DNA virus and oncolgoy

Mini-review

DNA viruses in human cancer:An integrated overview on fundamental mechanisms of viral carcinogenesis

Deilson Elgui de Oliveira

*

Department of Pathology,Botucatu School of Medicine,State University of Sao Paulo (UNESP),Brazil

Received 5January 2006;received in revised form 17May 2006;accepted 18May 2006

Abstract

The ?rst experimental data suggesting that neoplasm development in animals might be in?uenced by infectious agents were published in the early 1900s.However,conclusive evidence that DNA viruses play a role in the pathogenesis of some human cancers only emerged in the 1950s,when Epstein–Barr virus (EBV)was discovered within Burkitt lymphoma cells.Besides EBV,other DNA viruses consistently associated with human cancers are the hepatitis B virus (HBV),human pap-illomavirus (HPV),and Kaposi sarcoma herpesvirus (KSHV).Although each virus has unique features,it is becoming clearer that all these oncogenic agents target multiple cellular pathways to support malignant transformation and tumor development.

ó2006Elsevier Ireland Ltd.All rights reserved.

Keywords:Viral carcinogenesis;DNA viruses;Cancer;Epstein–Barr virus;Kaposi’s sarcoma-associated virus;Human papillomavirus;Simian Virus 40virus;Hepatitis B virus

1.Introduction

In 1908,Vilhelm Ellermann and Oluf Bang,two Danish pathologists doing research on leukemia in chickens,provided important evidence that malig-nant tumors in animals could be caused by the so-called ‘‘?lterable agents’’[1].Three years later,Peyton Rous reported that another avian malignan-cy,a sarcoma developing in Plymouth Rock fowls,could be readily reproduced by inoculating cell-free tumor extracts in healthy animals [2].

The ‘‘?lterable agents’’are now recognized as retroviruses,and many others associated with

tumors in a variety of animals were discovered in the last century.Because human tumors were not believed to be contagious,the ?rst studies on viral carcinogenesis were received with skepti-cism.Additionally,the putative infectious neo-plasms of chickens were not considered to be valid models for analogous human diseases.More-over,in 1907Ciu?o reported that benign tumors could be,in fact,transmissible among humans [3].The debate about the association of infectious agents with human tumors remained in a ‘‘latent state’’until 1964,when the Epstein–Barr virus (EBV)was discovered within malignant cells from Burkitt lymphoma (BL)[4].

In 1990,it was estimated that 15.6%of cancer inci-dence (about 1,450,000cases)would be associated

0304-3835/$-see front matter ó2006Elsevier Ireland Ltd.All rights reserved.doi:10.1016/j.canlet.2006.05.010

*

Fax:+551438152348.

E-mail address:elgui@https://www.doczj.com/doc/344738742.html,

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Cancer Letters 247(2007)

182–196

https://www.doczj.com/doc/344738742.html,/locate/canlet

with microbial and parasitic agents[5].Developing countries have higher incidences of virus-induced cancers,which can actually represent a remarkable public health issue.Besides its epidemiological rele-vance,as elegantly stated previously,‘‘viral carcino-genesis provided the breakthroughs that crystallized current concepts of cancer development,and revealed mechanisms for the orchestration of normal cell growth control’’[6].

Here,we aim to review the state-of-art of DNA virus-induced carcinogenesis,focusing on major concerns addressed in the literature.The role of RNA viruses in human carcinogenesis has been dis-cussed elsewhere[6].

2.Cancer in brief

The term cancer encompasses a great variety of entities,many with unique etiology,pathogenesis, natural history,treatment response,and cure rates. According to Hanahan and Weinberg,most cancers are seldom self-su?cient in growth stimuli;they become insensitive to anti-growth signals;they are capable of causing tissue invasion(and few will be able to cause metastasis as well);their proliferative potential is unrestricted,they cause sustained angio-genesis locally,and their cells are resistant to apop-tosis.Noteworthy,these‘‘hallmarks of cancers’’are present in variable degrees in di?erent diseases con-sidered,among di?erent cases of the same cancer, and even in a given malignant tumor after time[7].

When transformed cells develop into malignant tumors,they have already had acquired some of the above-mentioned hallmarks,which will be strengthened during tumor progression.Besides, new features will be acquired,and malignant tumors will become more heterogeneous and aggressive as a consequence.Interestingly,some of the above fea-tures are also required for e?cient viral replication, and few viruses become very skilled in providing them to infected cells.As these features impair cell stability and tissue homeostasis,infection by onco-genic viruses o?ers a prone-to-transformation sce-nario,in which the development of a malignant tumor is likely to happen or,at least,it is boosted.

3.DNA viruses in human cancer

To date,few DNA viruses are consistently asso-ciated with human neoplasms,notably the hepatitis B virus(HBV),human papillomavirus(HPV),EBV, and Kaposi sarcoma herpesvirus(KSHV).General information about these pathogens is provided in Table1.

Chronic infection with HBV is an important risk factor for development of hepatocellular carcinoma, a malignant tumor frequently observed in some countries of Asia and Africa[16].On the other hand,speci?c HPV genotypes are responsible for benign neoplasms and some carcinomas,including cancer of uterus[17],some penile and upper aerodi-gestive tract carcinomas[18,19].Major achieve-ments in the prevention of virus-induced cancers may be attributable to strategies to control infection in human populations.For instance,HBV vaccina-tion has dramatically decreased the number of hepa-tocellular carcinomas,and it is expected that a new HPV vaccine will have a profound impact on the prevention of cervical cancer[20],and perhaps a sig-ni?cant decrease in other HPV-related neoplasms.

EBV and KSHV are members of the Herpesviridae family of viruses,which encompass many of the most complex animal viruses described so far.Both can establish long-term viral infections in their

Table1

DNA viruses with well-documented association with human cancers

Virus Viral taxonomy Discovery Genome Associated human cancers Review Hepatitis B virus Hepadnaviridae1967[8,9]dsDNA(partial)

3.2kb,4genes

Hepatocellular carcinoma[10]

Human papillomavirus Papillomaviridae1983[11]DsDNA8kb Squamous cell carcinomas in anogenital

and head and neck sites

[12]

Epstein–Barr virus/ human herpesvirus4Herpesviridae

(gammaherpesvirus;

lymphocryptovirus)

1964[4]DsDNA172kb,

90genes

Burkitt lymphoma,undi?erentiated

nasopharyngeal carcinoma,Hodgkin

lymphoma,non-Hodgkin lymphoma in

immunosuppressed patients

[13]

Kaposi sarcoma-associated herpesvirus/human herpesvirus8Herpesviridae

(gammaherpesvirus;

radnovirus)

1994[14]DsDNA165kb,

90genes

Kaposi sarcoma,primary e?usion

lymphoma,

[15]

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target cells.EBV is the most important etiological factor in classic BL,and it is also detected in undif-ferentiated nasopharyngeal carcinomas(NPC)[21], in a subset of Hodgkin lymphomas(HL),and in some cases of non-Hodgkin lymphomas,notably in immunosuppressed patients[22].Few other puta-tive associations between EBV infection and human cancers are currently under evaluation[23].On the other side,in the last10years much e?ort has been devoted to the study of KSHV,the last human DNA tumor virus identi?ed so far.KSHV is now consistently detected in all forms of Kaposi sarcoma (KS),in HIV-positive primary e?usion lymphomas (PEL),and in Multicentric Castleman disease (MCD)[24].

Despite the lack of conclusive data about other DNA tumor viruses in the development of human cancers,studies on adenoviruses and two small DNA viruses,the simian virus40(SV40)and poly-omaviruses,have been providing fundamental information to clarify the molecular mechanisms of virus-induced cell transformation.Adenoviruses are widespread in the human population,and the infection is often asymptomatic.However,it may cause respiratory illnesses in children,conjunctivitis, and gastroenteritis.More than50serotypes of human adenoviruses have been described,but none is consistently associated with cancer in humans. However,few human adenoviruses do cause malig-nant tumors in rodents(e.g.,Ad12,Ad18,and Ad31),and their oncogenic properties rely on two main viral genes:E1A,which disrupts the function of important tumor suppressing proteins,and E1B,which inhibits apoptosis in many di?erent ways[25].

SV40and the polyomavirus,on the other hand, usually infect monkeys and mice,respectively. Because cells from SV40or polyomaviruses natural hosts are very permissive to the lytic viral cycle,they are not transformed during infection.However,in non-permissive or semi-permissive animal cells,the expression of viral products–namely the large T antigen of SV40and the middle T antigen of poly-omaviruses–can be su?cient to produce malignant cells.In fact,SV40may transform human?bro-blasts in vitro,and it was demonstrated that this virus also transforms human mesothelial cells[26]. The association between SV40infection and the development of pleural mesotheliomas is currently under debate due to con?icting data in the literature [27–29].Noteworthy,important epidemiological evidence that the virus at least act as a co-factor in the development of asbestos-associated human mesotheliomas has recently emerged[30].

It is not surprising that the?rst successful attempt to convert human normal cells into pheno-typically malignant ones with a minimum set of genetic elements employed components of a DNA virus(namely,SV40-encoded proteins),in addition to the H-RAS oncogene and the hTERT gene, which encodes the catalytic subunit of human telo-merase[31].As will be discussed in the following sections,DNA tumor viruses developed many dif-ferent strategies to control key cellular processes, including cell proliferation,di?erentiation,cell sig-naling,senescence,and apoptosis(Fig.1).

4.Viral infection and genetic instability

Genetic instability is one of the foremost charac-teristics shared by cancers,observed either at the chromosomal or gene levels in malignant cells. Chromosomal imbalances,namely aneuploidy,have long been detected in malignant cells using conven-tional microscopy and karyotyping.On the other hand,a lesson has been taken from studies on inher-ited cancer syndromes in which dysfunction of DNA-repair genes causes genome-wide mutational events.Whether gene mutations or aneuploidy is the leading phenomenon in carcinogenesis remains an unsolvable dilemma,since both play a variable role in the development of di?erent cancers.

Lately,aneuploidy has caught more attention due to new data suggesting that it could be one of the earliest events in the development of some inher-ited as well as sporadic cancers.Aneuploidy itself was proposed as the leading gear in the genetic instability engine[32].In this regard,disruption of the mitotic spindle apparatus may be responsible for the establishment of chromosomal imbalances, which allows further mutations in critical genes to happen.Noteworthy,chromosomal abnormalities are well-known consequences of telomere dysfunc-tion,and it seems that,at least in some cancers,neo-plastic cells experience a variable period of telomere shortening before they become immortal.This results in progressive genetic instability,which becomes more and more severe after cell transfor-mation.The genetic instability also plays a role in tumor progression,providing cell heterogeneity that allows the emergence of malignant clones that have acquired advantageous features,such as low nutri-ent demand,auto-su?ciency in growth signals,high proliferation rate,low immunogenicity,etc.

184 D.Elgui de Oliveira/Cancer Letters247(2007)182–196

HPV-infected cells from precancerous lesions of the cervix often show aberrant mitotic ?gures [33].The HPV E7oncoprotein from high-risk HPV genotypes contributes to the induction of genomic instability by inducing centrosome duplication errors [34].Interestingly,recurrent gains of 3q were observed in cervical and vaginal mucosa during the transition of high-risk HPV-associated severe dys-plasia to invasive carcinoma [35].HBV X protein is responsible for a similar mechanism of genetic instability:it causes the formation of abnormal cen-trioles,followed by multipolar mitosis and chromo-somal imbalances,which are assumed consequences of crm1sequestration,a nuclear export protein that keeps proper centrosome synthesis and segregation [36].Additionally,DNA tumor viruses cause genetic instability other than aneuploidy.For instance,HPV E6protein downregulates the enzyme O 6-methylguanine-DNA methyltransferase (MGMT),which participates in DNA repair and prevents mutation in many critical genes,including TP53[37].On the other hand,HBV X protein was found to interact with XAP-1/UVDDB,a putative DNA-repair protein that binds damaged DNA during nucleotide excision repair (NER)[38].In time,EBV LMP-1protein inhibits DNA repair in both p53-de?cient and p53-competent cells,and H1299p53-de?cient epithelial cells expressing LMP-1are more sensitive to UV radiation and bleomycin,two important DNA-damaging agents [39]

.

Fig.1.Overview of some critical cellular pathways and cellular proteins targeted by known human DNA oncoviruses.Multiple cellular processes are modi?ed by viral products before malignant transformation takes place.For instance,infected cells may become genetically instable over time because viral proteins interfere with DNA repair,cause unattended cell cycle progression or disrupt the mitotic spindle.Besides,imbalance towards antiapoptotic signals by oncoviruses makes infected cells resistant to death,and stabilization of telomeres provides them limitless replicative potential.Altogether,these phenomena favor the development and progression of virus-associated cancers.Refer to text for more details.

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5.Cell immortalization by DNA virus

Cell immortalization is another pivotal phenom-enon in carcinogenesis,also one of the major char-acteristics shared by malignant neoplasms.In order to produce malignant tumors,cells must have to bypass apoptosis de?agrated during cell crisis, the endpoint of cellular senescence,observed when a critical level of telomere shortening is reached. Moreover,major mechanisms that grant an increased lifespan to malignant cells are related to telomere maintenance and suppression of apoptosis, which will be discussed later.

