
• Reassortment of gene segments can occur and thus
  create hybrid viruses.

68

• Gene segment Protein location
  Function
• VP1
  (inner capsid)
  Polymerase
• VP2
  (inner capsid)
  Transcriptase
• VP3
  (inner capsid) mRNA
  capping
• VP4
  (outer capsid
• 
  spike at vertice)
  Activation by protease to VP5 and
• 
  
  VP8 in ISVP, HA and VAP
• NS53
  
  RNA binding
• VP6
  (inner capsid)
  
• 
  Groups (A-E) and
  
• 
  Subgroups (I,II) Major
  structural protein binds to NS28 at
  
• 
  
  ER and promote outer capsid assembly
• NS34
• NS35
• VP7
  (Outer capsid)
• 
  Serotypes ( 1-7)
  Type-specific antigen major, outer capsid
  
• 
  
  component

69

• Rotaviruses are found in many different mammals
  and birds.
• Rotavirus is stable at room temperature and to
  treatment with detergents, pH extremes of 3.5 to
  10 or even repeated freezing and thawing.
• Infectivity is enhanced by proteolytic enzymes
  such as trypsin.

70

• Human and animal rotaviruses are divided into
• 7 serotypes on the basis of antigenicity of VP7
  and VP4
• 5 groups on the basis of electrophoretic mobility
  of VP6 DNA
• 2 subgroups on the basis of antigenicity of the
  inner capsid protein VP6.

71
Caliciviruses and Related Agents

• Caliciviruses are 27-38 nm, non enveloped,
  icosahedral viruses with a () ss RNA genome
  (7500 bases).

72

• Norovirus (previously called Norwalk agent) was
  discovered by EM in stool from adults during and
  epidemic of an acute gastroenteritis in 1968 in
  Norwalk, Ohio.

73
Noroviruses

• Noroviruses (genus Norovirus, family
  Caliciviridae) are a group of related,
  single-stranded RNA, nonenveloped viruses that
  cause acute gastroenteritis in humans.
• Norovirus was recently approved as the official
  genus name for the group of viruses provisionally
  described as Norwalk-like viruses (NLV).
• Currently, there are at least five norovirus
  genogroups (GI, GII, GIII, GIV and GV), which in
  turn are divided into at least 31 genetic
  clusters.

74

• Astroviruses have a five or six-pointed star
  shape of 28-30 nm in diameter with an icosahedral
  symmetry.

75
The Retroviridae
76
(No Transcript)
77

• Genome
• - 2 identical molecules of ss RNA
• - Gene order is invariably gag- pro/ pol - env.
  
• - other genes are present in some viruses.
• Mode of Replication
• Classification
• - 6 genera, and human pathogens belong to 2
  genera HTLV-BLV and Lentiviruses.
• HTLV-1 (1981), HTLV-2 (1982)

78
Human Immunodeficiency Viruses (HIV)

• Pneumocystis carinii and Kaposis sarcoma among
  initial 4 H club of AIDS.
• Isolation of LAV by Montagnier In April 1983.
• A year later, Galo at NIH, isolated HTLV-3.
• HTLV-3 and LAV showed 98-99 identity.
• LAV-2 and HTLV-4 were isolated In 1986

79

• In 1986, the ICVT renamed the viruses HIV-1 and
  HIV-2.
• HIV-1 and HIV-2 are lentiviruses (lenti slow).
• HIV-1 and HIV-2 share about 40 of their genome
  sequence.
• Similarity is remarkable between HIV-1 and
  SIVcmp, and between HIV-2 and SIVsmm.

80

• Greatest sequence variation exists in the env.
  gene
• The genome is composed of 2 identical copies of
  9.749 kb ss RNA of positive polarity.
• A tRNA molecule is positioned near the 5- end of
  each strand, with 10-50 copies of reverse
  transcriptase. The tRNA is used as a primer for
  DNA synthesis.
• At each end, there are LTR sequences which
  contain promoters, enhancers, and other gene
  sequences for binding different cellular
  transcriptional factors.
• Although of positive polarity, HIV genome is not
  infectious

81

• cleavage
• P55 ? P17(MA), P24(CA), P9(NC), and
• P7 (?).
• P100 ? P10 (pro) P51/P66 (RT/ RNAse H),
• P32(1N).
• P160 ? gP120 (SU) and gP 41(TM).

82
HIV Genome
83
Other Genes of HIV

• tat Positive regulator of transcription
• rev Regulator of viral expression
• vif Affects viral infectivity
• vpr Positive regulator of transcription,
• augments virion production.
• vpu Down regulates CD4
• nef So-called negative-regulation factor. It
• augments viral replication and
  down
• regulates CD4

84
Antigenic Variation

• The reverse transcriptase is very error prone and
  lacks proof reading which contribute to HIV
  diversity.
• The immune response of the host is unable to
  completely curtail viral replication.
• Virus gene products may be relatively invisible
  to the immune response and the virus may be able
  to mask or change its antigenic specificity.

