“DETECTION OF EPSTEIN BARR VIRUS INFECTION IN HIV INFECTED INDIVIDUALS ON ANTIRETROVIRAL THERAPY USING
MOLECULAR METHOD AT A TERTIARY CARE CENTRE IN SOUTH INDIA ”
DISSERTATION SUBMITTED TO
In partial fulfillment of the requirement for the degree of DOCTOR OF MEDICINE IN MICROBIOLOGY
(Branch IV) M. D. (MICROBIOLOGY) Registration No.: 201714302
of
THE TAMIL NADU DR. M. G. R MEDICAL UNIVERSITY CHENNAI- 600032
DEPARTMENT OF MICROBIOLOGY TIRUNELVELI MEDICAL COLLEGE
TIRUNELVELI- 11
MAY 2020
BONAFIDE CERTIFICATE
This is to certify that the dissertation entitled “DETECTION OF EPSTEIN BARR VIRUS INFECTION IN HIV INFECTED INDIVIDUALS ON ANTIRETROVIRAL THERAPY USING MOLECULAR METHOD AT A TERTIARY CARE CENTRE IN SOUTH INDIA ” submitted by Dr.JAYASWARYA S.K to the Tamilnadu Dr. M.G.R Medical University, Chennai, in partial fulfillment of the requirement for the award of M.D. Degree Branch – IV (Microbiology) is a bonafide research work carried out by her under direct supervision & guidance.
Head of the Department, Department of Microbiology
Tirunelveli Medical College,
Tirunelveli
CERTIFICATE
This is to certify that the Dissertation “DETECTION OF EPSTEIN BARR VIRUS INFECTION IN HIV INFECTED INDIVIDUALS ON ANTIRETROVIRAL THERAPY USING MOLECULAR METHOD AT A TERTIARY CARE CENTRE IN SOUTH INDIA” presented here in by DR.JAYASWARYA.S.K is an original work done in the Department of Microbiology, Tirunelveli Medical College Hospital, Tirunelveli for the award of Degree of M.D. (Branch IV) Microbiology under my guidance and supervision during the academic period of 2017 -2020.
The DEAN
Tirunelveli Medical College,
Tirunelveli - 627011.
DECLARATION
I solemnly declare that the dissertation titled “DETECTION OF EPSTEIN BARR VIRUS INFECTION IN HIV INFECTED INDIVIDUALS ON ANTIRETROVIRAL THERAPY USING MOLECULAR METHOD AT A TERTIARY CARE CENTRE IN SOUTH INDIA ” is done by me at Tirunelveli Medical College hospital, Tirunelveli. I also declare that this bonafide work or a part of this work was not submitted by me or any others for any award, degree, or diploma to any other University, Board, either in or abroad.
The dissertation is submitted to The Tamilnadu Dr. M.G.R.Medical University towards the partial fulfilment of requirements for the award of M.D.
Degree (Branch IV) in Microbiology.
Place: Tirunelveli Dr. JAYASWARYA S.K,
Date: Postgraduate Student,
Register No: 201714302
ACKNOWLEDGEMENT
First and foremost, I thank the Almighty for His presence throughout my work.
Without the Grace of God nothing would have been possible
My Research project is made possible with the support of many people. I take this opportunity to express my gratitude towards them.
I am grateful to The Dean, Dr.S.M.Kannan M.Ch (Uro), Tirunelveli Medical College and Tirunelveli Medical College hospital, Tirunelveli for permitting me to carry out this study.
It is with great privilege and respect that I express my cordial and humble thanks to Dr.C.Revathy,M.D.,Professor and Head, Department of Microbiology, Tirunelveli Medical College, whose kindness, guidance and constant encouragement enabled me to complete this study.
I also thank Dr.P.Gnanaguru,M.D., Associate Professor, Department of Microbiology, Tirunelveli Medical College, for his support for my project.
I am very thankful to Dr.M.A.Ashiha Begum,M.D., Associate Professor, Department of Microbiology, Tirunelveli Medical College, for the encouragement throughout the period of study.
I am very grateful to Dr.V.P.Amudha M.D., Associate Professor, Department of Microbiology, Tirunelveli Medical College, for the constant support rendered throughout the period of study and encouragement in every stage of this work.
I sincerely thank my Co-guideDr.B.Cinthujah M.D, Senior Asst.Prof. Depatment of Microbiology, Tirunelveli Medical College without whose encouragement, motivation and guidance this work would not be possible
.
I express my sincere gratitude to Dr. I.M RejithaM.D., Senior Asst.Prof,Depatment of Microbiology, Tirunelveli Medical College for her encouragement, support, and corrective comments during the research period.
I am highly obliged to Senior Assistant ProfessorsDr.G.Velvizhi, Dr.S.Gowri, M.D, Dr.Kanagapriya,M.D, Dr.R.Nagalakshmi,M.D, Dr.K.Subha M.D., Dr.Suyambu Meenakshi,M.D, Department of Microbiology, Tirunelveli Medical College, for their evincing keen interest, encouragement, and corrective comments during the research period.
Special thanks are due to my co-postgraduate colleagues Dr.V.Ashwini, Dr.Roohee Zubaidha, Dr.G. Malathi, Dr.A.Sangeetha,for never hesitating to lend a helping hand throughout the study.
I express my sincere thanks to all my senior colleagues L.Gracia Paul, Dr.M.SaiShruti, Dr.E.Manimala, Dr.Maya Kumar, Dr.Uma Maheshwari.R for their helpful and supportive attitude in completing my project.
I would also wish to thank my junior post-graduate colleagues, , Dr.S.I.SaheedAskar, Dr.M.SriVidhya, Dr. R.Priyadharshini, Dr.V.Thanalingam, , Dr.Reachel Reena.D, Dr.Rajasri.A.G, Dr.Aparna.V.S, Dr.Kaviya.C, Dr.Latha Bharathi.Cfor their help, motivation and support.
Special thanks toMr.V.Chandran , K.Umayavel , Mrs Uma ,Miss Karputhamani for their helpful and supportive role in completing my project.
I am indebted to my parents Mr.S.Karmega kannan, Mrs.S.Bharathi,my brother Dr.B.K.Kharthickeswar for being my backbone and all the family members not only for their moral support but also for tolerating my dereliction of duty during the period of my study.
I also thank Dr.Bhavani MD(SPM) for her guidance in the statistical work.
I wish to thank Dr.Logeshwari.V, Dr.Sumathi.V, Dr.Amsanithya, Mrs.Anitha Bharathi S.A, for their friendly motivation and constant support.
.
CERTIFICATE – II
This is to certify that this dissertation work titled “DETECTION OF EPSTEIN BARR VIRUS INFECTION IN HIV INFECTED INDIVIDUALS ON ANTIRETROVIRAL THERAPY USING MOLECULAR METHOD AT A TERTIARY CARE CENTRE IN SOUTH INDIA ” of the candidate Dr.JAYASWARYA.S.K with registration Number 201714302 forthe award of M.D. Degree in the branch of MICROBIOLOGY (IV) . I personally verified the urkund.com website for the purpose of plagiarism Check. I found that the uploaded thesis file contains from introduction to conclusion pages and result shows 1 percentage of plagiarism in the dissertation.
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CONTENTS
l. N Title Page No.
