Multiplex PCR for detection and quantification of intra- and extra-cellular viral genomes Lara Isobel Compston, Daniel Candotti, Jean-Pierre Allain Cambridge Blood Centre, UK University of Cambridge

Introduction Interaction between viruses with the host is dependant on the interplay between factors related to both the virus and the host Target cells can be damaged either directly by the virus or by the immune response initiated, and an equilibrium needs to be reached between them Several viral survival strategies are the result of the virus/host interplay : 1) Clinical recovery due to successful development of humoral and cellular immune responses. 2) Latent viral infection (transient escape of immune response in primary infection and reactivation) 3) Establisment of chronic viral infection with partially effective immune response.

New model of viral infection Recent evidence has emerged that, in common with latent viruses, after recovered infections, common viruses are not eliminated from the host but contained efficiently, persisting in sanctuaries were they escape the immune response. Acute infection Sanctuaries: biological portfolio Recipients of TX or organ Clinical symptoms Clinical recovery By immune defenses Antibodies White cells No detectable virus in circulation Lymph nodes CMV, HHV-8, EBV, HIV Liver HBV, HAV, HCV Bone marrow HEV B19 Immunocompetent Maintain viral control Undetectable in blood Immunodeficient Age, chemo; transplant of BM or organs; HIV - Increased susceptibility to external pathogens - Reactivation of past infections ( viral load) - Severe symptoms

Aims of the study It was hypothesised that the generality of reactivation of common latent and persistent viral genomes may constitute an indicator of the overall immune status of the host. The objective was to systematically detect and quantify a panel of common viruses in persons who are either immunocompetent or present with varying degrees of immunodeficiency ranging from mild to severe.

Screening algorithm Multiplex real-time PCR undetectable Sample negative for specific virus Multiplex real-time PCR PCR signal for specific viruses No Yes Single virus qPCR Viral load quantification Final viral load result (>50 copies) ≤ 50 copies Confirmation of ≤ 50 copies Nested-PCR B19 / HBV/ HHV-8 EBV/CMV/ VZV GBV-C / HAV

Development of Standards for qPCR NIBSC standards: WHO international standard for Hepatitis B virus 97/746 WHO international standard for Hepatitis A virus 00/560 Plasmid standards: Constructed for the following targets: EBV and HHV-8 (cell culture) CMV (clinical sample) VZV (oligonucleotide construct of target region) B19 (received as a gift) PCR amplicons of target regions Plasmids purified and quantified by UV spectroscopy, plasmid concentration used to derive copy number by standard conversion for qPCR Clinical standards: A high titre clinical sample of GBV-C serially diluted and used as a standard Unknown initial viral load, therefore dilution giving 100% L.O.D designated as 10 AU (arbitrary units)

Dynamic range with NIBSC standards 95% C.I. values shown HBV Hepatitis A virus

NIBSC standards: WHO international standard for Hepatitis B virus 97/746 WHO international standard for Hepatitis A virus 00/560 Plasmid standards: Constructed for the following targets: EBV and HHV-8 (cell culture) CMV (clinical sample) VZV (oligonucleotide construct of target region) B19 (received as a gift) PCR amplicons of PCR target region Plasmids purified and quantified by UV spectroscopy, plasmid concentration used to derive copy number by standard conversion for qPCR Clinical standards: A high tire clinical sample of GBV-C serially diluted and used as a standard Unknown initial viral load, therefore dilution giving 100% L.O.D designated as 10 AU (arbitrary units)

Dynamic range with plasmid standards 95% C.I. values shown CMV VZV EBV HHV-8

Dynamic range with plasmid standards 95% C.I. values shown 50 % Limit of detection B19