85

• The envelope gene displays frequent mutations.
• HIV envelope glycoproteins have two unusual
  features.
• - They are extensively glycosylated
• - They contain hypervariable regions that permit
  the virus to present new antigenic configurations
  to the host.

86

• HIV can constantly vary its surface antigenic
  composition which may allow it to avoid
  inactivation.
• Such a mechanism hinders the development of an
  effective vaccine containing the surface
  glycoproteins.
• Major sequence differences exist between the two
  HIV types antibodies against the surface
  glycoprotein of HIV-1 only partially cross react
  with HV-2.

87
Arboviruses
88

• Epidemiologic classification, but taxonomically
  diverse.
• More than 400, all of which are RNA viruses and
  about 100 of them infect humans.
• They establish life-long infections of arthropods
  and humans are accidentally infected.
• They have strong dependence on climatic
  conditions.

89
Families

• Togaviridae VEE, EEE, WEE
• Falviviridae Dengue, YF, West Nile fever,
• JE, St. Louis
  Encephalitis, Kyasanur
• Forest Disease, Omsk
  H.F.
• Bunyaviridae CE, Rift valley fever, Crimean-
  Congo
• virus, Sand fly
  fever, Hantaan virus
• Arenaviridae Junin, Machupo, Sabia, Guanarito,
  
• Lassa
• Filoviridae Marburg and Ebola viruses

90
The Bunyviridae

• A supergroup of at least 300 viruses.
• Spherical, enveloped, 90-120 nm, with 3 segments
  of ambisense ssRNA.
• Four genera Bunyavirus, Phlebovirus, Niarovirus
  and Hantavirus.
• All, except Hantaviruses, are arthropod borne.
  Hantaviruses are rodent borne.

91
Arenaviruses

• Pleomorphic, enveloped viruses of 120 nm in
  diameter
• 2 circles of ambisense ss RNA
• A sandy appearance (arenosa sandy in Greek)
  because of the ribosomes in the virion.

92
Filoviruses

• Filamentous, enveloped, with 80nm in diameter.
• - ss RNA of helical symmetry
• They vary in length from 800 to 14.000nm.

93
Hepatitis Viruses
94

• A large number of viruses can cause hepatitis
  (EBV, CMV, VZV, HSV, YF, Lassa virus etc).
• There are other viruses, however, that only cause
  hepatitis.
• At least six viruses, A through E and a newly
  discovered virus GB, are considered hepatitis
  viruses.
• Hepatitis viruses differ greatly in their
  taxonomy, structure, mode of replication and mode
  of transmission as well as in the course of the
  disease they cause.

95
Hepatitis A Virus

• It is the cause of infectious hepatitis, a term
  that was coined in 1912 to describe the epidemic
  form of the disease.
• The virus was first isolated in 1973 by Feinstone
  et al using IEM.
• Previously classified as enterovirus 72, HAV has
  been put in a separate genus hepatovirus

96
Differences Between HAV and Enteroviruses

• HAV nucleotide and amino acid sequences are
  dissimilar to enteroviruses.
• HAV is difficult to grow in cell culture and it
  usually replicates very slowly causing no CPE
  (nonlytic).
• HAV is stable at a PH of 1
• HAV has only one serotype and one neutralization
  site is dominant.
• Enterovirus -specific monoclonal antibody does
  not react with HAV.

97
Virus Structure

• HAV is a naked 27-32 nm icosahedral virus with a
  ssRNA genome of positive polarity.
• Its genome is 7.5 kb in length, polyadenylated at
  the 3- end and carries a protein (VPg) at the 5-
  end.

98
Hepatitis A Virus
99
Hepatitis B Virus
100
Hepatitis B Virus

• Hepatitis B virus is unusual among animal viruses
  in that
• Infected cells produce multiple types of
  virus-related particles.
• - 42 nm double-shelled particles (Dane
  particles)
• - 20 nm spheres, usually present in 104-106 fold
  excess over Dane particles.
• - Smaller quantities of filaments of 20 nm
  diameter and variable length.

101
(No Transcript)
102
Hepatitis B Virus
103

• The genome is partially double stranded.
• It replicates utilizing an RNA intermediate and
  has a reverse transcriptase.
• It is unusually stable for an enveloped virus.
• The Dane particle is the only infectious form.
• Envelope HBsAg.
• Core HBcAg.

104
(No Transcript)
105

• With less than 3200 nucleotides, HBV has the
  smallest genome of any human virus.
• HBV genome is relaxed circular partially duplex
  DNA species, whose circularity is maintained by
  5- cohesive ends.
• The genome has a coding organization that is
  highly compact and over half of the sequence is
  translated in more than one frame.

106

• HBV uses its genome economically by encoding
  different proteins within the same region of DNA
  in different reading frames.
• About half of the genomes nucleotides are used
  to code simultaneously for different proteins,
  and all code for at least one protein.
• The regulatory signals overlap with coding
  sequences and are not separate regions.

