1. Completed genomes: viruses
Chapter 16:
Completed genomes: viruses
Jonathan Pevsner, Ph.D.
Bioinformatics and Functional Genomics
(Wiley-Liss, 3rd edition, 2015)
You may use this PowerPoint for teaching purposes

2. Learning objectives After studying this chapter you should be able to:
define viruses;
explain the basis of the classification of viruses;
describe the genomes of HiV, influenza, measles, Ebola, and herpesviruses;
describe bioinformatics approaches to determining the function of viral genes and proteins;
describe key bioinformatics resources for studying viruses; and
compare and contrast DNA and RNA viruses.

3. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

4. Introduction to viruses
Viruses are small, infectious, obligate intracellular parasites. They depend on host cells to replicate. Because they lack the resources for independent existence, they exist on the borderline of the definition of life.
The virion (virus particle) consists of a nucleic acid genome surrounded by coat proteins (capsid) that may be enveloped in a host-derived lipid bilayer.
Viral genomes consist of either RNA or DNA. They may be single-, double, or partially double stranded. The genomes may be circular, linear, or segmented.

5. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

6. Introduction to viruses
Viruses have been classified by several criteria:
-- based on morphology (e.g. by electron microscopy)
-- by type of nucleic acid in the genome
-- by size (rubella is about 2 kb; HIV-1 about 9 kb;
poxviruses are several hundred kb). Mimivirus
(for Mimicking microbe) has a double-stranded
circular genome of 1.2 megabases (Mb).
-- based on human disease

7. The International Committee on Taxonomy of Viruses
(ICTV) offers a resource for classification

8. Virus taxonomy from the ICTV website
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Year

9. Classification of viruses
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10. Classification of viruses
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11. Classification of viruses based on nucleic acid composition
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12. Mutation rate as a function of genome size
hammerhead
viroid CChMVd
tobacco mosaic virus, human rhinovirus, poliovirus, vesicular stomatitis virus, bacteriophage Φ6, measles virus
C. elegans, Mus, Drosophila, human
E. coli
bacteriophages λ, T2, T4; herpes simplex virus
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13. Human disease relevance of viruses
Vaccine-preventable viral diseases include:
Hepatitis A
Hepatitis B
Influenza
Measles
Mumps
Poliomyelitis
Rubella
Smallpox
Source: Centers for Disease Control website

14. Human disease relevance of viruses
Disease Virus
Hepatitis A Hepatitis A virus
Hepatitis B Hepatitis B virus
Influenza Influenza type A or B
Measles Measles virus
Mumps Rubulavirus
Poliomyelitis Poliovirus (three serotypes)
Rotavirus Rotavirus
Rubella Genus Rubivirus
Smallpox Variola virus
Varicella Varicella-zoster virus
Source: Centers for Disease Control website

15. Vaccine-preventable viral diseases
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From

16. Seven viruses that cause cancer in humans
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17. Viral genomes page (NCBI)
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18. Viral metagenomics
Viral metagenomics refers to the sampling of representative viral genomes from the environment.
A typical viral genome is ~50 kilobases (in comparison, a typical microbial genome is ~2.5 megabases). A sample is collected (e.g. seawater, fecal material, or soil). Cellular material is excluded. Viral DNA is extracted, cloned, and sequenced.
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19. Henle-Koch postulates: criteria needed to establish a causal relationship between a microbe and a disease
[1] The parasite occurs in every case of the disease in question and under circumstances which can account for the pathological changes and clinical course of the disease.
[2] It occurs in no other disease as a fortuitous and nonpathogenic parasite.
[3] After being fully isolated from the body and repeatedly grown in pure culture, it can induce the disease anew.
These postulates (especially [3]) are very hard to prove with viruses. Huebner suggested alternate postulates.
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20. Huebner’s postulates: criteria needed to establish a causal relationship between a virus and a disease
[1] The virus must be a “real” entity established by passage in culture.
[2] The virus must originate from human specimens.
[3] Active infection should produce an antibody response.
[4] A new virus should be fully characterized and compared with other agents.
[5] The virus must be constantly associated with a specific illness.
[6] Human volunteers inoculated with the newly recognized agent in double‐blind studies should reproduce the clinical syndrome.
[7] Epidemiological studies should identify patterns of infection and disease.
[8] A specific vaccine should prevent the disease, establishing an agent as the cause.
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21. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

22. Bioinformatic approaches to viruses
Some of the outstanding problems in virology include:
-- Why does a virus such as HIV-1 infect one species
(human) selectively?
-- Why do some viruses change their natural host?
In 1997 a chicken influenza virus killed six people.
-- Why are some viral strains particularly deadly?
-- What are the mechanisms of viral evasion of the host
immune system?
-- Where did viruses originate?

