XMRV nytt: Systematisk studie av Zhang et al juni 2011 på hyppighet av XMRV forekomst i humane cellelinjer og bevis for en horisontal virusspredning til andre cellekulturer/cellelinjer

Forskere fra universitetsmiljøer i Texas, Baltimore og Houston i USA har gjennomført en systematisk undersøkelse på tumor cellelinjer hvor det ble sett på xenograft tumor cellelinjer fra syv forskjellige og uavhengige laboratorier og 128 non- xenograftede tumor cellelinjer. Formål med undersøkelsen er å se på hyppighet av gammaretrovirus XMRV/P-MLV forekomsten i humane cellelinjer og potensielle bevis for en horisontal virusspredning til andre cellekulturer/cellelinjer.

Studien har tittel “Frequent detection of infectious xenotropic murine leukemia virus (XMLV) in human cultures established from mouse xenografts” av forfattere som: Yu-An Zhang, Anirban Maitra, Jer-Tsong Hsieh, Charles M. Rudin, Craig Peacock, Collins Karikari, Rolf A. Brekken, Victor Stastny, Boning Gao, Luc Girard, Ignacio Wistuba, Eugene Frenkel, John D. Minna and Adi F. Gazdar.

Denne studien er publisert og hentet fra:

LANDES BIOSCIENCE Volume 12, Issue 7   October 1, 2011

Sammendrag/abstrakt (fritt oversatt til norsk):

Formål: En systematisk undersøkelse av hyppigheten av xenotrofisk murine leukemia virus (XMLV) forekomsten i humane cellelinjer som kommer fra cellelinjer mus xenograft og søke etter mulige beviser for en horisontal virusspredning til andre cellelinjer brukt i kultur.

Metode: Det ble undersøkt xenograft tumor cellelinjer fra syv forskjellige og uavhengige laboratorier og 128 non- xenograftede tumor cellelinjer.

Cell line DNA was examined for mouse DNA contamination, and by three Taqman qPCR assays targeting the gag, env or pol regions of MLV. Sequencing was used for viral strain identification.

Cellelinjenes DNA ble undersøkt for forekomst av eventuelle kontaminering av DNA fra mus ved hjelp av tre Taqman qPCR screenings metoder som finner gag, env eller pol regioner i viruskappen til MLV. Gensekvensene ble undersøkt for å identifisere virusvariantene.

Supernatant væske ble testet for revers transkriptase (RT) aktivitet.

Resultat: Seks av 23 (26%) av xenograft cellekulturene som var fri for DNA fra mus, var sterkt positive for MLVs og gensekvensene ble funnet til å ha større enn 99 % likhet for kjente MLV varianter.

Fire av fem tilgengelige supernatante væsker fra disse virale positive kulturene var sterkt positiv for RT aktivitet. Tre av disse væskene ble ytterligere undersøkt for å bekrefte frigjøring av vironer/virus til andre humane cellekulturer.

Resultater fra undersøkelsen av de 78 non-xenograft deriverte cellelinjene som befinner kultiverings fasiliteter som inneholder xenograft deriverte cellelinjer ble: 13 (17 %) av var positive for MLVs inkludert XMRV (som er en virusstamme/variant som først ble identifisert i humant vev). Til sammenligning forble alle 50 kulturene i xenograft frie fasiliteter funnet negative for virale gensekvenser.

Konklusjon: Humane cellekulturer som blir derivert etter xenograft fra mus inneholder og frigjør smittsom XMRV. Laboratorier som arbeider med xenograft deriverte humane cellekulturer bør være seg bevisst over forurensingen/kontaminering med potensielt fare for biologisk smittefarlig materiale (biohazard) fra humantrofiske virus med opprinnelse fra mus og evnen varianter av MLVs har til å bli horisontalt spredt og infisere andre kulturer.

Hele studien kan du lese her (pdf-fil) Zhang et al 2011 systematisk studie av cellelinjer som detekterer smittsomme XMRV juli 2011

Noen interessante utdrag fra studiens diskusjonsdel (utheving gjort av meg):

Reports of XMLV strains being present in human xenograft

cultures appeared in the 1970s.10,12,33 While several reports

have documented this phenomenon, most reports are sporadic

case reports without systematic analysis of true frequency.

Two reports examined multiple xenograft cultures12,33 and found frequencies

of 7 and 67% respectively. However, because of the

relatively crude detection methods available at that time and failure

to check for mouse cell contamination, the true frequencies

cannot be determined from these reports. Because of our initial

failure to culture SCLC tumors directly from tumor samples, we

developed long-term cultures from xenografted tumors.34 In the

mid 1980s we detected (by electron microscopy) large numbers

of retroviral particles released by SCLC cell line NCI-N417. We

destroyed stocks of this cell line and persuaded the American

Type Culture Collection to stop distribution. However, prior

to this finding, the cell line had been distributed extensively

to the scientific community and we obtained a culture from

Gerold Bepler, Moffitt Cancer Center, Tampa, FL. While we

did not report on the viral findings, others have done so14,15 and

we refer to this XMLV strain as N417 although it has also been

referred to as VB3.2.21 Recent reports of squirrel monkey retroviruses

(SMRV) infecting human cultures (by unknown routes

or sources) and their possible large scale horizontal spread have

also been published,35,36 and several human cell lines for human

immunodeficiency virus research were found to release infectious

XMLV.22,37 However, there are virtually no reports of contamination

of xenograft cultures published during the past 15 y

and most scientists appear unaware of the contamination problem

(authors’ observations). The purpose of our study was to

determine the frequency of xenograft culture contamination by

xenotropic viruses and their horizontal spread in multiple laboratories.