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 4
3 AIMS & OBJECTIVES 46
4 MATERIALS AND METHODS 47
5 RESULTS 58
6 DISCUSSION 75
7 SUMMARY 79
8 CONCLUSION 80
9 BIBLIOGRAPHY 10 ANNEXURE
i. Data Collection Proforma ii. Consent form
iii. Master Chart
ABBREVIATIONS
AIDS Acquired Immunodeficiency Syndrome BART BamHI-A rightward transcript
BL Burkitt Lymphoma
bp base-pair
C-terminal carboxyl-terminal CMV cytomegalovirus
CTL cytotoxic T lymphocytes CSW Commercial sex worker DNA deoxyribonucleic acid EA early antigen
EBER Epstein-Barr virus-encoded small RNA EBNA Epstein-Barr nuclear antigen
EBV Epstein-Barr virus
EDTA diaminoethanetetraacetic acid EFS event-free survival
ENKTL extra-nodal NK/T lymphoma FAM 6-carboxy-fluorescein
gp glycoprotein
HIV human immunodeficiency virus
IFN interferon
IRF interferon regulatory factor Kbp kilobase pair
kDa kiloDalton
KO knock-out
LCL lymphoblastoid cell line LCV lymphocryptovirus LMP latent membrane protein MAb monoclonal antibody
MHC major histocompatibility complex MSM male to male sex
miR microRNA
N-terminal amino-terminal
NFκB nuclear factor kappa B NK natural killer
NPC Nasopharyngeal carcinoma ORF open reading frames
oriP origin of plasmid replication PCR polymerase chain reaction
PTLD post-transplant lymphoproliferative disease RIC reduced-intensity conditioning
RNA ribonucleic acid RT reverse transcription
SLE systemic lupus erythtmatosus TCR T cell receptor
TNF tumour necrosis factor VCA viral capsid antigen vIL-10 viral IL-10
WHO World Health Organisation
INTRODUCTION
Communicable diseases are being considered to be the major public health burden year for more than thousand years1. Though there are many outbreaks due to various infections especially with viruses, Human Immunodeficiency Virus outbreak grew out to be a Pandemic causing Acquired Immunodeficiency Syndrome that resulted in more than 25 million deaths since its discovery in 1981 till 20062.Morbidity and Mortality due to those diseases increased in proportion with the population. The World Health Organization (WHO) had identified HIV/AIDS as one of the world’s first health emergency and an urgent threat to global public health. The Epidemic in Asian countries is contributed mainly by high risk behaviours like unsafe sexual practices with multiple partners, Intravenous Drug Usage etc.,2. Compared to Sub-saharan African countries, the prevalence of adult HIV in Asian countries like India is low. Approximately 80% of HIV infections in India are acquired heterosexually3.
Human Immunodeficiency virus (HIV) is an enveloped, positive-sense, single-stranded RNA virus of the lentivirus genus and Retroviridae family. HIV selectively infects and destroys CD4+ T lymphocytes, causing severe immunodeficiency in infected patients. The advanced stage of HIV disease is defined as Acquired Immunodeficiency Syndrome (AIDS), clinically characterized by CD4 counts less than 350 per mm3 of blood and/or overt clinical signs of opportunistic infections4.
In a healthy human body, microbes maintain an effective parity with the host, when a new microbe pervades, but for some, the host fails to readjust this parity, and these microbes become pathogens (copathogens). HIV-induced disruption of the equilibrium between a human host and its virome leads to two grave consequences:
1. Reactivation of ubiquitous viruses (copathogens), which start to replicate to higher levels, for example, HHVs
Of these Opportunistic viral infections, according to recent studies6, Epstein Barr virus infection is a major one.
Infection with EBV is widespread among different populations which occurs during early childhood in most parts of the world. Most of the time it is known to cause asymptomatic or mild infection only. In developing countries like India, the primary infection is acquired before the age of 3-5years in contrast to that of in the western world where the primary infection occurs late. It is the cause for Infectiuos mononucleosis mainly in adolescents and adults. It is manifested with fever, rash, pharyngitis, hepatomegaly and splenomegaly. In India, Infectious mononucleosis is an uncommon manifestation.
EBV establishes its latency after its primary infection and it can be detected both in
lymphoid tissue and peripheral blood. In immunosuppressed patients, the virus—host imbalance favors EBV reactivation which in turn induces lymphoproliferation. Therefore, it has been proposed that these benign proliferations may become malignant lymphomas if they are not well controlled by the host immune system, although this is a rare event7.
Evidence suggests that the expression of EBV in people with AIDS or those who can develop AIDS is in unusual patterns. These patterns include :
1. Increased titers of antibodies to EBV antigens
2. High levels of excretion of EBV in oropharyngeal secretions.
3. EBV-infected B cells in circulation - elevated
The first virus recognized to be the reason for cancer to occur in people is EBV; it causes both lymphomas and carcinomas8.
Risk factors for HIV-associated malignancy include a diagnosis of AIDS, low CD4 lymphocyte cell count, unsuppressed HIV replication, short antiretroviral therapy (ART) duration, and high EBV viral load. According to UNAIDS 2018, the third largest HIV epidemic in the world was in India.The prevalence of HIV among adults in India is 0.2- 0.3%.Though the epidemic of HIV slowed down overall between 2010-2017 with decline in new infection by 27% and AIDS related deaths being fell down by 56 %,new infections raised from 80000 to 88000 and AIDS related Death raised from 62000 to 69000 in 2017 which indicates that there is a clear delay in attaining prevention goals which are mainly attributed to Opportunistic infections.
HIV infection may result in earlier EBV reactivation and poor control of EBV replication and dissemination, which could in turn pose a risk of increased morbidity and mortality by developing lymphoproliferative disorders and carcinoma.As there are no much studies in India about EBV there is a paucity of data about the same,a study about EBV DNA detection in HIV infected individuals can give us better knowledge to understand whether the EBV reactivation in people with AIDS is significant.
REVIEW OF LITERATURE
History of Epstein Barr virus
The Discovery of Epstein-Barr virus was by Denis Burkitt and Anthony Epstein in March 1964(Zuck). Burkitt saw multifocal jaw tumors in two children in 1957 in Africa, the presentation of which was new to him and the same led him to study the prevalence of lymphoma in sub-Saharan Africa. He was surprised with the fact that theChildhood lymphomas were prevalent living in areas with Hyperendemic Malaria that made him to suspect infectious etiology9. Epstein after knowing about this succeeded in culturing the lymphoma cells from such tumor biopsies and identified a herpes virus in images of these lymphoma cells, taken from electron microscope10.This Burkitt`s lymphoma virus was found to differ from other known herpes viruses by Epstein, Burkitt and Achong as it was noninfectious for cultured cell lines and nonreactive with antibodies to other human herpes viruses.
EBV emerged as the first candidate human tumor virus and the prototype for the potentially oncogenic gamma-herpesviruses subfamily.
A technologist while handling the specimens before studying them under electron microscope in Henle laboratory, Philadelphia and she was also giving her lymphocytes for
Classification:
Group: Group I(ds DNA) Family: Herpesviridae
Subfamily Gammaherpesvirinae Genus: lymphocryptoivirus Species: Epstein Barr virus
The similarity between the EBV genome and that of other Gammaherpesviruses, G +C content of its genome and lymphotrophism, is the basis of this classification.
Morphology
The nucleic acid of EBV is a DNA core which is toroid in shape. DNA is double stranded.
