Epidemiology and pathogenesis of newly discovered viruses Evaluating their threat to human health

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Epidemiology and pathogenesis of newly discovered
viruses- - - Evaluating their threat to human
health

• Peter Simmonds
• Centre for Infectious Diseases, University of
  Edinburgh

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Virus discovery The impact of molecular methods

• Technology and bio-informatics
• Molecular methods for specific amplification and
  detection of viral genomes
• Completion of human genome sequencing
• Methods for finding sequences in one sample
  missing in the other
• Subtractive PCR (KSHV, TTV, GBV-A,B)
• Representation difference analysis (HCV, GBV-C)
• Amplification of DNA/RNA without knowing its
  sequence (following blind virus purification)
• Sequence-independent single primer amplification,
  SISPA (PARV4)
• Arbitrarily primed or random primed PCR
• PhiX29 DNA polymerase or rolling circle
  amplification
• Brute force nucleotide sequencing
• New technologies (Roche 454, Solexa) to sequence
  all DNA or RNA in a sample

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454 pyrosequencing

• Allows 400,000 random reads of input DNA/RNA
• Automated assembly of overlapping fragments
• Identification of sequences by BLAST
• Powerful enough to find viruses lurking in human
  cells

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What virus discovery programmes find

• DNA viruses that have always been there
• Persistent, non-pathogenic, high population
  frequency
• Polyomaviruses (WUPyV, KIPyV)
• Annelloviruses (TTV and related viruses)
• A range of circoviruses and even weirder ss
  circular viruses
• New herpesviruses
• DNA viruses with uncertain evolutionary
  histories
• Parvoviruses PARV4, HBoV
• New adenoviruses types
• RNA viruses
• New variants or genera in known virus families
• Members of putative new RNA virus families

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Evaluating a new virus clinical relevance

• Detection of virus in context of unexplained
  disease
• Yes SARS virus, KSHV, Merckel cell carcinoma
  PyV
• Possibly HBoV, WU and KI polyomaviruses, TTV
• Difficult Human cardiovirus, other new enteric
  viruses
• Could the virus fill a diagnostic gap?
• Yes - Virus detection in CSF in viral
  meningitis / encephalitis
• Difficult detection in faecal samples
• Frequency, incidence and transmission in the
  general population
• Age-related exposure
• Association with specific risk group /
  transmission routes
• Relationship to other viruses
• Related viruses with potentially similar
  characterstics
• Evidence for zoonotic transmission

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Novel Parvoviruses in humans

• Parvoviridae
• Wide range of diverse viruses infecting mammals
• Highly host-specific
• Acute resolving infections
• Highly transmissible, stable in environment
• Human Parvoviruses
• Human Erythrovirus (B19)
• PARV4
• Acute infection syndrome
• Little known about epidemiology
• Human Bocaviruses

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Human Bocavirus - Update

• HBoV genome
• Discovered by Allander et al.1 in respiratory
  samples from pooled human respiratory tract
  samples
• 5217 bases, single stranded DNA genome, three
  open reading frames
• Most closely related to bovine bocavirus and
  minute virus of canines
• HBoV found predominantly in young child age
  groups
• Specifically associated with LRTI infections,
  often as a co-infection
• Causes a systemic infection with viraemia
• Evidence for GI tract infection and faecal
  excretion
• HBoV Diagnosis
• Specific association with respiratory disease
  only with high viral load samples
• Seroconversion for IgG and IgM detection is
  acute, significant infections IgG reactivity
  non-durable
• Many infections occur without detection in NPA
  samples

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New human bocaviruses1

• Highly divergent HBoV variants found in faeces
• gt30 amino acid sequence divergence from HBoV
• Undetectable with conventional HBoV screening
  primers
• Entirely absent from respiratory samples in
  Edinburgh (0/5600) and Thailand (0/400)
• HBoV2 more prevalent in faecal samples than HBoV1
  (15/1500 compared to 6/1500)
• 1Kapoor et al., J.Inf.Dis (2008)

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Human Cosavirus
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Human Cosavirus5UTR Structure