Most human cancer cells have their telomeres lengthened by de novo expression of telomerase,an enzymatic complex formed by three major compo-nents:an RNA template of telomeric repeats (TTAGGG in vertebrates),the telomerase-associated protein(hTep-1),and a catalytic subunit that shows reverse transcriptional activity–the human telome-rase reverse transcriptase(hTert).Expression of TERT gene is rate limiting for telomerase activity, which is usually restricted to stem cells and germinal cells in non-neoplastic tissues.The RNA component is expressed constitutively and ubiquitously in nor-mal cells.Besides telomerase reactivation,telomeres may be subjected to the alternative lengthening of telomeres(ALT)pathway,a recombination-based mechanism for telomere lengthening.Although ALT activation may not share the same frequency of telomerase de novo expression during cancer development,ALT has gained considerable atten-tion due its anticipated role on cancer resistance to the new generation of drugs that target telomerase activity[40,41].

EBV has long been recognized for its ability to produce B-lymphoblastoid cell lines(BLCL)with increased lifespan in vitro.The virus uses the CD21transmembrane protein(C3d complement receptor)to infect B-lymphocytes,which allow both lytic and latent viral cycles.A subset of BLCL that has become immortalized by EBV in vi-tro is named post-immortal BLCL.EBV-infected BLCL generated from patients with accelerated ageing due to Werner’s Syndrome(WS)display impaired telomeres dynamics and fail to become immortalized[42].On the other hand,expression of WRN gene,which encodes the Wrn DNA heli-case(which is non-functional in WS),is often veri?ed in post-immortal EBV-infected BLCL. Therefore,active Wrn DNA helicase,which seems to work along telomerase for telomere lengthening,seems to be required for immortalization of EBV-infected BLCL[43].

It was proposed that EBV plays a role in the pathogenesis of classical BL because it causes intense proliferation of infected B cells,which remains unnoticed due to impaired immune surveil-lance.Immunological dysfunction has been attributed to Plasmodium infection in BL cases arising in the so-called Malaria belt area in Equatorial Africa. This scenario is favorable to accumulation of genetic abnormalities,including the t(8;14)translo-cations,which are frequently found in BL[44,45]. They cause juxtaposition of c-myc proto-oncogene (located on8q24)to one of the immunoglobulin heavy chain genes loci at14q.Consequently, EBV-infected B cells disclose upregulation of c-myc oncoprotein,and auto-sustained cell-prolifer-ation signals.Interestingly,it was previously reported that c-myc cooperates with the ubiquitous transcrip-tion factor Sp1to enhance TERT expression[46]. Indeed,telomerase activity is increased in nasopha-ryngeal carcinomas(NPC),and it can be achieved in vitro in NPC cells via c-myc transactivation by EBV latent membrane protein1(LMP-1)[47]. Additionally,increased telomerase activity in NPC cell lines may be due to LMP-1-mediated nuclear translocation of the reverse transcriptase subunit of human telomerase bound to NF-j B [48].

KSHV,on the other hand,transforms primary human endothelial cells in vitro,and only KSHV-infected cell cultures express telomerase,as accessed by the telomeric-repeat ampli?cation protocol (TRAP)[49].The KSHV latency-associated nuclear antigen1(LANA-1)binds Sp1to form a complex with an increased transcriptional activity in vivo. The LANA-1/Sp1complex transactivates the TERT promoter,thus contributing to telomere elongation and consequent cell immortalization due to increased telomerase expression in KSHV-infected cells[50].

In the context of HPV-associated malignancies, Zhang et al.evaluated the size of telomeres in cells from cervical pre-neoplastic lesions from16patients with high-risk HPV infection in a sequential fash-ion.Telomere shortening was predominantly found in early cervical intraepithelial neoplasia(CIN), whereas TERT expression was more evident in high grade CIN and invasive cervical carcinomas.These observations strongly suggest that cell immortaliza-tion and the acquisition of a malignant phenotype are closely associated phenomena,and indicate that

186 D.Elgui de Oliveira/Cancer Letters247(2007)182–196

the progressive genetic instability acquired during the telomere erosion phase in precancerous lesions may enhance the transformation of high-risk HPV-infected cervical cells[51].

In the last decade,it was demonstrated that the HPV-16E6protein induces TERT expression in human foreskin keratinocytes(HFK)[52].This abil-ity was originally described as independent of E6-mediated p53degradation.However,when HPV E6and E7oncoproteins are co-expressed in HFK cells,although they do show telomere losses,they still bypass crisis and become immortal.In a recent paper,McMurray and McCance[53]demonstrated that,regardless of telomerase activity,degradation of p53by HPV E6protein is a key event for immor-talization of keratinocytes in the presence of HPV E7.The combination of p53dominant negative mutant with HPV E7alone successfully produced immortalized cells,stressing the role of p53dysfunc-tion in this phenomenon[53].

Although HPV-16-infected keratinocytes had their lifespan extended by E6alone,pRb/p16INK4a inactivation is required in addition to TERT expres-sion for cells to become immortal[54].Interestingly, it seems that even such immortalized keratinocytes retain growth and di?erentiation control[55]. Therefore,although virus-induced cell immortaliza-tion provides the necessary progressive genetic instability that drives the acquisition of a malignant phenotype,additional oncogenic events are often needed(e.g.,activation of protoncogenes and inac-tivation of other tumor suppressor genes driven by the progressive genetic instability),in order for immortalized cells to transform.

6.DNA virus-induced malignant transformation

The expression of many proteins encoded by tumor suppressor genes is known to be critical for the maintenance of genome?delity and regulation of cell signaling and proliferation.When critical genes are disrupted,or became abnormally activated (e.g.,tumor suppressor genes and protoncogenes, respectively),the defective cells may evade biologi-cal security systems that avoid the emergence of transformed clones.Although di?cult to be identi?ed in vivo,transformed cells are characterized in vitro by low nutrient demand,morphological changes,high and unrestricted proliferation rate, anchorage-independent growth,and eventually tumorigenesis and metastasis capabilities,which can be assessed in animal models.

It seems reasonable that,during their evolutionary interaction with host cells,DNA tumor viruses whose products could target some key cellular regulatory proteins achieved an important adaptative advantage over those that could not.In this regard,many di?er-ent strategies for inhibition of p53tumor suppressor protein have been identi?ed in DNA tumor viruses so far.Noteworthy,p53was discovered in co-precip-itation experiments with the SV40large T antigen [56].Inactivation of p53-dependent pathways in virus-infected cells may have many consequences, notably unchecked cell cycle progression,progressive genetic instability and resistance to apoptosis.

E6proteins from high-risk HPV genotypes(e.g., HPV-16and HPV-18)cause proteasomal degrada-tion of p53,but E6proteins from low-risk geno-types(e.g.,HPV-6and HPV-11)fail to do it e?ciently.High-risk HPV E6proteins form a com-plex with the E6-AP protein,a member of the E3 family of ubiquitin ligases that only binds p53when it is associated with HPV E6.The E6/E6-AP com-plex drives p53to ubiquitin-mediated degradation, thus decreasing functional levels of p53in the infected cell[57].Additional cell transformation properties of high-risk HPV E6proteins rely on their ability to target host PDZ domain-containing proteins for degradation[58].On the other hand, the retinoblastoma gene(RB)product(pRb), another important tumor suppressor protein,is also targeted by HPV E7proteins.The HPV E7protein disrupts pRb control over E2F proteins,an impor-tant family of transcription factors,allowing the activation of cyclin–CDK complexes,which ulti-mately causes unrestricted cell progression through-out G1to S phases of the cell cycle[52].

The p53protein is also targeted by KSHV pro-teins LANA1and LANA2.The former is constitu-tively expressed in spindle-shaped cells of KS,as well as in latently infected endothelial cells and monocytes.The latter have a more stringent expres-sion pro?le,and it was described as a B-cell-speci?c latent viral protein[59].LANA-1represses p53 transcriptional activity,and inhibits the p53-depen-dent cell death in di?erent KSHV-infected cell types. However,this e?ect is caused neither by modi?ca-tions in p53DNA-binding properties nor by protein degradation[60].On the other hand,suppression of p53activity by LANA-2confers increased survival of B cells,and this mechanism has a suspected role in KSHV-induced lymphomagenesis[53].

Contrary to the above mentioned,EBV oncopro-teins do not seem to interact directly with tumor

D.Elgui de Oliveira/Cancer Letters247(2007)182–196187

suppressor proteins.In fact,EBV nuclear antigen2 (EBNA-2),which is critical for EBV-induced immortalization of B-lymphocytes,upregulates wild-type(wt)p53through the activation of NF-j B transcription factor,and this e?ect can be achieved by EBNA-2alone or in combination with LMP-1[61].Such upregulation of wt p53may have di?erent consequences in vivo,since it was demon-strated that its levels in EBV-infected BL cells deter-mine whether they would be impelled to unrestricted proliferation,cell cycle arrest or apoptosis[62].

In addition to their unique viral oncoproteins,a set of KSHV and EBV products shares variable structural and functional similarities with human cell proteins.Interestingly,herpesviral genes that encode cellular homologues generally do not con-tain intronic sequences,which suggests that they were potentially acquired from host cells with the participation of mRNA intermediates,and perhaps retroviral cooperation[63].Another possibility is that some viral products acquired a variable,usually low,degree of similarity with human proteins by means of functional and structural convergence dur-ing evolution.From our knowledge,this hypothesis has not been properly addressed yet.

In the past years,the viral mimicry of key cellular proteins(usually referred to as‘‘molecular piracy’’) provided many valuable insights into the mecha-nisms of viral carcinogenesis.For instance,it was demonstrated that the sole constitutive expression of the KSHV G protein-coupled receptor gene (v-GPCR)in an engineered transgenic mouse model (but none of other KSHV genes that encode onco-proteins,including v-cyclin,v-?ip,and kaposin) promotes the formation of vascular tumors in vivo which resemble KS lesions in humans[64].Interest-ingly,v-GPCR was recognized as a lytic cycle gene, raising new questions about the importance of de novo expression of lytic genes in virus-induced carci-nogenesis,as well as the putative role of these genes in early phases of cancer development.It was later reported that the v-GPCR-mediated sarcomagenesis could be dramatically a?ected by drugs which inhibit the Akt-signaling pathway,prompting new promising strategies for treatment of KS[65].The role of viral homologues of cellular proteins will be discussed further in the following sections.

In time,it has been recently reported that the EBV and KSHV genomes encode micro RNAs (miRNAs)that may have important cellular genes as targets for silencing during latent infection[66, 67].These very promising?ndings open a new mechanistic perspective of viral participation in malignant transformation,since the downregulation of critical regulatory proteins in infected cells might, in part,be explained by viral miRNAs activity.

7.Viral control of apoptosis

Apoptosis is crucial for tissue homeostasis.Cell number in tissues is primarily dependent on cell pro-liferation and cell death by apoptosis.Both phenom-ena are usually dysfunctional in malignant tumors.

The two main mechanisms of apoptosis converge at the level of activation of e?ector caspases.The ?rst mechanism that causes caspase activation is triggered when apoptotic ligands(e.g.,Fas ligand and TNFs)bind to their receptors on the cell surface (CD95and TNFR,respectively).The death domains in the intracytoplasmatic portion of these receptors activate caspase8and initiate the caspase cascade.Moreover,this pathway is triggered by extrinsic signals.On the other hand,the second pathway is triggered by intrinsic signals,including telomere overshortening,DNA damage,disruption of mitochondrial membrane,etc.Ultimately,they cause cytochrome c release from mitochondria and the formation of the apoptosome complex,which proteolytically activates downstream caspases. Caspases are responsible for most of the phenomena observed in the apoptotic cell,including chromatin cleavage,breakdown of structural nuclear and cyto-skeleton proteins,membrane changes,cell shrinkage and cell fragmentation into apoptotic bodies,etc. Activation and function of these cysteine-rich enzymes are tightly regulated by di?erent mecha-nisms,including post-translational modi?cations and direct inhibition by a family of inhibitory apop-totic proteins(IAPs).An alternative apoptotic cas-pase-independent pathway is described,and it is based on the release of apoptosis-inducing factor (AIF),a mitochondrial oxyreductase that causes DNA cleavage and chromatin condensation when it translocates to the cell nucleus[68].

Although EBV-infected BCBL are sensitive to apoptosis due to the scarcity of serum and growth factors in culture,actually they seem to be more resistant than antigen-stimulated primary B-lym-phocytes.In EBV latently infected cells,LMP-1is an important factor that confers resistance to apop-tosis.It has recently been reported that LMP-1 inhibits apoptosis of infected B-lymphocytes partly due to the inhibition of antiapoptotic BAX gene [69].EBV LMP-1mimics a constitutively activated

188 D.Elgui de Oliveira/Cancer Letters247(2007)182–196

CD40/TNF-receptor,turning on TRAFs/NF-j B, MAPK and JAK/STAT signal transduction cas-cades[70].LMP-1-expressing human vascular endo-thelial cells(HUVEC)show resistance to apoptosis, which was associated with the inhibition of caspase-3expression and upregulation of antiapoptotic genes,including BIRC3(encodes c-iap-2),and TRAF1[71].Interestingly,a homologue of the anti-apoptotic protein bcl-2is encoded in the EBV BHRF-1gene,which is usually expressed during the lytic cycle[72].

Similarly,KSHV ORF-16encodes a viral bcl-2 homologue,which is also predominantly expressed during lytic infection.To date,there are insu?cient data that explicitly implicate EBV BHRF-1or KSHV ORF16proteins in the pathogenesis of human cancers induced by EBV and KSHV,respec-tively.Actually,BHRF-1does not seem to be required for EBV-induced B-lymphocyte transfor-mation in vitro[73],and the protein has not been detected in the majority of cases of EBV-positive nasopharyngeal carcinomas and post-transplant lymphoproliferative disorders(PTLD)[74]. Although Liu et al.[75]reported BHRF-1tran-scripts in most of the44cases of NPC(and none of25non-NPC tissues)evaluated,the protein was consistently found by immunohistochemistry in only4/15cases of metastatic NPC.