Surrounding this is the Capsid with 162 capsomers. This nucleocapsid is present within an envelope that has glycoprotein spikes. Between the envelope and the nucleocapsid a protein tegument is present12.Major Capsid protein of 155 kDa, Minor Capsid protein of 30kDa,small capsid protein of 18 kDa, minor capsid protein binding protein of 40 kDa and portal protein of 68 kDa in particular ratios together constitute the Capsid of EBV and a virion has 12 such portal molecules. The tegument has been composed of proteins that are common to all herpes viruses and 2 proteins that are specific to gammaherpes virus subfamily. The common tegument proteins to all herpes viruses are BPLF1-large tegument protein of 350 kDa, BOLF1-large tegument protein binding protein of 140kDa, BBLF1-mMyristylated protein of 15 kDa,BGLF2- mMyristylated protein binding protein of 32 kDa,BVRF1- capsid associated
protein of 58kDa, BGLF1- packaging protein of 58 kDa,BSRF1- palmitylated protein of 27 kDa and BGLF4-TS kinase 0f 47kDa383. BNRF1-A Major tegument protein of size 140kDa, BLRF2 of size 19kDa,BRRF2 of 72 kDa,BDLF2 of 54 kDa and BKRF4 of 42 kDa are the specific tegument proteins of Gammaherpes virus. Some Cell proteins in the tegument that play important role in cytoplasmic reenvelopement are Hsp90,Cofilin, actin, enolase , HSP70 and β-tubulin .BLLF1-gp350,BKRF2-gL,BXLF2-gH,BDLF3-gp150,gB-N,BLRF1-gN,gB- C, BILF2 -gp78, BALF4-full length gB,BBRF3-gM and BZLF2-gp42 are the major components of glycoprotein present in the envelope.
Rather than virus BMRF2 enable the EBV-infected cells to adhere to epithelial cells by binding to epithelial cells13.
HIV HISTORY
The period of early 1970s and early 1980s in the United states of America and Europe saw many previously healthy patients came to treatment to the physicians for symptoms of immunologic dysfunction. It was found that this new unusual syndrome was characterised by generalised lymphadenopathy, opportunistic infections (typically Pnemocystiscariinipnemonia, Cytomegalovirus-associated retinitis, Cryptococcal meningitis)and unusual cancers like non-hodgkins lymphoma and Kaposi sarcoma. Peripheral blood examination of affected individuals showed a marked depletion of the CD4+ T-
partners and children of affected individuals. Based on pattern of symptoms and the spread of the disease it was suggested that the disease spread occur through a pathogen in contaminated blood or after sexual intercourse with affected individuals. Numerous microorganisms were proposed to be the etiologic agent for this type of acquired immunodeficiency syndrome or AIDS as the disease was called. In 1983 Montagnier and his colleagues at the Pasteur institute isolated an agent from the lymph nodes of an asymptomatic individual presented with complaints of generalized lymphadenopathy of unknown origin. the agent was subsequently cultured. During its replication in the cultured cells, the lymphadenopathy associated virus(LAV),the name given to the agent, released high titres of progeny virions.The virions showed Magnesium dependent Reverse transcriptase activity and electron microscopic features of retroviruses but unlike the typical retroviruses of vertebrate origin, these Lymphadenopathy associated Viruses(LAV) were highly cytopathic to peripheral blood mononuclear cells with CD4+ T-lymphocytes in the cell cuture being specific target15.At the national institute of health, Gallo and coworkers isolated Retrovirus from an AIDS patient and named it Human T-cell Leukemia virus type-III (HTLV-III) to differentiate it from non- cytopathic HTLV-I. The team also produced the first convincing serological evidence which linked the LAV like Retroviruses with the various Immunodeficient individuals of risk groups. Later levy et al. isolated similar retroviruses from both patients with aids and healthy individuals of risk groups and named it Aids-associated retroviruses(ARV). It was then suggested that the virus could induce both symptomatic and asymptomatic infections.
The new retrovirus associated with AIDS was subsequently named Human Immunodeficiency Virus or HIV-1. In 1986 a related human immunodeficiency virus, later named as HIV-2 , was isolated from individuals in West African countries similar to the HIV- 1 causing AIDS in United States, Europe and Central Africa.HIV-2 in comparison with HIV- 1 caused much slower decline in CD4+ T lymphocytes, longer asypmptomatic infections and lower mortality rates16.
Classification:
Family: Retroviridae Genus: lentivirus
Species: Human Immunodeficiency virus
Pathogenesis of HIV:
Genome structure and Latency of EBV:
The EBV genome comprises a linear double-stranded DNA, approximately 184kb long17, wrapped around a toroid-shaped protein core18. The genome is divided into long unique sequence domains by 3kbp internal direct reiterations19.The prototype B95-8 strain, the first virus to be fully cloned and sequenced21, is unusual as it has a 11.8kb deletion17. The complete sequences of two more strains of EBV have recently been determined: GD120, derived from a NPC tumour in a Chinese patient; and AG87621, derived from a Ghanian BL patient. The ends of the genome are flanked by 4-12 tandem copies of the 538bp terminal repeats22 (TR) which mediate episomalcircularisation. The precise number of TRs is determined during viral replicationwhich serves as a invaluable marker of clonality, as the progeny of latently infected cells retain anidentical number of TRs to the parental cell23. Since the prototype sequencing was achieved by a BamHI fragment cloned library, genes and genomic locations are often referred to by specific BamHI fragments - alphabetically in order of descending fragment size.
EBV can potentially encode approximately 80 proteins, some of which remain uncharacterised24.There are 2 major strain subtypes of EBV, referred to as type 1 and type 2, which can be further subdivided into different EBV strains25. All EBV isolates are highly homologous and share a common genome organisation26. The predominant sequence differences between type 1 and type 2 viruses relates to genes encoding the Epstein-Barr
another. Rather, it appears that the majority of EBV gene polymorphisms discernible in healthy virus carriers occur at a similar frequency in EBV+ tumours from the same geographical region28.
Latent infection in transformed primary B cells
The Latency I, II and III nomenclature was originally established following key observations on the pattern of EBNA and LMP protein expression in BL and LCLs 30. Exclusive expression of EBNA1, the viral protein essential for replication and maintenance of its own episome, was considered the most restricted form of latency: i.e. Latency I observed in BL. However, in non-dividing cells such as memory B lymphocytes in healthy carriers31 EBNA1 appears not to be critical, thus the term Latency 0 is applied to such cells where no viral antigen expression is seen. These terms will be used for discussion of viral gene expression within this thesis, although caveats relating to such nomenclature, particular when applied to an in vivo context, do exist. For example, considerable variation is seen to exist in B cell lymphomas of the immunocompromised, both between tumours32 and - at the single cell level33 - within a malignant population. Nonetheless, an accurate picture of latent gene expression within a tumour can usually be established by combining an assessment of viral proteins at the single cell level alongside quantitative RT-PCR to analyse the range of latent transcripts expressed. Following in vitro infection of primary B lymphocytes, circularised viral genomes are appreciable within the nuclei 12 to 16 hours after virus binding. At around the same time, the W promoter(Wp), present in the BamHI-W long internal repeat element, initiates rightward transcription34. The B cell specificity of this promoter is facilitated by B cell lineage specific activator protein (BSAP, or Pax5) binding sites upstream of Wp. Wp promotes the transcription of the initial viral RNAs, which are differentially spliced to generate mRNAs coding for the EBNA-LP and EBNA2 proteins.