NIBSC standards: WHO international standard for Hepatitis B virus 97/746 WHO international standard for Hepatitis A virus 00/560 Plasmid standards: Constructed for the following targets: EBV and HHV-8 (cell culture) CMV (clinical sample) VZV (oligonucleotide construct of target region) B19 (received as a gift) PCR amplicons of PCR target regions Plasmids purified and quantified by UV spectroscopy, Plasmid concentration used to derive copy number by standard conversion for qPCR Clinical standards: A high titre clinical sample of GBV-C serially diluted and used as a standard Unknown initial viral load, therefore dilution giving 100% L.O.D designated as 10 AU (arbitrary units)

Dynamic range with clinical standards 95% C.I. values shown GBV-C

Multiplex assays for DNA viruses relevant in Africa B19 singleplex = limit of detection; 50 IU Hepatitis B singleplex = limit of detection; 50 IU Rsq 0.995 Rsq 0.992 Ct of 50 IU = 34.95 Ct of 50 IU = 35.02 Multiplex: limit of detection; B19 500 IU, HBV 50 IU, HHV-8 10 copies HHV-8 singleplex = limit of detection;10 copies B19 Rsq 0.939 Ct of 500 IU: 34.59 Rsq 0.977 HBV Rsq 0.996 Ct of 50 IU: 35.43 HHV-8 Rsq 0.953 Ct of 10 copies: 33.67 Ct of 10 copies = 34.04

Multiplex assay for DNA viruses relevant worldwide EBV Singleplex: limit of detection; 214 copies CMV Singleplex: limit of detection; 350 copies Rsq 0.995 Rsq 0.941 Ct of 214 copies = 35.28 Ct of 350 copies = 34.46 Multiplex: limit of detection; VZV 217 copies, EBV 214 copies, CMV 350 copies VZV Singleplex: limit of detection; 217 copies EBV Rsq 0.995 Ct of 214 copies = 34.53 Rsq: 0.981 VZV Rsq 0.997 Ct of 217 copies = 39.24 CMV Rsq 0.998 Ct of 350 copies = 37.4 Ct of 217 copies = 36.59

RNA virus Duplex Hepatitis A singleplex: limit of detection; 40 IU Rsq 0.853 Duplex: Limit of detection; GBV-C 10 AU, HAV 40 IU HAV Rsq 0.636 Ct of 40 IU = 35.79 Ct of 40 IU = 37.59 GBV-C Rsq 0.880 Ct of 100 AU = 37.0 GBV-C singleplex: limit of detection; 10 AU Rsq 0.942 Ct of 100 AU = 37.42

qPCR results in Ghanaian blood donors CMV VZV EBV HHV-8 HBV B19 GBV-C HAV 1 10 100 1000 10000 100000 1000000 1.0x10 07 Limit of detection

Background serology and viraemia in Ghanaian blood donors VZV CMV EBV HHV-8 HBV B19 GBV-C HAV 46.3 93.4 97.1 22.8 72.4 13.4 58.4 0.49 3.9 0.99 10 20 30 40 50 60 70 80 90 100 Virus Viraemia Ab % Positive

Summary A range of different types of standards where utilised for qPCR, which had similar dynamic ranges, repeatability and reproducibly NIBSC standards Plasmid standards Clinical standards Triplex assays where developed and optimised to match the sensitivity of the singleplex PCR This was achieved, except for B19 ( one-log decrease in sensitivity) The HAV assay could not be utilised as a quantitative assay despite extensive optimisation The Ghanaian blood donor population, while having high seroprevalence for each virus examined indicating previous exposure, had only very low frequency and level of viraemia No viraemia was detected with HHV-8, VZV and HAV despite high background seroprevalence Against this background studies of reactivation in various situations of immunodeficiency can now be conducted

Acknowledgements Division of Transfusion Medicine (University of Cambridge) Jean-Pierre Allain Daniel Candotti Komfo Anokye Teaching Hospital (Kumasi, Ghana) Ohene Opare-Sem Francis Sarkodie Laboratoire de Virologie (Hôpital Armand Trousseau) A. Garbarg-Chenon