Nevertheless,a putative participation of EBV BHRF-1and KSHV ORF16products in viral car-cinogenesis cannot be ruled out completely.In a murine model,it was demonstrated that bcl-2 homologues from gammaherpesviruses are impor-tant for persistent replication and chronic disease in immunocompromised hosts,and for e?cient ex vivo viral reactivation from latency[76].Both EBV and KSHV bcl-2homologues are more likely to avoid premature apoptosis of infected cells dur-ing the intense viral replication and virion assem-bling that occurs during productive infection. Therefore,these proteins might be important in the very early steps of EBV and KSHV-induced car-cinogenesis.In time,as EBV and KSHV non-con-ventional latency programs do exist,it might be possible that the re-expression of these antiapopto-tic proteins grants additional survival advantage to latently infected malignant cells in vivo[77].Note-worthy,recent data further support the participa-tion of known lytic cycle viral oncoproteins in the pathogenesis of human neoplasia[78].

Regarding the modulation of apoptosis by onco-genic viruses,more data are available on the role of v-FLIP and LANA-1KSHV proteins,which are encoded by viral ORF K13and ORF73,respectively. Both are constitutively expressed in KSHV latently infected cells.KSHV v-FLIP binds Fas-associated death domain proteins and caspase8(formerly FLICE),and it may further inhibit apoptosis via activation of the NF-j B pathway[79,80].On the other hand,as stated before,it was demonstrated that LANA1-expressing cells are resistant to apop-tosis by mechanisms related to the suppression of p53transcriptional activity[64].

The net e?ect of viral infection modulating apop-tosis can be particularly important in a therapeutic perspective.Both untreated and etoposide-treated BJA-B cells(a B-cell lymphoma lineage)are signi?-cantly more resistant to apoptosis when infected by EBV,and this e?ect seems to be due to virus-in-duced upregulation of endogenous bcl-2and low activation of caspases[81].

8.Viral modulation of cell microenvironment

The modi?cation of cell microenvironment in human malignancies associated with DNA viruses may be exempli?ed by HL,KS,and PEL.In these diseases,cross-talking of malignant and non-malig-nant cells is clearly a pivotal element for tumor growth.Herpesviruses infection in HL,KS and PEL causes auto-sustained production and release of cytokines and growth factors within the tumor, both by infected and non-infected,neoplastic and non-neoplastic cells.

Classical HL has peculiar histopathological fea-tures:tumors are formed by few scattered malignant Hodgkin/Reed–Sternberg(H-RS)cells surrounded by a heterogeneous non-neoplastic component of reactive in?ammatory cells.EBV is detected in H-RS cells in roughly half of the classical HL cases. In these cases,the virus modulates the features of H-RS cells and stimulates their growth and survival [82,83].Both EBV-negative and EBV-positive H-RS cells produce a di?erent repertoire of cytokines that recruit in?ammatory cells(e.g.,lymphocytes,neu-trophils,eosinophils,and macrophages),which in turn contribute to immune deregulation and tumor development[84].However,in EBV-associated HL, the cytokine imbalance is achieved more e?ciently, possibly because of increased activation of the NF-j B-signaling pathway in H-RS cells expressing LMP-1.This causes overproduction of Th2cyto-kines(including IL-13,IL-5,and IL-6)and chemokines(TARC)in a scenario of defective

D.Elgui de Oliveira/Cancer Letters247(2007)182–196189

cell-mediated(Th1)immune response,a putative consequence of the increased production of human interleukin10(hIL-10)by both H-RS cells and the surrounding tissue[85,86].

In KS,on the other hand,the proliferation of malignant spindle-shaped cells and atypical endo-thelial cells is achieved due to intense activity of angiogenic factors.These molecules are readily produced by KSHV-infected cells,as well as non-infected in?ammatory cells[87].KSHV is responsible for a paracrine mechanism that causes increased synthesis and release of VEGF and induces adjacent cells to proliferate beyond their natural lifespan[88].Furthermore,the expression of the B variant of kaposin in KSHV latently infected cells stabilizes some cytokine mRNAs, including GM-CSF and IL-6,and enhances the MAPK-signaling pathway via activation of MK2 kinase.This mechanism may increase cytokines production,causing a bivalent autocrine/paracrine ampli?cation loop that activates both KSHV-in-fected cells and adjacent non-infected cells[89]. Similarly,malignant cells in PEL depend on the microenvironment for e?cient growth,as previ-ously demonstrated in a mouse xenograft model [90].These data further stress the importance of cellular signaling in the pathogenesis of KSHV-as-sociated malignancies.

9.Viral subversion of immunologic surveillance

Many virus-associated malignancies arise in a scenario of mild to severe immunosuppression, either primary or acquired.Not surprisingly,some viruses have developed their own mechanisms for evasion of immunologic responses to achieve persis-tent viral infection and maximum viral burden for e?cient propagation.

It should be noted that the existence of a latency biological cycle is itself an important mechanism of immunologic evasion.During latency,the expres-sion of viral products is decreased to a minimum required for virus maintenance,avoiding the presen-tation of foreign antigens that could elicit an e?ec-tive immunologic response against the infected cells[91].EBV and KSHV deserve special attention in this regard,because herpesviruses e?ciently establish viral latent states,and they have achieved a repertoire of successful strategies for immunologic evasion.

One important mechanism for evading immuno-logic response relies on downregulation of major histocompatibility complex(MHC)molecules. KSHV proteins MIR1and MIR2(encoded by ORFs K3and K5,respectively)accelerate MHC class I molecules endocytosis and degradation, therefore making infected cells unattended by cytotoxic T cells[92].In addition,it was also reported that MIR proteins could regulate CD1d expression and function in KSHV-infected cells,reducing activa-tion of CD1d-restricted T lymphocytes[93].Anoth-er mechanism for KSHV evasion of host immune responses is based on viral products mimicking the functions of host immunoregulatory molecules.In this regard,KSHV encodes three secreted chemo-kines,vCCL1(ORF K6),vCCL2(ORF K4),and vCCL3(ORF K4.1),which–unlike their cellular homologues–display strong immunologic-inhib-iting properties[91].

The product of the EBV BCRF-1gene is a viral protein homologue to hIL-10,a potent immunosup-pressive cytokine[94,95].Similar to hIL-10,the EBV viral interleukin10(vIL-10)performs a diverse sort of activities that suppress cell-based immuno-logic response,which is the most active to overcome virus-infected cells and malignant tumors.EBV vIL-10downregulates class I and II MHC molecules,as well inhibits the expression of IL-12and co-stimula-tory molecules required for proper cytotoxic T-cell activation.However,since the BCRF-1gene is expressed in the late lytic cycle of the virus,a possi-ble role of vIL-10in immunologic evasion is consid-ered only in tumors in which a signi?cant number of neoplastic cells have become productively infected, or in those cases where the transcription of the BCRF-1gene has become aberrantly activated. Not only EBV encodes its own hIL-10homologue, patients with EBV-associated disorders show increased production of endogenous hIL-10,which may be partly due to transcriptional regulation exerted by some viral products,including LMP-1, EBERs and the viral transcription factor BZLF-1, as veri?ed in BL cell lines[96–98].

HPV E5protein impairs intracellular maturation of MHC class II dimers in keratinocytes in vitro. Consequently,cell surface expression of MHC class II is decreased,and E5-expressing keratinocytes cannot work properly as antigen-presenting cells [99].Additionally,as a di?erent approach for subversion of the immunologic response,most of the genes downregulated by HPV are associated with the IFN-signaling pathway,including the Stat1 [100].HPV E6and E7proteins also modulate the transactivation of interferons by binding interferon

190 D.Elgui de Oliveira/Cancer Letters247(2007)182–196

regulatory factors3and1(IRF-3and IRF-1), respectively[101,102].In fact,IRF-1acts as a tumor suppressor gene and cooperates with TP53 in response to DNA damage,stimulating the expression of the CDK inhibitor p21Waf1/Cip1, downregulating antiapoptotic genes and upregulat-ing proapoptotic ones.Remarkably,the expression of many interferon-induced genes plays a role in both anti-viral and anti-tumor immunity,stimulat-ing the expression of MHC class I and II molecules, shutting down the cell cycle machinery and synthe-sis of macromolecules,as well as de?agrating apop-tosis of infected and malignant cells.Thus,cells that constitutively express E6and E7HPV oncoproteins have gained an e?cient mechanism for immunolog-ic evasion due to dysfunction of the MHC system and IFN-signaling pathways[103].

10.The‘‘hit-and-run’’hypothesis in viral carcinogenesis

Evasion from inherited immunologic responses is more e?ective in early phases of viral infection and tumor development because of the emergence of speci?c response against viral and tumor-associated antigens.Thus,a situation might exist that,once the malignant tumor is established,malignant cells that abrogate the expression of viral products,and per-haps lose the viral genome,would be selected due to a decreased immunogenicity that provides them with an additional adaptative advantage for surviv-al and growth.This is the basis of the‘‘hit-and-run’’hypothesis of viral carcinogenesis,which was specu-lated as a possible explanation for the lack of viral vestiges in putative virus-associated malignant tumors[104].

There are no conclusive data about true‘‘hit-and-run’’mechanism in the development of virus-associated human cancers.For instance,no evi-dence to support the‘‘hit-and-run’’hypothesis as an explanation for EBV-negative Hodgkin lympho-ma cases was found so far[105].On the other hand, a putative‘‘hit-and-run’’mechanism was previously proposed in a model of HBV-induced hepatocarci-nogenesis[106].In this model,impairment of DNA-repair pathways by HBV proteins in chroni-cally infected hepatocytes would cause progressive genetic instability during the regeneration cycles of the in?amed liver tissue.In the long term,accumu-lation of mutations in hepatocytes may trigger malignant transformation and tumor progression, even if cells have lost the viral genome.Interesting-ly,Hessein et al.[107]recently claimed that HBV integration and microsatellite analysis revealed peculiar pro?les of viral genome integration and progressive loss of viral genome in regenerative hepatic nodules.Although these results may strength the‘‘hit-and-run’’mechanism in the above-mentioned model,the experiments of Hessein et al.remain to be repeated and evaluated carefully by others before any conclusion can be drawn.

Experimental models of a‘‘hit-and-run’’mecha-nism of viral carcinogenesis do exist[108,109],but they are of limited value because they fail to address the in vitro scenario complexity.Therefore,consid-ering that it is not yet possible to either con?rm or rule out the existence of the‘‘hit-and-run’’mecha-nism of viral carcinogenesis in vivo,whether it plays a role in the pathogenesis of human cancers remains to be further investigated.

11.Viral cooperation in cancer development: dangerous liaisons revealed

In the last20years,much attention has been devoted to the observation that HIV-infected patients are in higher risk to develop some cancers, notably KS,cervical carcinomas,and some non-Hodgkin lymphomas[110].Noteworthy,cancers arising in the context of HIV-induced immunosup-pression are usually associated with infection by oncogenic viruses,notably KSHV,HPV,and EBV.

Overall,two scenarios of cooperation between HIV and other viruses during cancer development can be outlined.First,it is assumed that immuno-suppression per se favors the survival of altered cells due to impairment of immune surveillance.In healthy organisms,cells infected by oncogenic virus-es are targeted by the immune system because they express virus-derived antigens.Therefore,the emer-gence of malignant clones is avoided.Second,as immunocompromised patients are prone to multiple infections,it is expected that proteins from di?erent viruses may act simultaneously in infected cells or participate in a complex signaling network among adjacent infected and non-infected cells.

The cooperation between HIV and KSHV in cancer development has caught attention in the past years,mainly because this encounter is veri?ed in vivo(e.g.,in KS and PEL,at least),and some studies have demonstrated interference between proteins from both viruses in vitro.For instance, the strong oncogenic properties of KSHV v-GPCR can be enhanced in HIV/KSHV co-infected cells.

D.Elgui de Oliveira/Cancer Letters247(2007)182–196191

The expression of the HIV-1tat protein in GR3 cells,which constitutively express v-GPCR,is asso-ciated with accelerated tumorigenesis in nude mice, as well as with increased synthesis of VEGF-C mRNA and increased activity of NF-j B and NF-AT[111].On the other hand,KSHV vFLIP acti-vates HIV LTR transcription and functionally cooperates with tat to support the HIV biological cycle[112].

In approximately70%of PEL cases,malignant lymphoid cells are infected by both KSHV and EBV.Therefore,the interaction between these her-pesviruses is more than a possibility,and it may have important impact on the pathogenesis of PEL.Actually,EBV infection of di?erent KSHV-positive PEL cell lines in vitro resulted in increased tumorigenesis in SCID mice[113].In time,it has been demonstrated that CMV,another herpesviral pathogen commonly found in immunosuppressed individuals,activates KSHV lytic replication in vitro [114].Unfortunately,the detailed mechanism of this cooperation remains elusive,and further studies are necessary to elucidate whether it may have any role in the development of malignant tumors.