Transcription is undertaken by host cell RNA polymerase II. Twenty-four to 36 hours
following B cell infection, levels of EBNA-LP and EBNA2 protein are approximately equivalent to those seen in established LCLs34. These early latent proteins cooperate to initiate transcription from the upstream BamHI C promoter (Cp). The mutual exclusivity of Wp and Cp activity within a single cell results in Cp replacing Wp as the dominant promoter for transcription of the EBNAs. The switch from Wp to Cp results in expression of the remaining EBNA transcripts. All EBNA mRNAs have exons in common from the BamHI W viral genomic fragment but alternative splicing into one of 4 alternative acceptor sites determines whether EBNA3A, 3B, 3C or EBNA1 is translated, each of which encodes its own polyadenylation signal. Expression of these EBNAs occurs at approximately 24 hours following infection and reaches a plateau after 48-72 hours. A positive feedback loop enhancing Cp activity, initially mediated by EBNA2 and EBNA-LP and thereafter by EBNA1, is countered by the repressive action of the EBNA3 proteins35. In LCLs, transcription of EBV‟s latent membrane proteins are activated by EBNA2. The expression and regulation of LMP1 and LMP2 is discussed later under the individual latent gene headings. The non-coding, nonpolyadenylated EBER1 and EBER2 transcripts are the last of the latent transcripts to be expressed, reaching substantial expression levels 3 days after infection. Transcription is predominantly mediated by cellular RNA polymerase III and EBERs are the most abundantly expressed EBV RNAs in established LCLs. More recently a relatively large number of EBV-encoded microRNAs (miRs) have been identified36, encoded
expressed in all forms of latency and in lytic cycle, differing to the more restricted expression of the BHRF1-derived miRs. BHRF1 transcripts in latent infection are almost exclusively initiated from the Cp/Wp promoters36.
Latency III
Following infection of primary B lymphocytes in vitro, almost all nuclear genome- positive B cells promptly express EBNA2 which initiates and drives the virus‟ growth transforming programme, inducing cell cycle transit (usually within 48-72 hours); ultimately leading to an established LCL. Latently infected B cells express 6 EBV nuclear antigens (EBNAs), EBNA 1, 2, 3A, 3B, 3C and LP and 3 latent membrane proteins (LMPs), 1, 2A and 2B. More recently, a tenth latent antigen expressed from Wp-initiated transcripts - the BHRF1 protein (a Bcl-2 homologue with antiapoptotic function) - has also been associated with Latency III type infection37. In addition, small, non-polyadenylated EBER1 and EBER2 transcripts, the highly spliced BamHI-A right wardtranscripts (BARTs) and a number of EBV-encoded microRNAs (miRs) are expressed. Suchpattern of latent gene expression is referred to as Latency III or the ‘growth programme’.The latency III pattern of gene expression seen in LCLs is inextricably accompanied by an equally characteristic cellular phenotype; markers of B cell activation and adhesion, usually absent (or weakly expressed) on resting B cells, are readily identified; LCLs resemble lymphocytes proliferating in response to antigen, mitogen, or stimulation with CD40 and IL4538. Such a transformative impact of Latency III gene products on B cell phenotype implicated these viral proteins as key effectors of cell ‘immortalisation’ and, importantly, is analogous to EBV-driven B cell lymphoproliferations occurring in the context of impaired T cell immunity39.
Latency I
The characterisation of latent gene expression in EBV-associated tumours and cell lines established from BL biopsies highlighted two alternate patterns of Latency. EBNA-1 is the only protein consistently expressed in BL57, alongside the non-coding EBERs, BARTs and miRNAs. This restricted pattern is referred to as Latency I and is characterised by activity of the Q promoter (Qp), for EBNA1 transcription, and silence of Cp, Wp, and the LMP promoters. It should be noted, however, that isolated cells within a Burkitt tumour biopsy can express LMP1and EBNA240 similar to the in vitro phenomenon of BL cell lines that are seen to ‘drift’ towards a Latency III phenotype.
Latency II
An intermediate pattern of EBV gene expression was originally identified in undifferentiated nasopharyngeal carcinoma (NPC)41, and thereafter in EBV-associated gastric carcinomas42, Hodgkin’s lymphomas43, and extra-nodal NK/T cell lymphomas (ENKTL)44.Akin to latency I, EBNA1 is transcribed from a highly spliced Q-U-K transcript driven by Qp, a TATA-less promoter whose configuration resembles the promoters of housekeeping genes - suggesting that Qp may act as a default promoter for EBNA1 in the absence of a ‘growth program’. This Q-U-K-spliced structure of the EBNA-1 transcript has been demonstrated in HL, NPC, and ENKTL44. As in BL, EBNA 1 expression is accompanied by the EBER and BART transcripts, but in Latency II additional latent
LMP1 and LMP2A in the Reed-Sternberg cells of HL, whilst expression of the LMP2 proteins in ENKTL has not been elucidated.
EBV Latent gene products LMP 1
In LCLs, LMP1 is translated from an RNA transcribed from the BNLF1 gene at the right end of the virus genome45, initiating from a site (ED-L1) within the BamHI N-EcoRI D fragment of the EBVgenome45. The unprocessed transcript - which lies entirely within the antisense strand of the first intron of LMP2A - is spliced to a 2.4kb mRNA, with an ORF of 1.3kb which crosses twointrons45. Later analyses of a nude mouse-passaged NPC detected two 3` coterminalLMP1 transcripts of 2.8kb and 3.5kb, at apparently equal levels as assessed by Northernblotting. An alternative (TATA-less) LMP1 promoter (pL1-TR) was subsequently mapped within 20 bp of the start of the first TR - approximately 600 bp upstream of ED-L1 - and gives riseto the larger mRNA. Both transcripts utilise the same ATG initiator, hence the encoded LMP1protein is identical. The 3.5kb mRNA was also found to be expressed in LCLs (IB4) and the BLAkata line, although levels of the 2.8kb mRNA were substantially higher in these B lymphoid cells. By 36 hours following in vitro infection of primary B lymphocytes, LMP1 mRNA is measurable and, within the first few days, approaches a level similar to an established LCL. The nucleotide sequence of the ORF predicted that LMP1 would comprise six hydrophobic transmembrane domains and a 200 amino acid acidic carboxy terminus. A monoclonal antibody, S12, confirmed that LMP 1 is distributed throughout the cytoplasmic and plasma membrane where it forms distinct aggregates. Further characterisation of the structure confirmed that LMP1 is an integral membrane protein of63 kDa and delineated three distinct domains. The amino-terminal cytoplasmic tail (aa 1–23)functions to tether LMP1 to the plasma membrane and orientates the protein; six hydrophobic transmembrane loops (aa 24–1) are involved in homo-
oligomerisation and self-aggregation; an elongated carboxyterminal cytoplasmic region (amino acids 187–386) mediates most of the signalling activity of the molecule. Interest in LMP1 as a crucial growth transforming protein of EBV was initially stimulated by a study demonstrating that LMP1 functions as a classic oncogene in a rodent fibroblast transformation assay. Subsequent studies corroborated its transformative properties by showing a requirement for LMP1 in EBV-induced B cell transformation in vitro47. Mutational analyses indicated that LMP`s cytoplasmic C-terminus and transmembrane domain 1 (TM1)were essential for B cell transformation. Deletion of the cytoplasmic N- terminus adversely impacted, but did not prevent transformation. In vivo, transgenic mice expressing LMP1 under the Ig heavy chain promoter developed monoclonal/oligoclonal splenic B cell lymphomas expressing LMP1 – confirming the potent oncogenic potential of this viral protein.LMP1 has pleiotropic effects on cellular phenotype, resulting in the induction of cell-surface adhesion molecules and activation antigens48, and upregulation of anti-apoptotic proteins (eg BCL2). LMP1 functions as a constitutively activated member of the tumour necrosis factor receptor (TNFR) superfamily and activates a range of signalling pathways in a ligandindependentmanner49. Functionally, LMP1 resembles CD40 – a bona fide member of the TNFR superfamily - and can partially substitute for CD40, as shown in LMP1 transgenic mice, providing both growth and differentiation signals to B cells50.The activation of several downstream signalling pathways contributes to the multiple
the recruitment of a multiprotein catalytic complex containing the NF-κB-inducing kinase (NIK), the IκB kinases (IKKs) with consequent activation of both the classic IκB- dependent100 and independent51 NF-κB pathways. Other kinases are also recruited to LMP1 through interactions with TRAF molecules, including mitogen activated protein kinase kinase kinases (MAPKKKs), which in turn contribute to the activation of the NF-κB, MAPK and phosphatidylinositol 3-kinase (PI3K) pathways. Taken together, it is clear that LMP1 effectively utilises its position within the plasma membrane to influence a large number of cellular genes and signalling pathways to promote the transformation of B cells and epithelial cells.