23. Diversity and evolution of viruses
The unique nature of viruses presents special challenges
to studies of their evolution.
viruses tend not to survive in historical samples
viral polymerases of RNA genomes typically lack
proofreading activity
viruses undergo an extremely high rate of replication
many viral genomes are segmented; shuffling may occur
viruses may be subjected to intense selective pressures
(host immune respones, antiviral therapy)
viruses invade diverse species
the diversity of viral genomes precludes us from
making comprehensive phylogenetic trees of viruses

24. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

25. Bioinformatic approaches to HIV
Human Immunodeficiency Virus (HIV) is the cause of
AIDS. Some have estimated that 70 million people were infected with HIV (2017) and 35 million died.
HIV-1 and HIV-2 are primate lentiviruses. The HIV-1 genome is 9181 bases in length. Note that as of 2017 there are 700,000 Entrez nucleotide records for this genome (but only one RefSeq entry).
Phylogenetic analyses suggest that HIV-2 appeared as
a cross-species contamination from a simian virus,
SIVsm (sooty mangebey). Similarly, HIV-1 appeared
from simian immunodeficiency virus of the chimpanzee
(SIVcpz).

26. HIV phylogeny based on pol suggests five clades
1. Simian immunodeficiency virus from the chimpanzee Pan troglodytes (SIVcpz) with HIV-1
Hahn et al., 2000

27. HIV phylogeny based on pol suggests five clades
2. SIV from the sooty mangabeys Cerecocebus atys (SIVsm), with HIV-2 and SIV from the macaque (genus Macaca; SIVmac)
Hahn et al., 2000

28. HIV phylogeny based on pol suggests five clades
3. SIV from African green monkeys (genus Chlorocebus)(SIVagm)
Hahn et al., 2000

29. HIV phylogeny based on pol suggests five clades
4. SIV from Sykes’ monkeys, Cercopithecus albogularis (SIVsyk)
Hahn et al., 2000

30. HIV phylogeny based on pol suggests five clades
5. SIV from l’Hoest monkeys (Cercopithecus lhoesti); from suntailed monkeys (Cercopithecus solatus); and from mandrill (Mandrillus sphinx)
Hahn et al., 2000

31. Evolutionary relationships of primate lentiviruses
Full-length Pol protein sequences were aligned and a tree was created using the maximum‐likelihood method. There are five major lineages (arrows 1–5).
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32. Evolutionary relationships of primate lentiviruses
The HIV‐1/SIVcpz lineage is displayed based on a maximum‐likelihood tree using Env protein sequences. Three major HIV‐1 groups (M, N, O; arrows 6–8) are distinguished.
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33. Bioinformatic approaches to HIV: NCBI
NCBI offers a retrovirus resource with reference genomes and protein sets, and several tools (alignment, genotyping).

34. Retrovirus resources (NCBI)
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35. Bioinformatic approaches to HIV: LANL
Los Alamos National Laboratory (LANL) databases provide a major HIV resource.
See
LANL offers
-- an HIV BLAST server
-- Synonymous/non-synonymous analysis program
-- a multiple alignment program
-- a PCA-like tool
-- a geography tool

36. Map of HIV infection subtypes (worldwide)
LANL geography tool
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Map of HIV infection subtypes (worldwide)

37. LANL map of HIV infection subtypes (Europe)
LANL geography tool
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LANL map of HIV infection subtypes (Europe)

38. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

39. Influenza virus
Influenza virus leads to 200,000 hospitalizations and ~36,000 deaths in the U.S. each year.
Influenza viruses belong to the family Orthomyxoviridae.
The viral particles are about nm in diameter and can be spherical or pleiomorphic. They have a lipid membrane envelope that contains the two glycoproteins: hemagglutinin (H) and neuraminidase (N). These two proteins determine the subtypes of Influenza A virus.
Influenza A