12.Concluding remarks

Major advances in the prevention,diagnosis, and treatment of human cancers came along with a better understanding of their etiology,pathogen-esis,and natural history.Thus,it is mandatory to properly validate any suspected causal link between viruses and human tumors.Unfortunately, it is not a trivial task.When considering a virus as a possible etiological agent for a given cancer, it has long known that the classical Henle–Koch postulates on infectious causes of disease are not ful?lled,and even the use of the most updated guidelines is challenging[115,116].On the other hand,increasing data on viral biology suggest that only few viruses can indeed be considered carcino-gens for humans.In this regard,all the informa-tion available so far raises little doubt,if any, that multiple cellular pathways are usually target-ed by viral proteins in vivo before the infected cell becomes malignant.

The above-discussed molecular mechanisms show that infection by DNA oncogenic viruses vir-tually impairs all critical cellular processes during cancer development.Moreover,if one considers the classical multistep model of carcinogenesis,a plausible role of viral infection throughout cell initi-ation and promotion to tumor progression might be observed.From this perspective,ongoing and future research on viral carcinogenesis is expected to pro-vide additional breakthroughs in our knowledge on cancer biology.

References

[1]V.Ellerman,O.Bang,Experimentelle Leukamie bei Hun-

ern.Zentralbl.Bakteriol.Parasitenkd.Infectionskr.Hyg.

Abt.Orig.46(1908)595–609.

[2]P.Rous,Transmission of a malignant new growth by

means of a cell-free?ltrate,J.Am.Med.Assoc.56(1911) 198.

[3]G.Ciu?o,Innesto positivo con in?ltrado di verrucae

volgare,G.Ital.Mal.Venereol.48(1907)12–15.

[4]M.A.Epstein,B.G.Achong,Y.M.Barr,Virus particles in

cultured lymphoblasts from Burkitt’s lymphoma,Lancet15 (1964)702–703.

[5]P.Pisani,D.M.Parkin,N.Munoz,J.Ferlay,Cancer and

infection:estimates of the attributable fraction in1990, Cancer Epidemiol.Biomarkers Prev.6(1997)387–400. [6]J.S.Butel,Viral carcinogenesis:revelation of molecular

mechanisms and etiology of human disease,Carcinogenesis 21(2000)405–426.

[7]D.Hanahan,R.A.Weinberg,The hallmarks of cancer,Cell

100(2000)57–70.

[8]B.S.Blumberg, B.J.Gerstley, D.A.Hungerford,W.T.

London,A.I.Sutnick,A serum antigen(Australia antigen) in Down’s syndrome,leukemia,and hepatitis,Ann.Intern.

Med.66(1967)924–931.

[9]A.M.Prince,An antigen detected in the blood during the

incubation period of serum hepatitis,Proc.Natl.Acad.Sci.

USA60(1968)814–821.

[10]R.Bonilla Guerrero,L.R.Roberts,The role of hepatitis B

virus integrations in the pathogenesis of human hepatocel-lular carcinoma,J.Hepatol.42(2005)760–777.

[11]M.Durst,L.Gissmann,H.Ikenberg,H.zur Hausen,A

papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from di?erent geo-graphic regions,https://www.doczj.com/doc/344738742.html,A80(1983) 3812–3815.

[12]M.E.Scheurer,G.Tortolero-Luna,K.Adler-Storthz,

Human papillomavirus infection:biology,epidemiology, and prevention,Int.J.Gynecol.Cancer15(2005) 727–746.

[13]L.S.Young,A.B.Rickinson,Epstein–Barr virus:40years

on,Nat.Rev.Cancer4(2004)757–768.

[14]Y.Chang,E.Cesarman,M.S.Pessin,F.Lee,J.Culpepper,

D.M.Knowles,P.S.Moore,Identi?cation of herpesvirus-

like DNA sequences in AIDS-associated Kaposi’s sarcoma, Science266(1994)1865–1869.

[15]D.C.Edelman,Human herpesvirus8–a novel human

pathogen,Virol.J.2(2005)78.

[16]M.C.Yu,J.M.Yuan,Environmental factors and risk for

hepatocellular carcinoma,Gastroenterology127(2004) S72–S78.

[17]F.X.Bosch,S.de Sanjose,Chapter1:Human papilloma-

virus and cervical cancer–burden and assessment of causality,J.Natl.Cancer Inst.Monogr.31(2003)3–13.

192 D.Elgui de Oliveira/Cancer Letters247(2007)182–196

[18]A.L.Bezerra,A.Lopes,https://www.doczj.com/doc/344738742.html,ndman,G.N.Alencar,H.

Torloni,L.L.Villa,Clinicopathologic features and human papillomavirus DNA prevalence of warty and squamous cell carcinoma of the penis,Am.J.Surg.Pathol.25(2001) 673–678.

[19]S.Syrjanen,Human papillomavirus(HPV)in head and

neck cancer,J.Clin.Virol.32(2005)S59–S66.

[20]E.L.Franco, D.M.Harper,Vaccination against human

papillomavirus infection:a new paradigm in cervical cancer control,Vaccine23(2005)2388–2394.

[21]P.Busson,C.Keryer,T.Ooka,M.Corbex,EBV-associated

nasopharyngeal carcinomas:from epidemiology to virus-targeting strategies,Trends Microbiol.12(2004)356–360.

[22]G.Niedobitek,L.S.Young,H.Herbst,Epstein–Barr virus

infection and the pathogenesis of malignant lymphomas, Cancer Surv.30(1997)143–162.

[23]S.L.Glaser,J.L.Hsu,M.L.Gulley,Epstein–Barr virus and

breast cancer:state of the evidence for viral carcinogenesis, Cancer Epidemiol.Biomarkers Prev.13(2004)688–697. [24]D.V.Ablashi,L.G.Chatlynne,J.E.Whitman Jr., E.

Cesarman,Spectrum of Kaposi’s sarcoma-associated her-pesvirus,or human herpesvirus8,diseases,Clin.Microbiol.

Rev.15(2002)439–464.

[25]A.J.Berk,Recent lessons in gene expression,cell cycle

control,and cell biology from adenovirus,Oncogene24 (2005)7673–7685.

[26]M.Bocchetta,I.Di Resta, A.Powers,R.Fresco, A.

Tosolini,J.R.Testa,H.I.Pass,P.Rizzo,M.Carbone, Human mesothelial cells are unusually susceptible to simian virus40-mediated transformation and asbestos cocarcino-genicity,https://www.doczj.com/doc/344738742.html,A97(2000)10214–10219.

[27]P.G.Cerrano,B.Jasani,R.Filiberti,M.Neri,F.Merlo,

S.De Flora,L.Mutti,R.Puntoni,Simian virus40 and malignant mesothelioma,Int.J.Oncol.22(2003) 187–194.

[28]H.D.Strickler,J.J.Goedert,S.S.Devesa,https://www.doczj.com/doc/344738742.html,hey,J.F.

Fraumeni Jr.,P.S.Rosenberg,Trends in U.S.pleural mesothelioma incidence rates following simian virus40 contamination of early poliovirus vaccines,J.Natl.Cancer Inst.95(2003)38–45.

[29]R.A.Vilchez,J.S.Butel,Emergent human pathogen simian

virus40and its role in cancer,Clin.Microbiol.Rev.17 (2004)495–508.

[30]A.Cristaudo,R.Foddis,A.Vivaldi,R.Buselli,V.Gattini,

G.Guglielmi,F.Cosentino,F.Ottenga,E.Ciancia,R.

Libener,R.Filiberti,M.Neri,P.Betta,M.Tognon,L.

Mutti,R.Puntoni,SV40enhances the risk of malignant mesothelioma among people exposed to asbestos:a molec-ular epidemiologic case-control study,Cancer Res.65 (2005)3049–3052.

[31]W.C.Hahn,C.M.Counter,A.S.Lundberg,R.L.Beijers-

bergen,M.W.Brooks,R.A.Weinberg,Creation of human tumour cells with de?ned genetic elements,Nature400 (1999)464–468.

[32]P.Duesberg, C.Rausch, D.Rasnick,R.Hehlmann,

Genetic instability of cancer cells is proportional to their degree of aneuploidy,https://www.doczj.com/doc/344738742.html,A95(1998) 13692–13697.

[33]C.P.Crum,H.Ikenberg,R.M.Richart,L.Gissman,

Human papillomavirus type16and early cervical neoplasia, N.Engl.J.Med.310(1984)880–883.[34]S.Duensing,K.Munger,Human papillomavirus type16

E7oncoprotein can induce abnormal centrosome duplica-tion through a mechanism independent of inactivation of retinoblastoma protein family members,J.Virol.77(2003) 12331–12335.

[35]K.Heselmeyer,M.Macville,E.Schrock,H.Blegen,A.C.

Hellstrom,K.Shah,G.Auer,T.Ried,Advanced-stage cervical carcinomas are de?ned by a recurrent pattern of chromosomal aberrations revealing high genetic instability and a consistent gain of chromosome arm3q,Genes Chromosomes Cancer19(1997)233–240.

[36]M.Forgues,M.J.Di?lippantonio,S.P.Linke,T.Ried,

K.Nagashima,J.Feden,K.Valerie,K.Fukasawa, X.W.Wang,Involvement of Crm1in hepatitis B virus X protein-induced aberrant centriole replication and abnormal mitotic spindles,Mol.Cell.Biol.23(2003) 5282–5292.

[37]K.S.Srivenugopal,F.Ali-Osman,The DNA repair protein,

O(6)-methylguanine-DNA methyltransferase is a proteolyt-ic target for the E6human papillomavirus oncoprotein, Oncogene21(2002)5940–5945.

[38]S.A.Becker,T.H.Lee,J.S.Butel,B.L.Slagle,Hepatitis B

virus X protein interferes with cellular DNA repair,J.

Virol.72(1998)266–272.

[39]M.T.Liu,Y.R.Chen,S.C.Chen,C.Y.Hu,C.S.Lin,Y.T.

Chang,W.B.Wang,J.Y.Chen,Epstein–Barr virus latent membrane protein1induces micronucleus formation, represses DNA repair and enhances sensitivity to DNA-damaging agents in human epithelial cells,Oncogene23 (2004)2531–2539.

[40]M.A.Dunham, A.A.Neumann, C.L.Fasching,R.R.

Reddel,Telomere maintenance by recombination in human cells,Nat.Genet.26(2000)447–450.

[41]S.Chang,C.M.Khoo,M.L.Naylor,R.S.Maser,R.A.

DePinho,Telomere-based crisis:functional di?erences between telomerase activation and ALT in tumor progres-sion,Genes Dev.17(2003)88–100.

[42]H.Tahara,Y.Tokutake,S.Maeda,H.Kataoka,T.

Watanabe,M.Satoh,T.Matsumoto,M.Sugawara,T.Ide, M.Goto,Y.Furuichi,M.Sugimoto,Abnormal telomere dynamics of B-lymphoblastoid cell strains from Werner’s syndrome patients transformed by Epstein–Barr virus, Oncogene15(1997)1911–1920.

[43]M.Sugimoto,H.Tahara,M.Okubo,T.Kobayashi,M.

Goto,T.Ide,Y.Furuichi,WRN gene and other genetic factors a?ecting immortalization of human B-lymphoblas-toid cell lines transformed by Epstein–Barr virus,Cancer Genet.Cytogenet.152(2004)95–100.

[44]R.Rochford,M.J.Cannon,A.M.Moormann,Endemic

Burkitt’s lymphoma:a polymicrobial disease?Nat Rev.

Microbiol.3(2005)182–187.

[45]C.A.van den Bosch,Is endemic Burkitt’s lymphoma an

alliance between three infections and a tumour promoter?, Lancet Oncol5(2004)738–746.

[46]S.Kyo,M.Takakura,T.Taira,T.Kanaya,H.Itoh,M.

Yutsudo,H.Ariga,M.Inoue,Sp1cooperates with c-Myc to activate transcription of the human telomerase reverse transcriptase gene(hTERT),Nucleic Acids Res.28(2000) 669–677.

[47]J.Yang,X.Deng,L.Deng,H.Gu,W.Fan,Y.Cao,

Telomerase activation by Epstein–Barr virus latent mem-brane protein1is associated with c-Myc expression in

D.Elgui de Oliveira/Cancer Letters247(2007)182–196193

human nasopharyngeal epithelial cells,J.Exp.Clin.Cancer Res.23(2004)495–506.

[48]L.Ding,L.L.Li,J.Yang,Y.G.Tao,M.Ye,Y.Shi,M.

Tang,W.Yi,X.L.Li,J.P.Gong,Y.Cao,Epstein–Barr virus encoded latent membrane protein1modulates nuclear translocation of telomerase reverse transcriptase protein by activating nuclear factor-kappaB p65in human nasopha-ryngeal carcinoma cells,Int.J.Biochem.Cell Biol.37 (2005)1881–1889.

[49]O.Flore,S.Ra?i,S.Ely,J.J.O’Leary,E.M.Hyjek,E.

Cesarman,Transformation of primary human endothelial cells by Kaposi’s sarcoma-associated herpesvirus,Nature 394(1998)588–592.

[50]S.C.Verma,S.Borah, E.S.Robertson,Latency-associ-

ated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus up-regulates transcription of human telome-rase reverse transcriptase promoter through interaction with transcription factor Sp1,J.Virol.78(2004)10348–10359.

[51]A.Zhang,J.Wang,B.Zheng,X.Fang,T.Angstrom,C.

Liu,X.Li,F.Erlandsson,M.Bjorkholm,M.Nordenskj-ord,A.Gruber,K.L.Wallin,D.Xu,Telomere attrition predominantly occurs in precursor lesions during in vivo carcinogenic process of the uterine cervix,Oncogene23 (2004)7441–7447.