Factors regulating LMP1 expression
The dominant (2.8kb) LMP1 mRNA found in LCLs is expressed from the ED-L1 site upon transcriptional activation by the EBNA-2 protein, which interacts with cellular transcription factors such as the repressor RBP-J kappa (CBF1 - involved in the Notch signalling pathway) and PU-1(Spi1). Both cellular proteins bind to distinct sites in the LMP-1 promoter52. By contrast, regulation of expression of LMP1 from the alternate, EBNA2- independent, L1-TR promoter is less well understood. pL1-TR is transcriptionally active in NPC cell lines, NPC tumour tissue53and Hodgkin lymphoma tissue108 and appears to be the dominant LMP1 promoter in these tumours, although the PCR methods in these studies were not quantitative53. Sadler et al found that transcription was initiated from within the first TR but not from within distal TR elements and suggested that unique sequences adjacent to the first TR may contribute to promoter activity54.Chen et al found that both LMP1 promoters responded to activation of the JAK/STAT pathway, with evidence of functional STAT- binding motifs at both sites53. This feature is in common with STAT-mediated positive regulation of Qp-driven transcription of EBNA1111 and, specifically, STAT3 is implicated as a biologically relevant candidate for activation of the Qp and L1-TRpromoters53. Other
cellular factors such as Sp1 and the unfolded protein response (UPR)protein XBP-1 are also thought to contribute to promoter activity. It seems likely that the EBNA2-independent pL1- TR promoter is transcriptionally dominant inLMP1-expressing cells of EBV+ NK and T cell lymphoproliferations, but this remains to be formally shown. LMP1 expression in ENKTL cell lines can be regulated by cytokines such as IL10 (a potent activator of STAT3), IL15 , although such cytokines were not sufficient for their proliferation in the absence of IL-2.
Recent work on Latency I BL lines found that IL-21 could potently induce LMP1 expression (in the absence of EBNA2), with the majority of the LMP-1mRNA originating from the ED- L1 promoter. Thus, it is feasible that rather than LMP-1 being a stable characteristic of a
’Latency II’ EBV infected cell, expression may be dynamic and influenced by extracellular signals.
LMP1 expression and function in T cells
Studies examining the role of LMP1 expression in T and NK cells are restricted to in vitro models expressing LMP1 in EBV-negative T cell lymphoma lines, whereas NK cells have not been studied in this context. Following from an initial observation that in vitro infection of a T cell lymphoma line with EBV resulted in up-regulation of TNF-α expression55, it was subsequently shown that the candidate protein was LMP1. Further work from the same group, investigating the pathogenesis of EBV-HLH, indicated that LMP-1-expressing T cells were relatively resistant to TNF-α-induced apoptosis likely due, in part, to downregulation of
expression in T cells, thereby inducing SLAM mediated enhancement of ERK/IFN signalling. This LMP1-induced transcriptional suppression of SAP is mediated via activating transcription factor-5 (ATF5). Such deregulation of SAP/SLAM/ERK by LMP1 and enhanced IFN secretion by T cells prompts comparison with the pathogenesis of X-linked lymphoproliferative disease, where mutations in the SAP gene similarly results in Th1 cytokine production.
EBNA1
EBNA1 is expressed in all virus-infected cells, with the exception of Latency 0 memory B Lymphocytes58, where it functions to maintain and replicate the episomal viral genome – achieved through sequence-specific binding to the plasmid origin of viral replication, OriP57. The interaction of cellular DNA, EBNA1 and oriP results in a single replication of the EBV genome during Sphase, followed by equal segregation of replicated EBV genomes to progeny cells during celldivision59.
The amino- and carboxy-terminal domains of EBNA 1 are separated by a variably- sized Gly-Ala repeat sequence, the chief function of which appears to be stabilisation of the mature protein, preventing its proteasomal breakdown degradation and/or suppressing EBNA1 Mrna translation initiation. EBNA1 is not critical for B-cell transformation in vitro, but seems to substantially promote the efficiency of this process. A more direct effect on lymphomagenesis is inferred by the ability of B-cell directed EBNA1 expression to produce B-cell lymphomas in transgenic mice60and by evidence of pro-survival effects in BL cells in vitro Expression of EBNA1 is augmented by a number of cellular factors, including the interferon regulatory factors, IRF1 and 2 and the cell cycle E2F transcription factors; all of which bind to distinct regions close to the (Fp/Qp) EBNA1 transcription initiation site. In restricted forms of latency, EBNA1 restrains its own transcription by binding downstream of the transcriptional startsite. The function of EBNA1 in EBV-associated NK and T
lymphoproliferations has been largely uninvestigated. An isolated report showed that partial silencing of EBNA1 expression in an ENKTL line retarded cell proliferation, associated with an accumulation of cells in G0/G1 phase61,although the mechanism was not clearly delineated.
EBNA 2
An early clue to the essential role of the EBNA2 protein in B cell transformation came from the inability of the EBNA2 deletion mutant virus, the P3HR1 strain62, to establish transformed primary Bcells. Reinstatement of the deleted DNA region restored the virus
‘transformative properties, delineated its functional elements and confirmed EBNA2’s critical role in B cell immortalisation in vitro63.EBNA2 functions as a potent transcriptional activator of both viral and cellular genes and upregulates specific B cell antigens such as CD21 and CD23, in addition to LMP1 and LMP2.The protein contains a number of distinct structural elements, most notably the RBP-Jκ binding domain, the acidic activation domain and the homotypic association domains - all considered essential to the transformation and transcriptional properties of EBNA257.
EBNA2 is not expressed in EBV+ NK and T cell lymphoproliferations or other tumours with restricted patterns of latency, where the Wp/Cp promoters are dormant.
Similarly, other Latency III-associated proteins (LP, EBNA3A,B,C, BHRF1) arealso not expressed in T/NK lymphoproliferations.