107

• Four ORFs are present in the DNA.
• - ORF P encodes the viral polymerase and the
  terminal
• protein found on minus strand
  DNA.
• - ORF C encodes the core protein (C antigen)
• - ORF S/pre-S encodes the HBsAg.
• - ORF X encodes a protein that enhances the
• expression of heterologous and
  homologous
• genes.

108

• The reading frame for HBsAg was shown to have two
  in-frame initiation codons which result in three
  products L, M, and S.
• The bulk of HBsAg is the S protein, M protein
  accounts for 5-15 of it and L for 1-2.
• The S (gp 27, 24-27 kD) glycoprotein is
  completely contained in the M (gp36 33-36 kD)
  glycoprotein which is contained in the L (gp42
  39-42 kD) glycoprotein.

109

• The coding organization of the core protein is
  similar, two in-frame AUGs were found in the core
  ORF.
• Initiation at the upstream AUG gives rise to a
  C-related protein that is secreted from infected
  cells into circulation (HBeAg).
• Three major mRNAs are produced
• 1) 2100 b mRNA HBsAg (S, M)
• 2) 2400 b mRNA HBsAg (L)
• 3) 3500b mRNA HBcAg, HBeAg, polymerase and a
  protein
• primer for DNA
  replication and it acts as a
• template for
  genome replication

110
(No Transcript)
111

• In addition to a minor mRNA (700 b) which codes
  for X protein (a transactivator of transcription
  and a protein kinase).
• HBsAg contains the group specific antigenic
  determinant termed a and type specific
  determinants termed d or y and w or r
• Type specific determinants behave like mutual
  alleles. Combinations result in four possible
  antigenic subtypes (adw, adr, ayw, ayr).

112
HBV Variation

• Eight genetic groups (A-H Genotypes) and a
  possible 9th type (I?) and Within genotypes 24
  subtypes have been described which differ by 4-8
  of the genome.
• HBV Antibody Escape Mutants
• Substitution of arginine for glycine at aa 145.
• Immunity to vaccine does not neutralize mutant.
• Failure to detect HBsAg in donated blood
• HBV Precore Mutants
• Detected in patients with severe chronic liver
  disease and who may have failed to respond to
  interferon therapy (increased pathogenicity)
• Polymerase Variants ( drug resistance)

113
Replication
114

• Virus particles acquire an envelope in the
  endoplasmic reticulum or proximal Golgi
• The surface antigen is glycosylated in the Golgi
  apparatus.
• Virions are then secreted via the constitutive
  pathway of vesicular transport.

115
Hepatitis D virus (Delta virus)

• HDV is not a true virus but a defective virus or
  a natural satellite of HBV
• Delta antigen is present in two forms, small
  (short) with 24 kd and large (long) with 27kd.
• The short form which is more abundant is required
  for RNA replication whereas the long form
  suppresses viral RNA replication and is required
  for packaging of the HDV genome by HBsAg.

116
(No Transcript)
117

• HDV replicates only in HBV-infected cells and
  direct pathologic changes are limited to the
  liver, the only organ in which HDV has been shown
  to replicate.
• HDV itself seems to be cytopathic and HDV antigen
  (delta) may be directly cytotoxic.

118
Hepatitis C virus
119

• Was discovered in 1989 (post transfusion
  hepatitis).
• Flaviviridae, genus Hepacivirus (HCV and GBV-C).
• Enveloped, 55-65 nm.
• Six genotypes with several subtypes for each
  genotype
• ss RNA genome
• - 9.4 kb
• - over 98 contains protein coding sequence.
• - a single large ORF

120

Hepatitis C Virus
capsid
Envelope protein
protease/ helicase
RNA polymerase
RNA dependent
c22
c 33
c-100
5
3
core
E1
E2
NS2
NS3
NS4
NS5
hypervariable region
121

• The genome codes for nine proteins 3 structural
  and 6 nonstructural.
• The structural proteins are core protein (P22),
  E1(gp76), and E2 (gp35) which are envelope
  glycoproteins.
• The nonstructural proteins are NS2, NS3, NS4A,
  NS4B, NS5A, and NS5B.
• HCV has circumvented the cap requirement by
  evolving an Internal Ribosome Entry Site (IRES)
  at its 5- end.

122
Hepatitis E Virus

123

• Recognized as a distinct disease in 1980.
• Virion is 32-34 nm in diameter, nonenveloped,
  with an icosahedral symmetry.
• Although it was originally classified in
  the Caliciviridae family, the virus has since
  been classified into the genus Hepevirus, but was
  not assigned to a viral family.
• The genome is approximately 7200 bases in length,
  is a polyadenylated single-strand RNA molecule
  that contains three discontinuous and partially
  overlapping ORFs

124

• ORF1 encode methyltransferase, protease,
  helicase, and replicase
• ORF2 encode the capsid protein 
• ORF3 encodes a protein of undefined function.
•  An in vitro culture system is not yet available.