Development and Evaluation of a Highly Sensitive_ Multiplexed Real

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Development and Evaluation of a Highly Sensitive_ Multiplexed Real Powered By Docstoc
					Epidemiology and pathogenesis of newly discovered viruses
                            ---
          Evaluating their threat to human health




                        Peter Simmonds
         Centre for Infectious Diseases, University of Edinburgh
                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
         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
        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
        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
        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
                       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
                        New human bocaviruses1

   Highly divergent HBoV
    variants found in faeces
   >30% 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)
Human Cosavirus
              Human Cosavirus
               5’UTR Structure


   1056 base 5’UTR
   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
           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 >50% 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)
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)
         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, < 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 (< 5 years of age)
   High genetic diversity, possibly multiple rounds of infection with
    different serotypes.
   Invariably undetectable in CSF samples of meningitis/encephalitis cases
                 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
    References:1Allander et al., J.Virol., 81; 4130-36 (2007); 2Gaynor et al., PLoS Pathogens 3: e64 (2007)
            Clinical Characteristics of Positive Subjects


Disease        Total [WU/KI]            Age         M/F    Immuno-           Co-detected
                                                          suppressed           viruses
LRTI           8 [5/3] / 114 (7%)   1 (0.13-1.5 )   5/3      0/8       RSV (2), AdV (3), HBoV (1)

URTI            5 [2/3] / 75 (7%)   14 (0.7-34 )    4/1      3/5       AdV (2), HBoV (1)

None           6 [3/3] / 56 (11%)    5 (1.5-15 )    4/2      5/6       AdV (1), B19 (1)



          LRTIs
               Young, invariably another respiratory pathogen detected (RSV,
                AdV, HBoV)
          URTI and None
               Older, almost all immunosuppressed (8/11)
               ALL, BMT transplant, neutropoenia, Gaucher’s disease,
            Polyomaviruses and Immunosuppression

                                 Group          n        KIPyV   WUV   JCV   BKV
   Increased detection in   Control            30        1       -     -     -
    HIV infection
                             HIV Neg. IDU       19         -      -    1      -
   Most marked in MSMs
                             IDU Pre-AIDS       6          -      -     -     -
        May be more
         immunosuppressed    IDU AIDS           11         -      1    2      1
         than IDUs           MSM AIDS           31        3       3    1      9



   Greater frequency of                 PV+    PV-
    WUV and BK               AIDS        20         22
    reactivation in AIDS     No AIDS        2       47
                             p < 10-6
                        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
Mutation in the TCR
             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
                                                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)
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
          Group                  B19            PARV4           HBoV
          Control             8/8 (100%)          0/8           0/8
    IDU, HIV-uninfected       8/12 (67%)       1/12 (8%)        0/9
     IDU, HIV-infected        12/24 (50%)     17/24 (71%)       0/24
    MSM, HIV-infected         7/13 (54%)         0/14           n.t.
      Haemophiliacs            1/2 (50%)       1/2 (50%)        n.t.

Plasma samples
    DNA assayed from 40 μl plasma – sensitivity 25 DNA copies / ml
                    Group              B19              PARV4
                    Control            0/40             0/40
               HIV-infected            0/36             0/36
           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 1:100 dilution
       Reactivity calculated as OD of VP2 well – mock-infected control
            Anti-PARV4 Detection Frequencies

   Group      Potential Exposure                     HCV HIV Anti-PARV4
    IDU       HCV-infected, HIV co-infected          +    +     8/12
              HCV-infected, HIV uninfected           +    -      ??
    MSM       Gay men HIV-infected, HCV-neg.          -   +     1/15
    Het.      HIV-infected by heterosexual contact    -   +     0/13
  Control     Adult attendees of orthopaedic          -   -     0/50
              outpatients: low risk factors


Hemophiliacs Exposed to non-virally inactivated      +    +     11/20
             FVIII & FIX                             +    -     4/15
Hemophiliac Brother or sister of haemophiliac         -   -     1/35
  siblings  raised in same household
            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
           Development of a Strategic Archive
                  in 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 (n≈7500) and CSF (n≈2300) samples and
        NAs; 2500 surveillance faecal samples and plasma
       Future targeted archiving of samples from defined risk groups
        (IDUs, MSMs) and disease presentations
     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

				
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