EBNA LP
EBNA-LP, encoded by the leader of each of the EBNA mRNAs, encodes a protein of variable size according to the number of BamHW repeats within a given viral isolate64. The major role of EBNA-LP is to specifically potentiate the effect of EBNA2-mediated transcriptional regulation65 of both viral (eg LMP1) and cellular genes – the latter exemplified by cyclin D2-mediated induction of G0 to G1 cell cycle transit.
EBNA 3 family
Each of these large, related, multifunctional proteins has a highly charged N terminus, dimerisation domains and a nuclear localisation region. Despite expression of relatively few EBNA3 mRNA transcripts per infected cell, the stability of all 3 proteins results in intranuclear accumulation66. Degradation of the EBNA3s leads to the presentation of highly immunogenic peptides by surface MHC class I, serving as attractive targets for CD8+
CTLs67.
The EBNA3 proteins impart significant influence on EBV-mediated viral and cellular transcription. A common conserved region allows binding of RBP-Jκ, resulting in competition with EBNA2 and Notch for RBP-Jκ and thus negatively regulating EBNA-2 activity68. EBNA3A and EBNA3C are essential for B-cell transformation in vitro69 ,whilst EBNA2B appears not be critical in this system. In addition to their key role as transcriptional regulators, accumulating evidence implicates theEBNA3 family in EBV-mediated lymphomagenesis. For exampleEBNA3C is seen to cooperate with RAS in rodent-fibroblast transformation assays, deregulating cell-cycle checkpoints.
Other proteins expressed in latent infection BHRF1
Although primarily a lytic antigen, BHRF1 (a homologue of the cellular BCL-2 protein) has recently been identified in the context of latent infection in LCLs and a subset of BL cells. The 17kDa protein is thought to be non-essential for virus replication but may contribute to cellular transformation in vitro70. Notably, it is consistently expressed in BL cells where the Wp promoter(rather than Qp) is activated – termed Wp-restricted latency.
Such expression is thought to confer a survival advantage to the tumour cells.
BARF1
The BARF1 ORF encodes a soluble molecule of 33kDa that is quickly secreted into the medium of cultured cells71. Initially thought to be exclusively expressed during early lytic cycle, its presence in the context of NPC and BL – seemingly in the absence of replicative infection – prompted consideration of a putative oncogenic function71. Of interest, BARF1 transcripts were identified in cell lines derived from patients with extra-nodal NK/T lymphoma (ENKTL) and chronic active EBV (CAEBV), without evidence of full lytic cycle, at levels comparable to LCL. By contrast, levels of BHRF1 mRNA in these T and NK lines were considerably lower than the LCL control.
Non-coding transcripts EBERs
In addition to protein-coding transcripts, all types of latent EBV-infected cells contain abundant non-polyadenylated, non-coding transcripts generated by viral hijack of cellular RNA polymerase III. EBER1 (166 nucleotides) and EBER2 (172 nucleotides) form stem- loop structures by intermolecular base-pairing, resulting in double-stranded RNA (dsRNA)- like molecules which are the most highly expressed viral transcripts in latently infected cells72; estimated at 107 RNA copies per B cell. That the EBERs are so widely and abundantly expressed in latently-infected cells, provides a highly sensitive and specific diagnostic indicator of EBV infection ex-vivo: both at the single cell level via in-situ hybridization and by RT-Q-PCR of RNA derived from tissue biopsies and cell lines.
Although seemingly ubiquitous in latent infection, irrespective of cell type, an isolated report has shown that cells undergoing replicative infection may not express EBERs – at least in the context of oral hairy leucoplakia73. Some controversy exists as to whether EBERs are required for transformation of B cells in vitro. Initial work indicated that they were dispensable whilst more recent studies found that loss of EBER2 but not EBER1 considerably impaired the B lymphocyte transformation ability of EBV.
With some difficulty, B cells could be transformed with the EBER KO recombinants but growth of the resultant EBER KO LCLs was sluggish although, interestingly, could be enhanced by IL-6 supplementation. EBERs have also been shown to induce anti- inflammatory cytokines, such as IL1074 and type I interferon75, mediated through RIG-1 (retinoic acid-inducible gene-1).Recently, a paracrine effect of EBERs was demonstrated, inducing signalling through toll-like receptors (TLR) and the subsequent release of type I IFN and proinflammatory cytokines such as TNFα. This ‘immune activating’ effect may be relevant to the immunopathology of diseases such as IM, CAEBV and HLH, in which high
levels of EBER1 were found in ex vivo serum samples from most cases examined76.Whilst the EBERs seem to have some influence upon cellular phenotype and play a role in immunomodulation, the mechanistic details are not well understood and their role in disease pathogenesis remains unclear. In particular, their impact on the phenotype of EBV-associated NK and T cell lymphoproliferations has not been investigated.
BARTs
Originally identified in NPC tissue, an intricate cluster of non-polyadenylated RNAs are transcribed from the BamHI A region77and are collectively termed BamHI A rightward transcripts (BARTs). The primary transcripts contain a number of potential ORFs but uncertainty still exists as to whether or not these rightward transcripts encode protein, but they do yield a number of EBV miRs. Similarly to the EBERs they are found to be widely expressed across the spectrum of EBV-associated malignancies and in peripheral blood Blymphocytes78. The biological function of the BARTs is not clear, but it has been proposed that they may potentially regulate gene transcription or modulate kinase activity.
EBV-encoded micro-RNAs
Fully processed miRNAs (miRs) are short (21–24 nucleotides), single-stranded RNAs derived from double-stranded RNA precursors that post-transcriptionally regulate gene expression. This is most commonly achieved by binding to sequences within the 3`
untranslated region (with partial or complete complementarity) of target mRNAs and thus
region of the BARTs). Todate, aside from the prototype B95.8 EBV strain22 (which contains a 11.8 kbp deletion in the BART region), 25 EBV-encoded pre-miRNAs - resulting in 4 mature BHRF1 miRNAs and 40BART miRNAs - have been identified80.Analysis of NPC biopsies has shown high-level expression of most EBV miRNAs derived from the BART miRNA cluster, whilst the BHRF1 miRNAs are not expressed in primary NPC tissue.
As the Qp promoter is located between the BHRF1 and EBNA1 ORFs, viral RNA initiating from Qp cannot yield any of the BHRF1 miRNAs. Current data is consistent with the notion that expression levels of BART miRNAs mirror that of the BART mRNA transcripts. Studied in the context of NPC, EBV miRs can evidently target other EBV transcripts including LMP1 and LMP2Aas well as cellular genes. It should be noted, however, that high expression of a given miR may not consistently equate to the expected target effect in all tumours81.
In the context of NK/T lymphoproliferations, although certain cellular miRs have been proposed toconfer a pro-survival advantage to ENKTL cells in vitro, the expression and function of EBV miRs in this spectrum of disease has not yet been investigated.
EBV infection in vivo B cells and epithelial cells
EBV transmission between humans usually occurs by contact with oral secretions.
The virus replicates within the oropharynx resulting in active shedding of virus in the vast majority of seropositive individuals54,. It remains somewhat unclear whether oropharyngeal epithelial cellsor B lymphocytes are the primary site of replication, although infected B cells identified in IM tonsils were seen to be predominantly latent, with no clear evidence of full viral replication82.