40. Influenza virus
Since 1976, the H5N1 avian influenza virus has infected at least 232 people (mostly in Asia), of whom 134 have died.
A major concern is that a human influenza virus and the H5N1 avian influenza strain were to combine, a new lethal virus could emerge causing a human pandemic. In a pandemic, 20% to 40% of the population is infected per year.
►The 1918 Spanish influenza virus killed tens of millions of people (H1N1 subtype).
►1957 (H2N2)
► 1968 (H3N2)
► Asia (H5N1)
► 2009 (H1N1, “swine flu”)

41. Influenza virus There are three types: A, B, C
► A and B cause flu epidemics
► Influenza A: 20 subtypes; occurs in humans, other animals.
For example, in birds there are nine subtypes based on the type of neuraminidase expressed (group 1: N1, N4, N5, N8; group 2: N2, N3, N6, N7, N9). The structure of H5N1 avian influenza neuraminidase has been reported (Russell RJ et al., Nature 443:45, 2006).
► Influenza A genome consists of eight, single negative-strand RNAs (from 890 to 2340 nucleotides). Each RNA segment encodes one to two proteins.

42. examples of family Orthomyxoviridae complete genomes
Influenza viruses:
examples of family Orthomyxoviridae complete genomes
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43. Genes in a representative Influenza A virus complete genome (A/Puerto Rico/8/34(h1N1))
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44. Schematic of the eight segments from a typical Influenza A virus
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45. Chronological summary of influenza A strains
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46. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

47. Measles virus
Measles virus is one of the deadliest viruses in human history
Leading cause of death in children in many countries
Morbillivirus of the Paramyxoviridae family (includes mumps and respiratory syncytial virus)
Reference genome (see NCBI): accession NC_
Nonsegmented, negative‐sense RNA genome protected by nucleocapsids and an envelope.
Genome has 15,894 bases and six genes that encode eight proteins.
Six genes are designated N (nucleocapsid), P (phosphoprotein), M (matrix), F (fusion), H (hemagglutinin), and L (large polymerase).
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48. Eight proteins encoded by six genes of the measles virus genome
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49. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

50. Ebola virus
Ebola is a filovirus that is transmitted between people by contact with body fluids.
First reported outbreak occurred in 1976.
Largest outbreak began in 2014, centered initially in West Africa.
The virus causes hemorragic fever that is often fatal.
Ebola virus is an enveloped, single‐stranded RNA negative‐strand virus of the family Filoviridae.
The Zaire Ebola virus reference genome is 18,959 bases in length (accession NC_ ), with seven genes encoding nine proteins.
Bioinformatics resource include a UCSC Ebola virus browser.
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51. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

52. Herpesvirus Herpesviruses are double-stranded DNA viruses that
include herpes simplex, cytomegalovirus, and Epstein-Barr.
The genomic DNA is packed inside an icosahedral capsid; with a lipid bilayer the diameter is ~200 nanometers.

53. Herpesvirus Phylogenetic analysis suggests three major groups
that originated about MYA.
Mammalian herpesviruses are in all three subfamilies. Avian and reptilian herpesviruses are all in the Alphaherpesvirinae.

54. Herpesvirus: three main groups
Millions of years before present

55. Herpesvirus taxonomy
McGeoch et al. (Virus Res. 117:90-104, 2006) describe a new herpesvirus taxonomy.
Family Herpesviridae
Subfamilies Alpha-, Beta-, Gammaherpesvirinae
New family Alloherpesviridae (piscine, amphibian herpesviruses)

56. Phylogeny of the herpesviruses:
comparison to the evolution of host genomes
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57. Phylogeny of the herpesviruses:
comparison to the evolution of host genomes
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58. Out‐of‐Africa hypothesis: herpesviruses infected marine invertebrates such as oyster ~500 MYA, and various members of the Herpesviridae (listed to the right) established host species specificity.
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59. Herpesvirus taxonomy
Genome sizes range from 124 kb (simian varicella virus from Alphaherpesvirinae) to 241 kb (chimpanzee cytomegalovirus from Betaherpesvirinae).
► GC content ranges from 32% to 75%.
► Protein-coding regions occur at a density of one gene per 1.5 to 2 kb of herpesvirus DNA.
► There are immediate-early genes, early genes (nucleotide metabolism, DNA replication), and late genes (encoding proteins comprising the virion).
► Introns occur in some herpesvirus genes.
► Noncoding RNAs have been described (e.g. latency-associated transcripts in HSV-1).