[52]A.J.Klingelhutz,S.A.Foster,J.K.McDougall,Telomerase

activation by the E6gene product of human papillomavirus type16,Nature380(1996)79–82.

[53]H.R.McMurray,D.J.McCance,Degradation of p53,not

telomerase activation,by E6is required for bypass of crisis and immortalization by human papillomavirus type16E6/ E7,J.Virol.78(2004)5698–5706.

[54]T.Kiyono,S.A.Foster,J.I.Koop,J.K.McDougall,D.A.

Galloway,A.J.Klingelhutz,Both Rb/p16INK4a inactiva-tion and telomerase activity are required to immortalize human epithelial cells,Nature396(1998)84–88.

[55]M.A.Dickson,W.C.Hahn,Y.Ino,V.Ronfard,J.Y.Wu,

R.A.Weinberg, D.N.Louis, F.P.Li,J.G.Rheinwald, Human keratinocytes that express hTERT and also bypass

a p16(INK4a)-enforced mechanism that limits life span

become immortal yet retain normal growth and di?erenti-ation characteristics,Mol.Cell.Biol.20(2000)1436–1447.

[56]https://www.doczj.com/doc/344738742.html,ne,L.V.Crawford,T antigen is bound to a host

protein in SV40-transformed cells,Nature278(1979)261–263.

[57]M.Sche?ner,N.J.Whitaker,Human papillomavirus-in-

duced carcinogenesis and the ubiquitin-proteasome system, Semin.Cancer Biol.13(2003)59–67.

[58]R.A.Watson,M.Thomas,L.Banks,S.Roberts,Activity

of the human papillomavirus E6PDZ-binding motif correlates with an enhanced morphological transformation of immortalized human keratinocytes,J.Cell Sci.116 (2003)4925–4934.

[59]C.Rivas, A.E.Thlick, C.Parravicini,P.S.Moore,Y.

Chang,Kaposi’s sarcoma-associated herpesvirus LANA2is

a B-cell-speci?c latent viral protein that inhibits p53,J.

Virol.75(2001)429–438.

[60]J.Friborg Jr.,W.Kong,M.O.Hottiger,G.J.Nabel,p53

inhibition by the LANA protein of KSHV protects against cell death,Nature402(1999)889–894.

[61]W.Chen,N.R.Cooper,Epstein–Barr virus nuclear antigen

2and latent membrane protein independently transactivate

p53through induction of NF-kappaB activity,J.Virol.70 (1996)4849–4853.

[62]W.Chen,S.Huang,N.R.Cooper,Levels of p53in

Epstein–Barr virus-infected cells determine cell fate:apop-tosis,cell cycle arrest at the G1/S boundary without apoptosis,cell cycle arrest at the G2/M boundary without apoptosis,or unrestricted proliferation,Virology251(1998) 217–226.

[63]J.Nicholas,Evolutionary aspects of oncogenic herpesvi-

ruses,Mol.Pathol.53(2000)222–237.

[64]S.Montaner,A.Sodhi,A.Molinolo,T.H.Bugge,E.T.

Sawai,Y.He,Y.Li,P.E.Ray,J.S.Gutkind,Endothelial infection with KSHV genes in vivo reveals that vGPCR initiates Kaposi’s sarcomagenesis and can promote the tumorigenic potential of viral latent genes,Cancer Cell3 (2003)23–36.

[65]A.Sodhi,S.Montaner,V.Patel,J.J.Gomez-Roman,Y.Li,

E.A.Sausville, E.T.Sawai,J.S.Gutkind,Akt plays a

central role in sarcomagenesis induced by Kaposi’s sarcoma herpesvirus-encoded G protein-coupled receptor,Proc.

https://www.doczj.com/doc/344738742.html,A101(2004)4821–4826.

[66]S.Pfe?er,M.Zavolan,F.A.Grasser,M.Chien,J.J.Russo,

J.Ju, B.John, A.J.Enright, D.Marks, C.Sander,T.

Tuschl,Identi?cation of virus-encoded microRNAs,Sci-ence304(2004)734–736.

[67]X.Cai,S.Lu,Z.Zhang,C.M.Gonzalez,B.Damania,B.R.

Cullen,Kaposi’s sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells,Proc.

https://www.doczj.com/doc/344738742.html,A102(2005)5570–5575.

[68]N.Joza,S.A.Susin,E.Daugas,W.L.Stanford,S.K.Cho,

C.Y.Li,T.Sasaki,A.J.Elia,H.Y.Cheng,L.Ravagnan,

K.F.Ferri,N.Zamzami, A.Wakeham,R.Hakem,H.

Yoshida,Y.Y.Kong,T.W.Mak,J.C.Zuniga-P?ucker,G.

Kroemer,J.M.Penninger,Essential role of the mitochon-drial apoptosis-inducing factor in programmed cell death, Nature410(2001)549–554.

[69]T.Grimm,S.Schneider, E.Naschberger,J.Huber, E.

Guenzi,A.Kieser,P.Reitmeir,T.F.Schulz,C.A.Morris, M.Sturzl,EBV latent membrane protein-1protects B cells from apoptosis by inhibition of BAX,Blood105(2005) 3263–3269.

[70]A.G.Eliopoulos,L.S.Young,LMP1structure and signal

transduction,Semin.Cancer Biol.11(2001)435–444. [71]A.Xiong,R.H.Clarke-Katzenberg,G.Valenzuela,K.M.

Izumi,https://www.doczj.com/doc/344738742.html,lan,Epstein–Barr virus latent membrane protein1activates nuclear factor-kappa B in human endothelial cells and inhibits apoptosis,Transplantation 78(2004)41–49.

[72]S.Henderson,D.Huen,M.Rowe,C.Dawson,G.Johnson,

A.Rickinson,Epstein–Barr virus-coded BHRF1protein,a

viral homologue of Bcl-2,protects human B cells from programmed cell death,https://www.doczj.com/doc/344738742.html,A90 (1993)8479–8483.

[73]A.Marchini,B.Tomkinson,J.I.Cohen,E.Kie?,BHRF1,

the Epstein–Barr virus gene with homology to Bc12,is dispensable for B-lymphocyte transformation and virus replication,J.Virol.65(1991)5991–6000.

[74]J.Nicholls,E.Kremmer,C.A.Meseda,M.Mackett,P.

Hahn,M.L.Gulley,A.Brink,L.J.Swinnen,J.Greenspan, Y.De Souza,F.Grasser,J.Sham,M.H.Ng,J.R.Arrand, Comparative analysis of the expression of the Epstein–Barr virus(EBV)anti-apoptotic gene BHRF1in nasopharyngeal

194 D.Elgui de Oliveira/Cancer Letters247(2007)182–196

carcinoma and EBV-related lymphoid diseases,J.Med.

Virol.65(2001)105–113.

[75]M.Y.Liu,Y.Y.Shih,L.Y.Li,S.P.Chou,T.S.Sheen,C.L.

Chen,C.S.Yang,J.Y.Chen,Expression of the Epstein–Barr virus BHRF1gene,a homologue of Bcl-2,in nasopharyngeal carcinoma tissue,J.Med.Virol.61(2000) 241–250.

[76]S.Gangappa,L.F.van Dyk,T.J.Jewett,S.H.Speck,H.W.

Virgin4th,Identi?cation of the in vivo role of a viral bcl-2, J.Exp.Med.195(2002)931–940.

[77]E.Kie?,T.Shenk,Modulation of apoptosis by herpesvi-

ruses,Semin.Virol.8(1998)471–480.

[78]E.Seto,L.Yang,J.Middeldorp,T.S.Sheen,J.Y.Chen,M.

Fukayama,Y.Eizuru,T.Ooka,K.Takada,Epstein–Barr virus(EBV)-encoded BARF1gene is expressed in naso-pharyngeal carcinoma and EBV-associated gastric carci-noma tissues in the absence of lytic gene expression,J.Med.

Virol.76(2005)82–88.

[79]H.Matta,Q.Sun,G.Moses,P.Chaudhary,Molecular

genetic analysis of human Herpesvirus8-encoded viral FLICE inhibitory protein-induced NF-j B activation,J.

Biol.Chem.278(2003)52406–52411.

[80]H.Matta,P.M.Chaudhary,Activation of alternative NF-

kappa B pathway by human herpes virus8-encoded Fas-associated death domain-like IL-1beta-converting enzyme inhibitory protein(vFLIP),https://www.doczj.com/doc/344738742.html,A101 (2004)9399–9404.

[81]A.Blood,C.J.Edwards,H.H.Ishii,B.K.Pat,G.Bryson,

T.B.Sculley,G.C.Gobe,Epstein–Barr virus-mediated protection against etoposide-induced apoptosis in BJA-B

B cell lymphoma cells:role of Bcl-2and caspase proteins,

Arch.Virol.149(2004)289–302.

[82]H.Herbst,T.Ra?,H.Stein,Phenotypic modulation of

Hodgkin and Reed–Sternberg cells by Epstein–Barr virus, J.Pathol.179(1996)54–59.

[83]K.R.Baumforth,J.R.Flavell,G.M.Reynolds,G.

Davies,T.R.Pettit,W.Wei,S.Morgan,T.Stankovic, Y.Kishi,H.Arai,M.Nowakova,G.Pratt,J.Aoki, M.J.Wakelam,L.S.Young,P.G.Murray,Induction of autotaxin by the Epstein–Barr virus promotes the growth and survival of Hodgkin lymphoma cells,Blood106 (2005)2138–2146.

[84]E.Maggio,A.van den Berg,A.Diepstra,J.Kluiver,L.

Visser,S.Poppema,Chemokines,cytokines and their receptors in Hodgkin’s lymphoma cell lines and tissues, Ann.Oncol.13S1(2002)52–56.

[85]H.Herbst,H.D.Foss,J.Samol,I.Araujo,H.Klotzbach,

K.Krause,A.Agathanggelou,G.Niedobitek,H.Stein, Frequent expression of interleukin-10by Epstein–Barr virus-harboring tumor cells of Hodgkin’s disease,Blood 87(1996)2918–2929.

[86]B.F.Skinnider,T.W.Mak,The role of cytokines in classical

Hodgkin lymphoma,Blood99(2002)4283–4297.

[87]B.Ensoli,M.Sturzl,Kaposi’s sarcoma:a result of the

interplay among in?ammatory cytokines,angiogenic fac-tors and viral agents,Cytokine Growth Factor Rev.9 (1998)63–83.

[88]L.Wang,N.Wakisaka,C.C.Tomlinson,S.M.DeWire,S.

Krall,J.S.Pagano,B.Damania,The Kaposi’s sarcoma-associated herpesvirus(KSHV/HHV-8)K1protein induces expression of angiogenic and invasion factors,Cancer Res.

64(2004)2774–2781.

[89]C.McCormick, D.Ganem,The kaposin B protein of

KSHV activates the p38/MK2pathway and stabilizes cytokine mRNAs,Science307(2005)739–741.

[90]M.R.Staudt,Y.Kanan,J.H.Jeong,J.F.Papin,R.Hines-

Boykin,D.P.Dittmer,The tumor microenvironment con-trols primary e?usion lymphoma growth in vivo,Cancer Res.64(2004)4790–4799.

[91]P.S.Moore,Y.Chang,Kaposi’s sarcoma-associated

herpesvirus immunoevasion and tumorigenesis:two sides of the same coin?Annu Rev.Microbiol.57 (2003)609–639.

[92]L.Coscoy,D.Ganem,Kaposi’s sarcoma-associated her-

pesvirus encodes two proteins that block cell surface display of MHC class I chains by enhancing their endocytosis, https://www.doczj.com/doc/344738742.html,A97(2000)8051–8056.

[93]D.J.Sanchez,J.E.Gumperz,D.Ganem,Regulation of

CD1d expression and function by a herpesvirus infection,J.

Clin.Invest.115(2005)1369–1378.

[94]K.W.Moore,P.Vieira,D.F.Fiorentino,M.L.Trounstine,

T.A.Khan,T.R.Mosmann,Homology of cytokine syn-thesis inhibitory factor(IL-10)to the Epstein–Barr virus gene BCRFI,Science248(1990)1230–1234.

[95]D.H.Hsu,R.de Waal Malefyt,D.F.Fiorentino,M.N.

Dang,P.Vieira,J.de Vries,H.Spits,T.R.Mosmann,K.W.

Moore,Expression of interleukin-10activity by Epstein–Barr virus protein BCRF1,Science250(1990)830–832. [96]M.Vockerodt,B.Haier,P.Buttgereit,H.Tesch,D.Kube,

The Epstein–Barr virus latent membrane protein1induces interleukin-10in Burkitt’s lymphoma cells but not in Hodgkin’s cells involving the p38/SAPK2pathway,Virol-ogy280(2001)183–198.

[97]N.Kitagawa,M.Goto,K.Kurozumi,S.Maruo,M.

Fukayama,T.Naoe,M.Yasukawa,K.Hino,T.Suzuki,S.

Todo,K.Takada,Epstein–Barr virus-encoded poly(A)(-) RNA supports Burkitt’s lymphoma growth through inter-leukin-10induction,EMBO J.19(2000)6742–6750. [98]S.Mahot,A.Sergeant,E.Drouet,H.Gru?at,A novel

function for the Epstein–Barr virus transcription factor EB1/Zta:induction of transcription of the hIL-10gene,J.

Gen.Virol.84(2003)965–974.

[99]B.Zhang,P.Li,E.Wang,Z.Brahmi,K.W.Dunn,J.S.

Blum,A.Roman,The E5protein of human papillomavirus type16perturbs MHC class II antigen maturation in human foreskin keratinocytes treated with interferon-gam-ma,Virology310(2003)100–108.