• 1056 base 5UTR
• Contains a type II IRES
• Region of homology with FMDV and cardiovirus UTR
  sequences (grey)
• Conserved and novel structural features
• Collaborative studies to investigate IRES function

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Frequency of HCoSV and HEV detection in faeces
by RT-PCR

• Group HCoSV HEV
• Pakistan
• AFP 28/57 (49) 31/41 (76)
• Controls 18/41 (44) 25/41 (61)
• Edinburgh, UK
• Enteric bacteriology 2/1500 (0.1) 85/1500 (6)
• Minnesota, USA
• Child, gastroenteritis 1/100 (1)
• Very high frequencies gt50 in Egypt and Nigeria
• Current assessment
• As diverse as human enterovirus genus, scope for
  pathogenic serotypes / species irrespective of
  high frequency of infection
• Improved sanitation in Western countries may
  delay infection and create a different disease
  (eg. Poliovirus)

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Human Cardiovirus
From Drexler et al. EID 14 1398-405 (2008)

• Detected and cloned using SISPA from virus
  isolated from faeces of unexplained case of
  pyrexia (Jones et al., 2007)
• Falls in the Cardiovirus genus but distinct from
  TMEV and EMCV
• No close relationship with Vilyuisk virus
  (associated with neurodegenerative disease)

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Detection frequencies in different sample types

• Location, group Faeces Resp. CSF Source
• Canada - 3 / ?? - Boivin et al., 2008
• Germany
• Children age 1-12 4/51 - - Drexler et
  al., 2008
• Adults, 16-98 0/67 - -
• GP samples 1-97 0/538 - -
• Brazil 1/188 - - Drexler et al., 2008
• Edinburgh, UK 5/1500 0/3540 0/1575 Simmonds et
  al., unpubl.
• Bangkok, lt 5 years 4/450 0/400 - Chieochonsin,
  unpubl.
• California, USA 6/498 0/719 0/360 Chiu et al.,
  2008
• Positives invariably from young children (lt 5
  years of age)
• High genetic diversity, possibly multiple rounds
  of infection with different serotypes.
• Invariably undetectable in CSF samples of
  meningitis/encephalitis cases

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Novel Human Polyomaviruses

• Two related polyomaviruses
• Reported by two groups in 2007
• Cloned out of pooled respiratory samples1, 2
• Viruses named after lab/dept.
• KI Polyomavirus (KIPyV) 5040 bps
• WU virus (WUV) 5229 bps.
• Not closely related to other known polyomaviruses
• Show typical genome organisation
• Show 27 sequence divergence when aligned

References1Allander et al., J.Virol., 81
4130-36 (2007) 2Gaynor et al., PLoS Pathogens 3
e64 (2007)
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Clinical Characteristics of Positive Subjects

• LRTIs
• Young, invariably another respiratory pathogen
  detected (RSV, AdV, HBoV)
• URTI and None
• Older, almost all immunosuppressed (8/11)
• ALL, BMT transplant, neutropoenia, Gauchers
  disease,

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Polyomaviruses and Immunosuppression

• Increased detection in HIV infection
• Most marked in MSMs
• May be more immunosuppressed than IDUs
• Greater frequency of WUV and BK reactivation in
  AIDS

p lt 10-6
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Mutation in the TCR

• Transcription control region controls virus
  replication
• JCV TCR mutates and loses suppressive role in PML
• KIPyV and WUV TCRs poorly characterised, but
  similar arrangement of transcription sites and
  promoters likely
• Compared to rest of genome, frequent point
  mutations in WUV and KIPyV TCR
• Large number of mutations in WUV specifically
  found in severe immunosuppression
• Mutations around Ori but avoid transcription
  promoters

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Mutation in the TCR
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Biological differences between polyomaviruses

• Increased detection of WUV and BKV among
  immunosuppressed study subjects
• Levels of virus expression frequently extremely
  high
• Potentially damaging to target cells, although no
  specific disease associations identified
• Polyomavirus reactivation associated with
  development of specific mutations in the TCR
• Target tissues of WUV and KIPyV remain to be
  determined
• Further testing of autopsy tissue planned
• Not excreted in urine (unlike BKV and JCV)
• Greater frequency of detection in respiratory
  samples may be evidence for either earlier
  acquisition or a different route of transmission
  from JCV and BKV
• Study shows several similarities and differences
  between the two virus groups