60. Bioinformatic approaches to herpesvirus
Consider human herpesvirus 8 (HHV-8)(family Herpesviridae; subfamily Gammaherpesvirinae). Its genome is ~140,000 base pairs and encodes ~80 proteins. Its RefSeq accession number is NC_
We can explore this virus at the NCBI website.
Try NCBI  Genomes  viruses  dsDNA

61. HHV8 genome overview (NCBI)
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62. HHV8 proteins (graphic and tabular summaries)
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63. Viruses can acquire host genes
HHV-8 proteins include structural and metabolic proteins. There are also viral homologs of human host proteins such as the apoptosis inhibitor Bcl-2, an interleukin receptor, and a neural cell adhesion-related adhesin.
Mechanisms by which viruses may acquire host proteins include recombination, transposition, splicing. A DELTA BLAST search using HHV-8 interleukin IL-8 receptor as a query reveals several other viral IL-8 receptor molecules.

64. A viral protein (HHV‐8 ORF74) is a G‐protein coupled receptor that is homologous to a superfamily of mammalian G‐protein coupled receptors, including a high‐affinity interleukin 8 (IL‐8) receptor.
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Results of a DELTA BLAST search (query YP_ ) are shown.

65. Bioinformatic approaches to herpesvirus
Functional genomics approaches have been applied to human herpesvirus 8 (HHV-8). For example, changes in viral gene expression have been measured at different stages of infection. Conversely, gene expression changes have been measured in human cells following viral infection.

66. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

67. Mimivirus: mimicking microbe
Mimivirus is a member of the Mimiviridae family of nucleocytoplasmic large DNA viruses (NCLDVs).
It was isolated from amoebae growing in England.
The mature particle has a diameter of ~400 nanometers, comparable to a small bacterium (e.g. a mycoplasma). Thus, mimivirus is by far the largest virus identified to date.

68. Mimivirus: mimicking microbe
The mimivirus genome is 1.2 Mb (1,181,404 base pairs). It is a double-stranded DNA virus.
► Two inverted repeats of 900 base pairs at the ends (thus it may circularize)
► 72% AT content (~28% GC content)
► 1262 putative open-reading frames (ORFs) of length >100 amino acids. 911 of these are predicted to be protein-coding genes
► Unique features include genes predicted to encode proteins that function in protein translation. The inability to perform protein synthesis has been considered a prime feature of viruses, in contrast to most life forms.
See Raoult D et al. (2004) Science 306:1344.

69. Largest virus genomes
All are double-stranded DNA (no RNA stage). the order Megavirales has been proposed to reflect the genome size of at least 1 megabase.
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70. MUMmer: alignment of two genomes
Use MUMmer on the command line. Obtain viral genome sequences in the FASTA format (e.g. from NCBI).
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71. Comparison of megabase‐size viral genome sequences using MUMmer software
The Acanthamoeba polyphaga Mimivirus (reference, x axis) and Acanthamoeba castellanii mamavirus (y axis) genomes are largely collinear.
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72. Comparison of megabase‐size viral genome sequences using MUMmer software
Comparison of Acanthamoeba polyphaga Mimivirus versus Acanthamoeba polyphaga moumouvirus. Forward MUMs are indicated in red, while reverse MUMs are colored blue. A prominent inversion is evident and a translocation (arrow 1).
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73. Virus resources available on web
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74. Outline Introduction Classification of viruses
Bioinformatics approaches to virology
Human immunodeficiency virus (HIV)
Influenza virus
Measles virus
Ebola virus
Herpesvirus: from phylogeny to gene expression
Giant viruses
Perspectives

75. Perspectives
There are relatively few species of viruses because of their specialized requirements for replication in host cells.
Thousands of viral genomes have been sequenced, and metagenomics projects are helping to define the diversity of viral genomes.
It remains a challenge to use bioinformatics knowledge to successfully create vaccines. Many viruses (such as measles) remain deadly to humans, and some such as influenza pose potentially catastrophic threats.
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