[100]Y.E.Chang,https://www.doczj.com/doc/344738742.html,imins,Microarray analysis identi?es interferon-inducible genes and Stat-1as major transcrip-tional targets of human papillomavirus type31,J.Virol.74 (2000)4174–4182.

[101]L.V.Ronco, A.Y.Karpova,M.Vidal,P.M.Howley, Human papillomavirus16E6oncoprotein binds to inter-feron regulatory factor-3and inhibits its transcriptional activity,Genes Dev.12(1998)2061–2072.

[102]J.S.Park, E.J.Kim,H.J.Kwon, E.S.Hwang,S.E.

Namkoong,S.J.Um,Inactivation of interferon regulatory factor-1tumor suppressor protein by HPV E7oncoprotein.

Implication for the E7-mediated immune evasion mecha-nism in cervical carcinogenesis,J.Biol.Chem.275(2000) 6764–6769.

[103]A.E.Koromilas,S.Li,G.Matlashewski,Control of interferon signaling in human papillomavirus infection, Cytokine Growth Factor Rev.12(2001)157–170.

D.Elgui de Oliveira/Cancer Letters247(2007)182–196195

[104]R.F.Ambinder,Gammaherpesviruses and‘‘Hit-and-Run’’oncogenesis,Am.J.Pathol.156(2000)1–3.

[105]A.Gallagher,J.Perry,J.Freeland,F.E.Alexander,W.F.

Carman,L.Shield,R.Cartwright,R.F.Jarrett,Hodgkin lymphoma and Epstein–Barr virus(EBV):no evidence to support hit-and-run mechanism in cases classi?ed as non-EBV-associated,Int.J.Cancer.104(2003)624–630. [106]J.S.Butel,T.H.Lee,B.L.Slagle,Is the DNA repair system involved in hepatitis-B-virus-mediated hepatocellular carci-nogenesis?,Trends Microbiol4(1996)119–124.

[107]M.Hessein,G.Saad el,A.A.Mohamed,A.M.Kamelel,

A.M.Abdel Hady,M.Amina,C.E.Rogler,Hit-and-run

mechanism of HBV-mediated progression to hepatocellular carcinoma,Tumori91(2005)241–247.

[108]Y.Shen,H.Zhu,T.Shenk,Human cytomegalovirus IE1 and IE2proteins are mutagenic and mediate‘‘hit-and-run’’oncogenic transformation in cooperation with the adeno-virus E1A proteins,https://www.doczj.com/doc/344738742.html,A94(1997) 3341–3345.

[109]M.Nevels,B.Tauber,T.Spruss,H.Wolf,T.Dobner,‘‘Hit-and-run’’transformation by adenovirus oncogenes,J.

Virol.75(2001)3089–3094.

[110]Y.Aoki,G.Tosato,Neoplastic conditions in the context of HIV-1infection,Curr.HIV Res.2(2004)343–349. [111]H.G.Guo,S.Pati,M.Sadowska,M.Charurat,M.Reitz, Tumorigenesis by human herpesvirus8vGPCR is acceler-

ated by human immunode?ciency virus type1Tat,J.Virol.

78(2004)9336–9342.

[112]Q.Sun,H.Matta,P.M.Chaudhary,Kaposi’s sarcoma associated herpes virus-encoded viral FLICE inhibitory protein activates transcription from HIV-1Long Terminal Repeat via the classical NF-kappaB pathway and functionally cooperates with Tat,Retrovirology2 (2005)9.

[113]P.Trivedi,K.Takazawa, C.Zompetta,L.Cuomo, E.

Anastasiadou, A.Carbone,S.Uccini, F.Belardelli,K.

Takada,L.Frati, A.Faggioni,Infection of HHV-8+ primary e?usion lymphoma cells with a recombinant Epstein–Barr virus leads to restricted EBV latency,altered phenotype,and increased tumorigenicity without a?ecting TCL1expression,Blood103(2004)313–316.

[114]J.Vieira,P.O’Hearn,L.Kimball,B.Chandran,L.Corey, Activation of Kaposi’s sarcoma-associated herpesvirus (human herpesvirus8)lytic replication by human cytomeg-alovirus,J.Virol.75(2001)1378–1386.

[115]D.N.Fredericks,D.A.Relman,Sequence-based identi?-cation of microbial pathogens:a reconsideration of Koch’s postulates,Clin.Microbiol.Rev.9(1996) 18–33.

[116]V.Brower,Connecting viruses to cancer:how research moves from association to causation,J.Natl.Cancer Inst.

96(2004)256–257.

196 D.Elgui de Oliveira/Cancer Letters247(2007)182–196

病毒DNA提取

小鼠血浆中HSV-1 DNA的提取 [目的] 掌握血浆中病毒DNA 提取的原理，熟悉其提取方法。 [原理] E.Z.N.A.?Viral DNA Kit 该试剂盒提供了从小于250ul血浆、血清、无细胞培养液等样品中快速简单地提取病毒DNA的方法，液体样品经Buffer BL和蛋白酶（OB）消化后，经乙醇调节结合条件，过柱吸附DNA，再经过两步快速洗涤后，最后用Elution Buffer (10mM Tris,pH8.5)溶解基因组DNA。纯化的DNA可直接用于PCR，Southern杂交，酶切等实验。 [试剂及仪器]旋涡混合振荡仪；微量DNA定量仪；台式离心机；65℃水浴锅。 1.5ml无菌EP管（2个/人）；EP抗凝管(含100ul抗凝剂)（K）；1000ul枪及枪头；。Hibind DNA 结合柱(1个/人)；2ml收集管（3个/人）；OB蛋白酶（OB）；Buffer B L（含线性丙烯酰胺）(BL)；乙醇(乙)；Buffer HB(HB)；DNA Wash Buffer（W）；Elution Buffer(EB)。 [实验步骤] 一、血浆样品的制备： 1.用微量加样器取100ul抗凝剂加入1.5mlEP管中。 2.右手抓起小鼠尾巴，用左手固定动物，压迫眼球，尽量使眼球突出，右手用镊子迅速摘除 眼球，将流出的血液滴入含有抗凝剂的1.5ml EP管中，迅速混匀后，10000rpm，离心3min。 上层黄色透明的即为血浆。 二、DNA的提取 1.取血浆250ul（如不足250ul，用Elution Buffer补足250ul）于1.5ml无菌EP管中。 2.加入10ul OB蛋白酶和250ul BL Buffer（含4ul线性丙烯酰胺用于填补病毒DNA在吸附 柱上的本底吸附），于旋涡混合振荡仪上最大速度振荡15sec。 3.65℃孵育10min。孵育过程中，用旋涡混合振荡仪混匀一次。 4.加入260ul乙醇，旋涡混合振荡仪最大速度振荡20sec。12000×g离心10sec，使盖子上的 液体沉于管中。 5.将Hibind DNA结合柱装在2ml收集管中，将第4步离心的上清中所有液体（约760ul） 加入Hibind DNA结合柱装上，8000×g离心1min。卸下收集管，将收集管及其中的液体弃去。 6.将Hibind DNA结合柱装在另一个新的收集管中，向柱中加入500ul HB Buffer，8000×g 离心1min，卸下收集管，倒掉其中的液体后，将收集管重新装在柱子上。、 7.向柱中加入700ul DNA Wash Buffer，8000×g离心1min，卸下收集管，将收集管及其中 的液体弃去。 8.将Hibind DNA结合柱装在另一个新的收集管中，向柱中加入700ul DNA Wash Buffer， 8000×g离心1min，卸下收集管，倒掉其中的液体后，将收集管重新装在柱子上。 9.将空的Hibind DNA结合柱15000×g离心2min，以去掉其中残余的液体。卸下收集管， 将收集管及其中的液体弃去。----这一步非常重要 10.将Hibind DNA结合柱装在另一个新的无菌1.5ml EP管上（事先做好标记），加入50-100ul 65℃预热的Elution Buffer，室温静置5min后，8000×g离心1min，洗脱液中即含有病毒DNA。 三、DNA定量： 微量DNA定量仪。取1.5ul用于DNA定量。

单纯疱疹病毒疫苗讲义

生物制品学课堂作业课题：单纯疱疹病毒疫苗 组长：陈畑 组员：潘芸 金宏杰

日期：2015.04.03 分数： 单纯疱疹病毒疫苗讲义 各位同学，下午好，我们小组要讲的课题是单纯疱疹病毒疫苗。 1986年，日本的HSV（单纯疱疹病毒）抗体阴性者日渐增多，1~20岁阴性者达70一80%，40岁以上占10%，20多岁介于二者之间。与此同时，生殖器疱疹、新生儿疱疹、疱疹性脑炎等单纯疤疹病毒所引起的疾病逐渐增加。在美国，潜伏性(隐匿性)的生殖器疱疹患者达1000万人，每年原发性生殖器疤疹患者达30~50万人，这已成为严重的社会问题【森良一】。和CVM（巨细胞病毒）一样，HSV 在人群中感染相当广泛，HSVI人群感染率高达50% - 90%（多为隐性潜伏性感染）。随着我国经济、社会的发展，我们需要更多地关注这类只以人为宿主细胞的病毒，不管在预防上还是治疗上。【展示图片】 首先，我们来认识一下单纯疱疹病毒： 单纯疱疹病毒HSV（Herpes simplex virus）属α疱疹病毒亚科【有α、β、γ三亚科】，是疱疹病毒的典型代表病毒。HSV病毒颗粒（毒粒）呈球形，直径为120-150nm，完整病毒粒由核心、衣壳、皮层（内膜、被膜）及包膜（囊膜）组成，核心含双股DNA，缠绕成纤丝卷轴（圆柱状）；衣壳呈二十面体对称，由162个壳微粒组成，直径为100nm；衣壳外为一层皮层，最外为包膜上有刺突（或囊膜突起）；包膜表面含有12种糖蛋白【gB、gC、gD、gE、gG、gI、gH、gJ、gK、gL、gM和gN】【书本239】。 球形，120-150nm 核心: 线性dsDNA 衣壳: 20面体立体对称 皮层: 衣壳和包膜之间 包膜: 表面有糖蛋白刺突

病毒基因组DNA提取试剂盒使用说明书

病毒基因组DNA 提取试剂盒 Virus Genomic DNA Kit (目录号：HS0307) 产品包装 自备试剂 无水乙醇 储存条件 蛋白酶K 于-20℃，其他组分室温（15 ~ 25℃） 产品简介 本试剂盒适用于从新鲜或冷冻的血浆、血清和无细胞体液中提取高质量的病毒DNA 。无需使用苯酚、氯仿等有机溶剂抽提，独特的缓冲液/蛋白酶K 体系能迅速裂解病毒，使病毒蛋白与DNA 分离，在蛋白酶K 的作用下降解病毒蛋白，在高盐状态下将病毒DNA 选择性吸附于硅基质膜上，再通过快速的漂洗、离心步骤，去除蛋白等杂质，最后低盐的洗脱缓冲液将高纯度的病毒DNA 从吸附柱膜上洗脱下来。 本试剂盒操作简单、快速，所得病毒DNA 不含蛋白、核酸酶和其他杂质，可直接用于PCR 、RT-PCR 、Real-Time PCR 、印迹等分子生物学实验。 产品特点 1.简便快速，1小时内可获得高纯度的病毒基因组DNA 。 2.无需有机溶剂抽提，使用安全。 3.重复性好，产量高。 北京厚生博泰科技有限公司 Beijing Hooseen Biotech Co., Ltd.