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PARV41

• Discovered in plasma from an individual with an
  acute, undiagnosed post-transfusion reaction
• 5268 bases, single stranded DNA genome, two open
  reading frames
• Not closely related to any known genera of
  parvoviruses

• A very elusive virus
• Detected in only a single study subject in
  original study
• Infrequently found in pooled plasma from paid
  donors
• No known disease associations

1Jones et al., J.Virol. 102 12891-6 (2005)
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Autopsy samples

• 2 x 0.5 µg DNA assayed from lymph node/spleen and
  bone marrow
• Assay sensitivity 3 copies / million cells
• Highly concordant results between bone marrow and
  lymphoid tissues

Plasma samples

• DNA assayed from 40 µl plasma sensitivity 25
  DNA copies / ml

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Development of serology assay

• Expression of VP1/VP2 structural proteins
• Full length VP1/2 or VP2 sequence amplified and
  cloned into baculavirus (Autographa californica
  multiple nuclear polyhedrosis virus) expression
  vector
• Transfected into insect (Sf9) cells, and
  infectious virus passaged to increase titre and
  protein expression
• Virus-like particles observed from expressed VP2
  protein, antigen semi-purified by buoyant density
  centrifugation on sucrose.
• Anti-PARV4 ELISA
• Antigen and mock-infected Sf9 control used to
  coat ELISA plates
• Indirect ELISA format for IgG detection, screened
  at 1100 dilution
• Reactivity calculated as OD of VP2 well
  mock-infected control

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Anti-PARV4 Detection Frequencies
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PARV4 Models of Transmission

• Model 1 - Co-transmission with HIV
• Infection largely restricted to HIV IDUs, with a
  much lower frequency of infection in HIV-, HCV
  IDUs
• PARV4 (genotype 3) found in sub-Saharan Africans
  heterosexually infected with HIV
• However, entirely absent in HIV-positive MSMs,
  30 in HIV-negative haemophiliacs
• No evidence that immune status influences PARV4
  expression
• Model 2 Parenteral transmission of PARV4
• Infection restricted to IDUs, and virally exposed
  haemophiliacs
• However, problematic to explain low frequency of
  PARV4 infection in HIV- IDUs
• PARV4 may be inefficiently transmitted by
  parenteral routes only

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Development of a Strategic Archivein Clinical
Virology

• Need for archives
• Evaluation of emerging and newly discovered
  viruses
• Opportunity to devise more comprehensive,
  specific diagnostic methods to detect a wider
  range of viral pathogens
• Changed perception and regulation of clinical
  specimen testing
• Examples of current problems
• Decisions about introduction of HBoV screening
  without knowing its prevalence and disease
  associations
• Diagnostic gap in viral meningitis should other
  viruses, e.g. HPeV be screened?
• Rational choice of targets in large scale
  multiplexed diagnostic PCR testing
• Current status
• 3 years of respiratory (n7500) and CSF (n2300)
  samples and NAs 2500 surveillance faecal samples
  and plasma
• Future targeted archiving of samples from defined
  risk groups (IDUs, MSMs) and disease presentations

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Acknowledgements and Collaboration

• Virus Evolution Group, Centre for Infectious
  Diseases
• Colin Sharp, Elly Gaunt, Thaweesak Vee
  Chieochansin, Ines Robertson, Ashleigh Manning
• Specialist Virology Laboratory and associated
  laboratories, Royal Infirmary of Edinburgh
• Kate Templeton, Heli Harvala, Christopher Ludlam
• Department of Pathology
• Jeanne Bell, Iain Anthony, Frances Carnie
• Institute of Evolutionary Biology
• Paul Sharp, Andrew Rambaut
• Wellcome Sanger Centre, Hinxton, Cambridge
• Paul Kellam
• Blood Systems Research Institute, San Francisco,
  California
• Joe Victoria, Amit Kapoor, Eric Delwart