Evidence of virus replication has been observed in squamous epithelial cells of the tongue in patients with acquired immunodeficiency syndrome (AIDS) and occasionally in post-mortem tissue from immunocompetent virus-carriers. Viral shedding from the oropharynx is abrogated in acyclovir-treated patients, whilst the number of EBV-infected circulating B cells is unchanged by such treatment83. Taken together with data from ex vivo cultures of tonsillar epithelium, this suggests that infection of oropharyngeal epithelium is an integral part of the in vivo EBV–host interaction.
The process whereby oropharyngeal replicative infection results in latently infected B lymphocytes is not well understood, but tonsil sections from IM patients readily demonstrate EBER-expressing B lymphoblasts with a proportion of these cells co-expressing EBNA2 and LMP1229-230. Expression of these viral proteins is indicative of a growth-transforming infection akin to EBV-transformed lymphoblastoid cell lines in vitro, where the full spectrum of latent cycle genes (EBERs, BamHI A-encoded RNAs, EBNAs 1, 2, 3A, 3B, 3C, -LP, and LMPs 1 and 2) are expressed. In vivo, however, these EBV-transformed lymphoblasts appear to down-regulateviral antigen expression, resulting in a reservoir of EBER-positive antigen- negative memory B cells, considered to be in a resting state160,236 that persist within blood and oropharyngeal lymphoid tissues.
In normal adults, approximately 1 to 50 circulating B cells per million are infected with EBV, and the number of latently infected cells within a person remains stable over
T cells
Although distinctly B lymphotropic both in vivo and in vitro, the characterisation of a spectrum of EBV+ T and NK cell lymphoproliferative diseases85 inevitably signifies an in vivo infection event of their T and NK counterparts, although conflicting data exists regarding the frequency and nature of these occurrences.
In the context of primary EBV infection, Anagnostopouloset al examined tissue sections from tonsils excised during the acute phase of EBV-harbouring T cells were consistently identified in 9/9 cases using in-situ hybridisation for EBERs combined with antibodies directed against CD45RO and, more specifically, the TCR -chain. The number of infected T cells was stated to be considerably lower than EBER+ B lymphocytes within the same tonsil, though this observation was not formally quantified. The EBER+ T cells were located predominantly within the inter-follicular areas and morphological differences were evident within and between tonsils;
some T cells exhibited blastoid features whilst many appeared as small/medium- sizedlymphocytes. Similar findings of appreciable T cell infection in the context of primary infection were reported by a Japanese group who examined the lymph nodes of IM patients.
By contrast, other studies of tonsils and lymph nodes from patients with IM - applying either CD3 or CD4/CD8 antibodies - could not identify any EBER+T cells86. In keeping with the latter data, a further study of 14 IM tonsils found no evidence of T cell infection, although it should be noted that 30-50% of EBER+ tonsillar lymphocytes in this study could not be clearly assigned a B or T lineage, using CD20, CD79a, CD3 and CD45RO as markers.
In the case of EBV persistence, studies of primary and secondary lymphoid tissue have suggested rare instances of EBER+ T cells in vivo, more often (at least in tonsillar tissue) of CD4+ phenoype. Similarly, a study of reactive lymph node tissue from Peruvian patients, a region where the incidence of EBV-associated extra-nodal NK/T lymphoma
(ENKTL) is thought to be relatively high87, found in a minority of lymph nodes ascertained to contain EBER+ lymphocytes, that as many as 24% of EBER+ cells per section expressed CD3. This contrasted with a separate investigation of intramucosal lymphocytes in inflamed tissues where no definite evidence of T cell infection was seen. Noting the characteristic anatomical presentation of ENKTL in the nasopharynx, small studies from China examined both normal nasopharyngeal tissue (obtained post-mortem) and excised nasal polyps88. Very rare intraepithelial lymphocytes co-expressing EBER+CD3+ and LMP1+CD3+ were identified in a minority of cases and at a lower frequency than their EBER+B cell counterparts.
NK cells
EBV infection of NK cells in vivo has also been investigated using an IHC/ISH approach in IM and reactive tonsillar tissue. Trempatet al used an antibody directed to an NK-restricted epitopePEN589 to identify EBER+ NK cells in the context of primary infection. Of the 6 cases of IM lymph nodes and tonsils examined, smallEBER+PEN5+ cells were appreciable, albeit very infrequent; corresponding to 10-2 to 10-3 of all EBER+
lymphocytes. The vast majority of PEN5+ cells however were EBER negative, and none expressed EBNA2. Applying similar methodology, Hudnallet al undertook a comprehensive, semi-quantitative analysis of 20 EBV+ tonsils obtained from donors undergoing routine tonsillectomy. The majority (82%) of >1000 EBER+ cells weakly expressed CD20, while
Thus, within the limitations of dual IHC/ISH labelling on fixed tissue sections, the occurrence of EBV-infected T and NK cells in vivo, during primary infection and persistence, appears to be a relatively rare event.
EBV infection in vitro B cells
The basic understanding of B lymphomagenesis came from critical discovery of the ability of EBV to infect and transform Human B cells invitro.90B cells that were take from Peripheral blood, tonsillar or cord blood were readily infected. The tropism of EBV for B lymphocytes is achieved through the involvement of gp350, the major viral envelope glycoprotein with the C3d complement receptor , 145-kda transmembrane ,Cd21on the b cell surface91.Then, another envelope glycoprotein gp42 bind with cell surface HLA class II molecules, starting virus–cell fusion - a process that requires gp85/gp25 & the gp110 glycoprotein - and allow the viral entry. Cd21 expression during the development of b lymphocyte correlates with the effectiveness of viral binding and the substantial reduction in ebv infection can be achieved by blocking antibodies to Cd21, purified cd21 or by saturation of receptors of B lymphocyte with gp350/22093.eventhough gp350 stands for EBV`s primary mechanism for binding with B cell, a gp350 knock out (KO) recombinant EBV also has the ability to infect and transform those B cells, but with reduced efficiency. Inspite of efficient binding in vitro, only 10–15 % of genomes of bound virus make way to reach the cell and depending on the applied quantity of infection , 10-90% of B cells will attain a latently infected state. Though the exact mechanisms of EBV capsid dissolution and transit of its genome to the nucleus have not been studied completely, it is thought to be like other DNA viruses that the transport of EBV capsid to the pores of nucleus is mediated by the cytoskeleton94.Sequentially the linear viral DNA closes covalently to form a circular EBV genomes observed in cells infected with the virus. The subsequent growth-transforming effect
of EBV is mediated by the co-ordinated expression of nine latent proteins mediate the subsequent effect transformation of growth by EBV which finally result in the establishment of a persistently-lived lymphoblastoid cell line (LCL).
Epithelial cells
Cd21 is not expressed on epithelial cells and hence its infection by cell-free virus is least understood, but is believed to involve gp85/gp25 that behaves both as a ligand for binding (to an unidentified receptor) and in co ordination with gp110, as a fusion complex95. The role of gp42 in the infection of epithelial cell is not known certainly. virions produced in HLA class II-posi B cells that are HLA-II positive produce virions which has low gp42, due to sequestration into endosomes by nascent HLA class II molecules, and thus limit gp42 representation in virus particles. So, preferentially epithelium is targeted by gp42-low viruses that are produced in b cells, whereas efficient B cell infection is carried out by gp42-high viruses that are produced in epithelial cells and are well equipped to do so. Very low rates of infection of squamous epithelial cells occur on exposure of them to cell free virus in. When monolayers of epithelium were co-cultured with B cell lines that were EBV positive and with lytic cells, infection is augmented a little. The virus which is bound to the surface of primary B cells that has been exposed recently is extremely infectious for epithelium, elevating the efficacy of infection by 103-104times when compared to that of cell-free virus preparations.