4.所得病毒DNA纯度高，无污染物和抑制剂，方便下游应用。 注意事项 1.血清或血浆避免反复冻融，否则会使蛋白变性或产生沉淀，导致提取的DNA片段小，提取量下降。 2.如缓冲液Buffer GB、Buffer GD结晶或产生沉淀，可在56℃水浴溶解。 3.所有离心步骤均为室温下操作。 操作步骤 1. 取1.5 ml离心管（自备），加入20 ul的Proteinase K溶液。 2. 向离心管中加入200 ul血清或血浆，然后再加入200 ul Buffer GB，涡旋震荡15 sec。（注意：1、样本体积不足200 ul可以加入0.9% NaCl（自备）补足。2、为确保样本有效裂解，加入Buffer GB后，需将样本与Buffer GB充分混匀。） 3．56℃孵育15 min，短暂离心，将管壁上的溶液收集到管底。 4. 加入250 ul无水乙醇，涡旋震荡15 sec，室温放置5 min，短暂离心，将管壁上的溶液收集到管底。 (注意：如果环境温度超过25℃，无水乙醇应在冰上预冷后使用。) 5．将一个Spin Columns CG*放入Collection Tubes(2 ml)中，将上一步所得溶液转移到离心吸附柱中，10 000 rpm离心30 sec，弃收集管中的废液。 6. 向吸附柱内加入500 ul的Buffer GD，室温10 000 rpm离心30 sec，弃收集管中废液。（注意：Buffer GD中含有乙醇，用后及时盖紧，以防乙醇挥发） 7. 向吸附柱内加入500 ul的Buffer PW，室温10 000 rpm离心30 sec，弃收集管中废液。（注意：如需进一步提高DNA纯度，可重复步骤7一次。Buffer PW中含有乙醇，用后及时盖紧，以防乙醇挥发） 8．向吸附柱中加入500 ul无水乙醇，10 000 rpm离心30 sec，倒掉收集管中的废液，将吸附柱重新放回收集管中。 9．室温12 000 rpm离心3 min，甩干残留液体。 （注意：此步不能省略，否则残留乙醇会影响质粒的后续使用） 10. 将离心吸附柱置于一个新的1.5 ml塑料离心管（自备）中，小心打开吸附柱的盖子，室温放置3 min，使吸附膜完全变干。加入30 ~ 100 ul的洗脱液，室温放置2 ~ 5 min。12 000 rpm

单纯疱疹病毒抗体igg阳性是怎么回事

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均约4~5日，外阴部先有灼热感，旋即发生成群丘疹，可为一簇或多簇，继之形成水疱。数日后演变为脓疱，破溃后形成糜烂，自觉疼痛。大约有60%的原发患者，会在一年内再次复发，第1 年至少复发数次，第2年则逐渐减少。复发后一般症状比较轻，也没有淋巴结肿大，病程也短。 ★二、孕妇单纯疱疹病毒igg抗体偏高怎么办？ 单纯疱疹病毒对胎儿，与新生儿的影响都特别大，对胎儿致畸率非常高。许多报道指出，在怀孕早期感染(尤其是原发性感 染者)单纯疱疹病毒可通过胎盘引起胎儿病毒血症、自发性流产、死胎、先天畸形、宫内发育迟缓或早产，也可呈隐性感染或持续带病毒状态等。 如果检测结果为单纯疱疹病毒1型igg阳性只能说明感染过，说明过去，不能说明现在，反而，准妈妈要把注意力放在igm上，如果是igm阳性说明体内还有相关病毒，这时候可能要根据实际

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单纯疱疹病毒感染

单纯疱疹病毒感染 属于疱疹病毒科的以人类为自然宿主的疱疹病毒共有6型：单纯疱疹病毒Ⅰ型和Ⅱ型、巨细胞病毒、水痘-带状疱疹病毒、EB病毒及人疱疹病毒6型。本节主要讨论单纯疱疹病毒（Herpes simplex virus,HSV)对胎儿及新生儿的影响。 单纯疱疹病毒Ⅰ型多引起腰部以上的皮肤疱疹和眼、口腔疱疹。Ⅱ型多引起腰部以下的皮肤疱疹。与胎儿和新生儿有关的主要是Ⅱ型，但Ⅰ型有进也可引起先天怀和新生儿疱疹。 单纯疱疹病毒较易分离，接种于组织培养（原代兔肾细胞、鸡胚细胞、羊膜细胞或Hela细胞、WI-38细胞等）中24～48小时即可出现病变。直接荧光抗体法更可于2小时内得出结果，不但可确定为单纯疱疹病毒，而且可分清Ⅰ型或Ⅱ型。应用HSV-DNA酶切电泳图形分析法，不但可鉴别Ⅰ、Ⅱ两型，而且可检测出同型中不同株的差异，故可用于分子流行病学调查。 单纯疱疹病毒在人群中广泛存在，人是唯一的宿主。病人及带毒者是唯一的传染源。除皮疹疱液外，唾液、尿和粪便中也都含有病毒。传染方式很多，主要是通过皮肤、粘膜的直接接触。由于带毒者和隐性感染的广泛存在，加上传播方式很多，且无可靠的疫苗，故预防较难。单纯疱疹病毒主要侵犯起源于外胚层的组织，如皮肤、粘膜、眼和神经系统。人体的特异性细胞免疫对于HSV感染的限局和中止有重要作用，因此免疫功能正常者的原发感染常为局部感染，免疫未成熟的新生儿、免疫功能低下和高度营养不良者则可形成全身播散性感染。原

发性局部感染的患者中也有一部分伴有病毒血症。原发性感染后的体液免疫可清除体内大部病毒，而小部分病毒常潜伏在局部感觉神经节内，如三叉神经节、颈上神经节、迷走神经节、骶神经节等。在某些因素，如发热、日晒、创伤、月经、情绪激动、手术等刺激下，病毒可在神经节内被激活，沿轴突向其支配的周围组织扩散而引起复发，故复发总是在相同部位反复发生，而且在疱疹发生前常有局部皮肤的感觉异常。这种复发多不伴有病毒血症。 因此，如果孕妇罹患原发性疱疹病毒感染，病毒就有可能在病毒血症期间通过胎盘感染胎儿而形成先天感染。如果孕妇产道中疱疹病毒（原发感染或复发均可），则病毒可于分娩过程中感染新生儿而引起新生儿感染。无论是先天感染或新生儿感染，预后都较差。 （一）先天性单纯疱疹病毒感染 1、临床表现与诊断根据受染的胎龄不同可有不同的临床表现。如妊娠头8周受染则可发生先天畸形，如妊娠晚期受染则可与新生儿受染相似。一般主要有以下表现：小头、小眼、脉络膜视网膜炎、晶状体混浊、心脏异常（如动脉导管未闭）、颅内钙化、肢体异常（如短指或短趾）、癫痫发作、痉挛性肢体瘫痪、精神性运动发育迟缓（psychomotor retardation)、子宫内发育迟缓、体温不稳、脑发育不良、脑积水、精神神经障碍、角膜翳形成、肝脾肿大、肺炎、出生时或生后不久出现疱疹等。此外，流产、早产亦很常见。 关于诊断，由于临床表现与先天性弓形体病、先天性风疹及先天性巨细胞病毒感染都很类似，甚至有人建议统称为TORCH综合征（弓形体、

复发性单纯疱疹是什么【医学养生常识】

复发性单纯疱疹是什么 文章导读 众所周知，疱疹顾名思义就是人体某些部位长有疱疹，主要表现症状有：局部 皮肤皮肤瘙痒，红肿胀痛，长小疙瘩，而且疱疹易于感染蔓延。所以很多人患有的疱疹经 常性复发，让人头疼不已。而复发性单纯疱疹指的是局部皮肤会先发红发痒、之后出现水泡，也是疱疹的类型之一。 复发性单纯疱疹临床表现与诊断临床表现可分为三种类型。目前认为对全身性单纯疱疹 病毒感染的有效药物有两种，一是阿糖腺苷，一是acyclovir。从理论上干扰素也可能有效，但尚缺乏临床应用的报告。 临床 临床表现与诊断临床表现可分为三种类型： ①全身播散型：主为内脏受侵，表现为肝炎（血清转氨酶升高或/和黄疸、肝脾肿大）。肺炎（呼吸困难、紫绀）、弥散性血管内凝血（紫癜、血小板减少、血尿、血便）、心包炎、循环衰竭以及全身中毒症状（精神萎靡、吸乳差、呕吐、腹泻、惊厥、昏迷）等。此型可同时合并疱疹性脑炎，亦可不合并。皮肤、粘膜疱前也同样可有可无； ②中枢神经系统感染型：常表现为脑膜脑炎（昏迷、抽搐、病理反射、视神经乳头水肿、囟门隆起等，脑脊液常呈病毒性感染之改变）。此型可合并皮肤、粘膜疱疹，亦可不 合并； ③单纯疱疹型：仅于皮肤、眼睛或口腔等处出现单纯疱疹。疱疹部位有时与分娩时 先露部位有关，头位分娩者常在头部，臀位分娩者常在臀部及肛周。病变与成人同，偶可 变成脓疱及大疱。皮疹可迅速剥脱而造成诊断困难。 三型中以①②型较为常见，两型可占总数的3/4以上。病死率分另为80%及30%。幸存者也常有遗有严重的中枢神经系统损害和眼睛损害（较少）。 母亲产道有疱疹或单纯疱疹病毒阳性者诊断较易。发病后皮肤、粘膜伴有典型疱疹病变 者诊断也不太困难。但确诊还必须病毒学和血清学证明。对于没有皮疹的患者或皮疹非常 不典型的患者则主要靠病毒学与血清学的材料。如果病毒分离阳性或/和新生儿血液中出 现特异性IgM抗体，或一般抗HSV抗体在病程中有4倍以上的升高均可协助诊断。

RNA病毒基因组提取试剂盒使用说明

RNA病毒基因组提取试剂盒使用说明 货号：R2000 规格：50T/100T 保存：室温（15℃-25℃）干燥条件下可保存12个月，更长时间的保存可置于2℃-8℃。产品内容： 试剂盒组成R2000-50R2000-100 蛋白酶K1ml2ml 洗柱液50ml2ml 结合液25ml50ml×2 漂洗液15ml50ml RNase free ddH2O15ml15ml×2 RNase free吸附柱50个100个 RNase free收集管(2ml)50个100个 产品介绍： RNA病毒基因组提取试剂盒适合于从血清、细胞上清、淋巴液中提取RNA病毒基因组，不适合于细胞等组织内RNA病毒基因组的提取。使用本试剂盒提取的基因组RNA可用于RT-PCR实验。 操作步骤： 使用前请先在漂洗液中加入一瓶新开启的无水乙醇，加入到离瓶口约0.5-1cm距离，盖好摇匀。所有离心步骤均在2-8℃条件下进行。 1、取病毒上清液0.5ml，12000rpm离心5min，尽量吸尽上清使用，弃去沉淀（如无沉淀

可省去此步）。 2、向病毒上清中加入20ul10mg/ml的蛋白酶K，充分混匀，65℃消化10min，期间可颠倒离心管混匀数次。 3、吸附柱前处理：从包装中取出吸附柱，放入收集管中，加入700ul洗柱液，室温放置2分钟，2-8℃12000rpm离心2min，弃废液，将吸附柱放入收集管中待用。 4、向病毒上清中加入500ul结合液，充分混匀。再向管中加入400ul无水乙醇，充分混匀，此时可能会出现絮状沉淀，不影响RNA的提取，可将溶液和絮状沉淀都加入吸附柱中，静置2min。（吸附柱的最大容积为750ul，可分两次加入。一次吸附完离心后再将余下的混合液体加入柱中静置离心。） 5、12000rpm离心2min，弃废液，将吸附柱放入收集管中。 6、向吸附柱中加入700ul漂洗液(使用前请先检查是否已加入无水乙醇)，12000rpm离心1min，弃废液，将吸附柱放入收集管中。 7、向吸附柱中加入500ul漂洗液，12000rpm离心1min，弃废液，将吸附柱放入收集管中。 8、12000rpm离心2min，将吸附柱置于室温或50℃温箱放置数分钟，目的是将吸附柱中残余的漂洗液去除，否则漂洗液中的乙醇会影响后续的实验如酶切、PCR等。 9、将吸附柱放入一个干净的离心管中，向吸附膜中央悬空滴加50ul-100ul经65℃水浴预热的RNase free ddH2O，室温放置5min，12000rpm离心2min。即可得到高质量的病毒基因组RNA。 注意事项： 1、经常更换新手套。因为皮肤经常带有细菌，可能导致RNase污染。使用无RNase的塑料制品和枪头避免交叉污染。 2、样品应避免反复冻融，否则会导致提取的RNA提取量也下降。

病毒DNA提取

我要提取一种病毒的DNA，DNA是单链的，外面是病毒的蛋白质外壳包裹。 我的病毒样品是买来的灭活疫苗(vaccine)（干粉状），想用酚/氯仿来抽提DNA，请问病毒干粉要溶解在什么溶液里？ 回复 1、病毒干粉可以溶解在PBS、HBSS或者营养液如1640或MEM中。 2、振荡溶解之后，取上清加入等体积的消化缓冲液(0.5M EDTA pH8.0.1M Tris·HCl pH7.4, 10% SDS) , 再加入蛋白酶K (20mg/ml) 至终浓度50ug/ml, 于50℃消化2 h。 3、取出300ul 的消化产物, 加入等体积的酚抽提一次。 4、用等体积的酚∶氯仿∶异戊醇再抽提两次,。 5、加入1/10体积的3M 乙酸钠（pH 5.2）和2倍体积的无水乙醇沉淀 6、70% 乙醇洗涤 7、待沉淀干燥后溶于含RNase A (40ug/ml) TE(pH 8.0) 溶液中。 谢谢您回答我的问题，还想请问，从疫苗(vaccine)干粉中提取DNA，跑琼脂糖凝胶电泳，能看到明显的条带吗？病毒干粉要取多少量来提取？另外，我曾经把疫苗(vaccine)中的病毒干粉溶解在无菌高纯水中，结果水变成了红色，应该是有些疫苗(vaccine)佐剂在影响，请问要如何排除疫苗(vaccine)佐剂影响，红色的物质可能是什么？？ 红色的东西应该是保护剂的颜色，保护剂一般是蛋白或其他大分子一类的东西，对DNA提取可能会有一些干扰，如果有条件，可以细胞传代一次最好，不过可能影响也不是很大。疫苗(vaccine)干粉中提取的DNA跑琼脂糖凝胶电泳，能否看到明显的条带，主要与含有的病毒量、提取的DNA量有关。如果后续是扩增基因，微量的可能也就足够了。病毒干粉溶解具体看病毒说明书的推荐，如果没有一般用1-2ml的量来溶解，取多少量来提取DNA，按照说明书一般取300ul左右，如DNAzol。注意提取之前先离心，取上清提取DNA。 血清HBVDNA提取方法 裂解法： 试剂： TES：10mmol/LTris-HCl,pH8.0, 5mmol/L EDTA, 0.5%SDS 150μg/ml蛋白酶K 100μl血清加TES 方法： 65°C3小时 加等体积酚-氯仿-异戊醇,混匀,12000ｒ/ｍｉｎ离心10分钟,将上清吸入一新管 再用等体积氯仿-异戊醇提取一次,在上清中加入1/10体积的2ｍｏｌ/Ｌ乙酸钠和2倍体积的无水乙醇,-20℃放置30分钟(或过夜),沉淀ＤＮＡ,用70%乙醇洗沉淀1次,12000ｒｐｍ/ｍｉｎ离心10分钟,倾去上清,晾干,加100μｌＴＥ缓冲液溶解沉淀 煮沸法提取血清HBV DNA： 吸200ul 血清放入0.5ml EP管中，沸水煮10分钟，10000rpm离心5min

第三十章疱疹病毒

第三十章疱疹病毒 一、名词解释 1．潜伏感染2．整合感染3．巨细胞病毒4．人类疱疹病毒6型5．EV 6．异嗜性抗体7．VCA—IgA抗体8．永生化 二、填空题 1．常见的人类疱疹病毒包括──、──、──、──等。 2．疱疹病毒中最常引起胎儿先天性感染的是──病毒；与鼻咽癌有关的病毒──病毒。 3．可潜伏在三叉神经节的病毒是──，潜伏在骶神经节的病毒是──。 4．通过垂直传播引起胎儿畸形的疱疹病毒是──和──。 5．病毒感染细胞后可表现为──感染、──感染、──和──感染。 6．疱疹病毒的核酸类型是──，衣壳呈──，──包膜。 7．与宫颈癌关系密切的病毒有──、──和──。 8．HSV的传播途径主要有──、──和──。 9．HSV的原发感染类型主要是──感染，VZV的原发感染类型主要是──感染。 10．带状疱疹只发生于幼年得过──的成人，发病的主要原因──下降。 11．巨细胞病毒除通过垂直传播外，还可以通过──、──和──途径传播。 12．EBV的传播主要是通过──方式，也可以通过──途径感染。 13．HHV一6型的原发感染引起──，还可与──共同感染CD4+细胞。 14．EB病毒与──癌关系密切。若孕妇血清中检出巨细胞病毒IgM类抗体，提示胎儿有──。 15．HSV—I潜伏于──和──神经节中，HSV一Ⅱ潜伏于──中。HSV一Ⅱ感染与癌的发生有密切关系。 16．儿童期初次感染水痘一带状疱疹病毒，表现为──；成人复发者表现为──。 17．与EB病毒感染有关的疾病主要有──、──、──。 18．CMV的感染途径有──、──、──、──、──。 19．可感染免疫抑制状态下B细胞的病毒为──。 三、最佳选择题 1．可导致胎儿先天性畸形的一组病毒是( ) A．柯萨奇病毒、流感病毒、腮腺炎病毒B．风疹病毒、巨细胞病毒、单纯疱疹病毒 C．HIV、乙型脑炎病毒、丙型肝炎病毒D．巨细胞病毒、腺病毒、乙型肝炎病毒 E．EB病毒、麻疹病毒、脊髓灰质炎病毒 2．疱疹病毒不包括( ) A．HSV B．VZV C．CMV D．HBV E．EBV 3．不符合单纯疱疹病毒特点的一项是( ) A．患者和健康者为传染源B．主要经飞沫传染C．原发感染后可形成潜伏感染 D．婴幼儿感染HSV—I绝大部分无临床表现E．HSV一Ⅱ与宫颈癌有密切关系 4．对疱疹病毒的错误叙述是( ) A．均为双股DNA有包膜病毒B．主要通过接触传播 C．免疫力低下者在原发感染后形成潜伏感染 D．婴幼儿感染多为隐性感染 E．病毒基因与宿主基因的整合与致癌关系密切 5．下列病毒中可引起潜伏感染的是( ) A．麻疹病毒B．疱疹病毒C．风疹病毒D．乙型脑炎病毒E．乙型肝炎病毒

单纯疱疹病毒介绍

单纯疱疹病毒介绍 单纯疱疹病毒是什么 单纯疱疹病毒是什么？一起来了解下！单纯疱疹病毒（herpes simplex virus 简称HSV）存在于病人、恢复者或者是健康带菌者的水疱疤液、唾液及粪便中，传播方式主要是直接接触传染，亦可通过被唾液污染的餐具而间接传染。HSV感染现已成为世界上第四大传染病。 单纯疱疹病毒在全球广泛分布，人群中感染极为普遍，潜伏和复发感染者较多。患者和带毒者是该病的传染源。病毒可通过皮肤、粘膜的直接接触或性接触途径进入机体。单纯疱疹病毒感染新生儿、儿童和成人，通常分为原发感染和复发感染。 HSV对动物感染宿主范围较广。常用实验动物为家兔、豚鼠及小鼠等。HSV在多种细胞中能增殖，常用原代兔肾、人胚肾细胞以及地鼠肾等传代细胞培养分离病毒。感染细胞很快出现明显细胞病变，并出现嗜酸性核内包涵体。 单纯疱疹病毒1型和2型 单纯疱疹病毒，其包膜含有病毒抗原和细胞本身成分。知道的人们常常看到1型和2型的描述，究竟这两种代表什么呢？ 单纯疱疹病毒有两个血清型，即1型（HSV-1、HHV-1，α）和2型（HSV-2、HHV-2，α）。1型多引起腰以上皮肤疱疹、口腔疱疹及角膜结膜炎，2型多引起腰以下皮肤疱疹及外生殖器疱疹。 HSV-1多在幼儿期感染，主要通过接触传播，侵犯皮肤、粘膜和神经系统。原发感染后，病毒可潜伏终生，受诱发因素刺激后复发，因而潜伏感染和反复发作是本病的重要特征。HSV-2多在成年人，主要通过性交传播，侵犯泌尿生殖道。两者均产生疱性病变并有致癌的可能。除人外，还可感染多种实验动物和细胞培养，引起典型的细胞病变。 人是单纯疤疹病毒唯一的自然宿主，主要通过直接接触传播HSV一Ⅰ型尚可通过空气飞沫传播。HSV一Ⅱ型则主要通过性生活传播、在分娩时HSV-Ⅱ型可通过产道直接感染新生儿，无明显季节性，以散发为主，此病毒存在于病人恢复者或者是健康带菌者的水疱疤液唾液及粪便中。 单纯疱疹病毒感染症简介

用免疫胶体金层析法检测血液标本中2型单纯疱疹病毒抗体

用免疫胶体金层析法检测血液标本中2型单纯疱疹病毒抗体 发表时间：2012-08-09T17:17:52.390Z 来源：《中外健康文摘》2012年第18期供稿作者：王丹丹崔连东[导读] 近年来，通过对重组的HSV-2 糖蛋白研究，已经有检测该病毒特异性抗体的商品化的试剂盒供临床实验室应用。 王丹丹崔连东（辽宁省人民医院检验科辽宁沈阳110015）【中图分类号】R446.11【文献标识码】A【文章编号】1672-5085（2012）18-0057-02 【摘要】目的建立了一种特异性的用于免疫胶体金层析技术（GICA）检测单纯疱疹病毒2型（HSV-2）的免疫球蛋白IgG。方法共有239份血清和100份全血标本用免疫胶体金层析法进行检测，同时，血清的标本用ELISA方法作对照检测，全血标本分别用ELISA 和免疫印迹法（IB）确定。结果与ELISA 方法对比，GICA测定特异性的HSV-2抗体的敏感性为 100%（69/69，特异性为97.1%（171/176）；与HSV-1.轮状病毒和EB病毒抗体的交叉反应分别为2.7%（3/112），2.9%（1/34）和6.2%（1/16）；与带状疱疹病毒和人类巨细胞病毒无交叉反应；一定量胆红素、甘油三酯或血红蛋白对该试验无影响；不同的采血方法如毛细管法，EDTA或肝素抗凝的静脉采血法，及血清对实验结果无影响。结论用免疫胶体金层析技术（GICA）检测单纯疱疹病毒2型（HSV-2）的免疫球蛋白IgG是一种方便、简捷、快速和准确的试验方法。 【关键词】免疫胶体金层析法单纯疱疹病毒ELISA 免疫印迹单纯疱疹病毒（HSV）是一种常见的可引起人类多种疾病的世界范围内的病毒。HSV可分为两种类型：HSV-1和HSV-2。HSV-1通常感染人的舌、口、唇、筛窦和眼，而HSV-2 却与人类的生殖道和新生儿的感染相关。HSV可感染不同年龄的人群如新生儿、儿童和成年人。据统计，40岁的人群中，超过90%人血液中HSV-1 抗体呈阳性[1]。在美国，大多数的性生活活跃的年轻人均有生殖道疱疹病毒感染[2]。 HSV-2 的主要传播方式是通过性传播[3]。典型的症状为疱疹性生殖道炎症，其表现为生殖道局部的溃烂，同时可伴随发热、淋巴结炎和尿失禁。有些研究表明85%的原发疱疹性生殖道炎症是由HSV-2引起的[4]。由于很多的病例无明显的临床症状，因此，要有效地管理疱疹病毒的感染者，用血清学诊断来区分两种类型的病毒就显得尤为重要[5]。这两种类型的病毒的抗原有极高的相似性，然而，它们各自的糖蛋白却有很大的差异，可刺激机体产生不同的特异性抗体，并可用于商业上的诊断产品[6,7]。与蛋白印迹的方法相比，型特异性的血清学诊断方法也可以准确地诊断HSV-1和HSV-2的感染[8]。在临床实验诊断中，HSV-2的特异性血清诊断学方法主要是免疫印迹和酶联免疫吸附试验（ELISA）。这里我们介绍一种简便、快速，灵敏度高和特异性强的免疫胶体金层析技术，用于HSV-2的特异性糖蛋白抗体的检测。 1 材料和方法 1.1标本359例临床收集用于检测HSV-1.2抗体的患者血清，其参考的结果是通过ELSIA方法获得。同时，为了研究交叉试验，271份血清检测检测了巨细胞病毒(n=11)、带状疱疹病毒(n = 14)、EB病毒(n = 16),和轮状病毒(n=34)；为了研究干扰试验，在两个HSV-2抗体阳性和阴性的标本中加入甘油三酯、白蛋白、胆红素和血红蛋白，其血清终浓度分别为10mg/ml,60mg/ml、0.2mg/ml和l220mg/ml。为了研究不同的采血方法对胶体金层析法试验结果的影响，100名志愿者，分别用指尖毛细管采血法，EDTA及肝素抗凝静脉采血法和单纯血清采血法采血。试验采用双盲法，作为参考方法，HSV-2抗体检测采用商品ELISA试剂盒检测患者的血清标本。 1.2免疫胶体金层析法当标本中存在HSV抗体时，抗体先与 40nm 的胶体金结合鼠抗人IgG抗体结合，随着硝酸纤维素膜的层析作用，结合物向前移动。就可以与三个条带上分别标记了HSV-1糖蛋白抗原、HSV-1糖蛋白抗原和羊抗鼠抗体结合，相应的条带就会出现。当测试条带和控制条带均出现时，为阳性；当测试条带未出现而控制条带出现为阴性；仅有测试条带出现或未出现而控制条带未出现，表明检测结果无效。所有检测结果均要求第二个试验人员确认。其示意图如图1 图1 免疫层析法检测卡示意图 1.3 参考方法用市售的 ELISA和免疫印迹试剂盒方法作对照，操作方法严格按照说明书的要求。用相应的检测巨细胞病毒、带状疱疹病毒、EB病毒和轮状病毒的ELISA试剂盒对标本进行检测。 1.4 数据评价接受者操作特性曲线（ROC）、似然比、阳性阴性预测值（PPV、NPV）等用MedCalc V9.3.0.0 统计软件统计。 2 结果 2.1用GICA方法检测血清的结果 在总共359份血清标本中，用GICA法和ELISA法分别检测，有89例HSV-2抗体阳性，阳性率为25.2%（89/359）。两种方法均阳性的为89例; 均阴性为257例; 有6份标本ELISA检测结果在临界值的范围内。其结果见表1 。表1 CIGA与ELISA 法检测HSV-2抗体结果

血清中病毒DNA提取方法

血清中病毒DNA提取方法。 一煮沸法 从血清中提取病毒DNA，与其它方法比较，此法最为简单，并且很多文献都有报道。 鲁艳芹等在一篇名为《乙型肝炎病毒荧光定量PCR检测方法及专用试剂盒》专利(专利申请号为200610044679)中对六种方法提取血清中乙型肝炎病毒DNA的效率做了比较，得出结果是：NaOH裂解法最佳，其次是直接煮沸法，但该法不适合提取粘稠度较高的血样标本。说明直接煮沸法还是可以的。 另外，陈秀珍等人《采用不同方法提取血清及乳汁中HBV-DNA扩增结果分析》对抽提法、热处理、碱变性法提取乙肝标志物阳性出产妇血清及乳汁中HBV-DNA方法进行评价，发现血清标本HBV-DNA的提取，三种方法无明显差异。 陈秀珍等：取血清100ul，放入0.5ulEP管中，沸水中煮10分钟，10000r/m,离心5分钟，取上清备用。 专利：取血清100ul和100ulPBS，煮沸10分钟，12000rpm, 离心5分钟，取上清备用. 两篇文章所用的煮沸法大致一样,只是是否加PBS的差异。加PBS，这样使得血清在煮10分钟后，不会干掉，没有上清，另外，它不会干扰核酸的提取。加TE是否也可以呢？ 二裂解液煮沸法 郭龙华等《3种方法从血清标本中提取I--IBV DNA 的比较》

比较3种从血清标本中提取HBV DNA方法的效果，选出一种快速、简单、高效提取血清中HBV DNA的方法——裂解液煮沸法，此法提取的HBV DNA模板含量最高，与碱裂解法和蛋白酶K-苯酚抽提法之间的差异显著(P