T cells
After the discovery of B cells expressing a receptor for binding with , but before the molecular characterisation of Cd21 (Cr2) it was found that such receptor of virus was also present on a T cell leukaemia line, ‘Molt 4’ that was formed from the peripheral blood of a patient with T cell acute lymphoblastic leukaemia. Many attempts were made to infect MOLT 4 by which binding of EBV to the cell surface was proved but never the viral entry96.
Subsequently, Cd21 was expressed in variable amounts on a proportion of T cell lymphoma/leukaemia cell lines; Cd21 was negative in HSB-2, HUT-78, CEM and T-ALL-1.
It is worthy to note that rather than mature T cell tumors, Cd21-positive T cell lines were positive from lymphoblastic leukaemias97.
Significantly, consistently but variably Cd21 was found to get expressed on a proportion (8-18%) of primary human thymocytes, obtained from paediatric patients undergoing elective cardiac surgery. An inverse correlation between intensity of Cd21 and expression of Cd3 was also obvious in these primary t cells – proved that those T lineage cells were immature relatively. Still more convincing to know, infection of primary thymocytes in vitro when co-cultured with EBV was seen, with EBNA1 expression positively seen by immunoblotting after 12 days – though the efficacy of cd21+ t cells infection not clear. EBV infection augmented the Proliferation of thymocyctes and with IL2 treatment, a synergistic effect too was apparent. All these data, challenged the belief that restriction of tropism of EBV to B cells and epithelial cells and a restricted pattern of expression of viral genes was suggested. Further data about the pattern of expression of EBV gene in infected T cells invitro is limited and some suggested that EBV latency II type expression98
While the Cd21/CR2 expression on mature peripheral blood T lymphocytes is studied, conflicting reports are seen. Low intensity of expression of CR2 is seen in 30-40% of both
CD4+ and CD8+ T cells was reported by Fischer et al using antibodies to CR2.In another study a small proportion of T cells was found to get infected99.There was a consistent low level interaction that explained CR2-independent mechanism of infection whilst the epithelial cell infection
Intoto, the in vitro data consistently say that during development in the thymus, T cells transiently express Cd21 which is sufficient for binding of EBV and in some situations, viral entry is also permitted
NK cells
Invitro infection of such NK cells have not been reproduced by others, but if this infection causing efficiency is feasible, there exist the opportunities – at least in 48-72 hours – to examine the consequences carefully upon cell phenotype and viral and cellular transcription100.
Replication
Two types of lifestyles in replication are chosen by EBV
Latent replication
Lytic replication
EBV genomic DNA in latent state exist as circular closed plasmid and seems to behave like host chromosomal DNA .Recently it has been demonstrated the OriP-containing plasmids replication is dependent on : ORC2 & Cdt1 which are the chromosomal initiation factors.
The amplification is 100-1000 fold by the EBV genome in the viral productive cycle, by the machinery of Viral replication. Productive replication of DNA occurs at various sites of the nucleus that are called as replication compartments and the cell cycle progression is arrested by the lytic programme and the cellular environment is modified tremendously101
EBV is known to cause various diseases ranging from asymptomatic infections to malignancies like nasopharyngeal carcinoma, lymphoma etc.
Infectious mononucleosis:
Transmission of EBV in IM:
Hoagland`s clinical observation102 documented that primary infection among young adults and adolescents was through deep kissing and confirmed it by a prospective study among undergraduate students103.Though sexual transmission was also thought about, a study at University of Minnesota104 found that the risk was similar among the subjects who acquired primary infection due to kissing with or without penetrative sexual intercourse and the subjects who acquired primary infection who had no history of kiss or sex through out the under graduation.
Unusually it can be transmitted by transfusion of blood105,transplantation of solid organs106, transplantation of Hematopoietic stem cells107. But mode of infection of preadolescent children is known but they might have got the infection from their parents and siblings who shed virus in their oral secretions109. The incubation period of infectious mononucleosis had been found to be 32 to 49 days102.
The Infectious mononucleosis is clinically characterized by fever, fatigue, cervical lymphadenopathy and Pharyngitis108(Henry H Balfour Jr, Samantha K Dunmire and Kristin AHogquist) observed usually in late adolescents and young adults in developed countries and the mechanism is not clearly understood which may be due to:
1. In preadolescents, there is a lack of recognition of the syndrome ,which is not rare and
Recently it has been found by Azzi et al.111 that levels of CD56dim NKG2A+ killer-cell immunoglobulin-like receptors (KIR)− NK cells is significantly high in children`s peripheral blood than adolescents or adults which implicate that certain classes of NK cells as associated factors in the earlier control of EBV.
H jalgrim H et al.112 stated that IM in developed countries is more common than in developing countries as the primary EBV infection is acquired late in developed countries.
Clinically it is presented in two ways103:
Sudden onset of sore throat and swollen neck i.e, cervical lymphadenopathy that lasts for minimum 3 weeks
Gradual development of myalgia,malaise and fatigue
In minority of cases abdominal pain, splenomegaly,hepatomegaly nausea, vomiting, periorbital, eyelid edema and palatal petechiae are seen. Rash is observed only in patients who were given Penicillin derivatives.
Complications occur rarely in acute phase of primary infection but Airway obstruction due to Oropharyngeal inflammation, Streptococcal pharyngitis, Meningoencephaitis, hemolytic anemia and thrombocytopenia occur in 1% of patients.113.
Immune response:
The replication of virus is first detected in the oral cavity during the incubation period.103 where tonsilar epithelial cells and B cells are infected114. Before 2 weeks of onset of symptoms, EBV genome can be observed in blood. At the same time, gene expression profiling revealed a systemic type I interferon response in few individuals who subsequently present with infectious Mononucleosis115.
Acute illness is characterized by high viral loads in blood & oral cavity and IgM against EBV VCA, atypical expansion of CD8+ T cells. significance of this increase in CD8+
cells is explained by fatal outcome of the disease in people with T cell
dysfunction116.According to literatures there is no substantial increase in the CD4+ T cells, data supports the concept that CD4+ T cells are very important in controlling EBV infection and are known to appreciate lytic antigens through MHC II tetramers both in acute infection and lifelong maintained117.
IgG antibodies that are produced against EBV VCA is detectable only after first week of illness but persist for life. Immune response to p18 component of VCA is developed first followed by p23 component.
Immediate early, early and late early are the EBV antigens belonging to temporal classes of lytic gene products. Antibodies against immediate EA BZLF1 are found to appear quickly and remain. Antibodies to EAs p138,p54 are not constant. They appear early and disappear after convalescence but antibodies directed against EBNA1 develop usually after 3 months of onset of illness118but persist for life. when there is a delay in EBNA1 antibody response, a delayed response of CD4+ T cell to EBNA-1 is correlated.117
NK cells are gaining more importance than CD8+ T cells during IM because severe EBV related outcomes are reported in immunodeficiencies including T cell and NK cells and their cytolysis pathways.119 In control of early infection in oropharynx NK cells play a prominent role than in peripheral blood.
NK cells control EBV infection in B cells in two ways122
Direct cytolysis of infected cells