By sequencing the viruses so isolated we could determine

whether a single or multiple strains of XMLV were responsible

for contamination and horizontal spread.

*

For our initial study we obtained 26 xenografts cultures from

seven independent laboratories. Only two of the laboratory chiefs

that participated in this study were previously aware of the XMLV

contamination issue. We utilized three primer sets that detected

virtually all XMLV stains as well as most MLV strains, covering

the three major structural and functional regions of the mouse

retroviral genome. Our multiple qPCR approaches had advantage

to detect all possible MLV sequences (increased sensitivity) with

one or two additional probe confirmation (increased specificity)

as compared with a single qPCR assay. Nine of the samples were

positive by either two or all three of the probes used. However,

three of the nine positive samples contained varying amounts of

mouse DNA, presumably as a result of survival of mouse stromal

cells from the mouse xenograft. Apparently the mouse stromal

cells may persist for lengthy periods in culture, occasionally in

excess of one year. These three cultures were removed from further

study as the mouse genome contains multiple endogenous

MLV provirus sequences which make interpretation difficult.

*

Six of the remaining 23 xenograft cultures (26%) were positive for

one (or in one case, two) strains of XMLV or related viruses.

These six cultures came from six independent labs indicating the

widespread nature of the contamination. Of interest, our viral

sequencing homology analysis indicated that multiple strains of

XMLV were present in the six positive lines, demonstrating that

there are multiple strains of XMLV or MLV-related viruses capable

of infecting human cells individually or simultaneously after

xenografting.

*

Supernatant fluids were available from five of the six viral positive

xenograft cell lines. Four of these released large numbers of

potentially infectious viral particles, indicating possibilities of

horizontal spread to other human cultures as well as posing a biohazard

of unknown potential to laboratory personnel. We presume

that in one of these five cell lines the XMLV virus existed

in a latent form. Because of the possibility of spread to non-xenografted

human cultures, we examined cultures maintained in the

same culture facilities. A total of 78 cultures were obtained from

five of the laboratories. Thirteen of these cultures (17%) from four

of the laboratories were positive for XMLV. The viruses identified

as being responsible for horizontal spread were identical to those

identified in the positive xenograft cultures in the respective laboratories.

By contrast, 50 cell lines from the Gazdar lab maintained

in a facility free of xenograft cultures tested negative for virus.

These differences were significant, indicating the potential for

horizontal viral spread to other cultures maintained in the same

laboratory facility as xenograft cultures. In one case XMRV virus

infection to a non-xenograft colorectal carcinoma cell line RKO38

was demonstrated from an XMRV containing prostate xenograft

derived cell line 22Rv1 even though the two cell lines had been

maintained in the same culture facility for only a few days.

*

Our results indicate that human tumor cells frequently

become infected with MLV virus after xenografting and subsequent

culture. We have observed that mouse stromal cells may

persist in culture for lengthy periods. Mouse stromal cells, while

they contain abundant provirus forms of MLV, including ecotropic,

polytropic and xenotropic strains, seldom spontaneously

release large amounts of infectious virus (authors’ unpublished

findings). Virus infection of xenografted cells may require activation

of XMLV virus by chemical or immunological induction

in mouse and by prolonged mouse and human cell contact.

Viral transfer may occur in the mouse host or during subsequent

xenograft culture. Our findings of infectivity of XMLV-positive

supernatant fluids demonstrated that XMLV can readily infect

other human cultures without presence of mouse cells or other

aiding factors, indicating that these viruses are highly infectious.

*

In conclusion, our studies demonstrated that several MLV

strains were present in over one fourth of xenograft cell lines.

Infected cell lines were identified in most laboratories working

with or establishing xenograft cultures, indicating that such

contamination was widespread. Infected cultures usually release

large numbers of infectious virions, and intra-laboratory spread

of MLV virus to other cell lines maintained in the same facilities

may occur, confirming the highly infectious nature of MLV

virus. Retroviruses have been associated with multiple diseases

including solid and hematologic malignancies, AIDS as well as

with non-malignant diseases. The high susceptibility of human

cells to infection with XMLV, the high levels of reverse transcriptase

activity present in culture supernatant fluids and the

demonstrated infectivity of the shed virions suggest that such

viruses may present potential biohazards to laboratory personnel

involved in cell culture facilities or to those handling human

xenografts. In addition, the effects of the integrated provirus or

the released virions on the biology of infected tumor cells are

unknown. Provirus integration into the genome is not random,

and occurs preferentially at transcription start sites, CpG islands,

DNase-hypersensitive sites and gene-dense regions, suggesting

that provirus integration may influence transcription in the host

cell.

*

Thus laboratories handling or culturing human xenografts

should monitor for the presence of MLV, and should consider

monitoring personnel for viral antigens or antibodies to them.

Laboratories working with xenograft cultures should have full

knowledge and understanding of the potential biological and

biohazardous risks and should not distribute or publish their

findings without full disclosure of the virus status of their xenograft-

derived materials.

***

Kommentar:

Konklusjon: Humane cellekulturer som blir derivert etter xenograft fra mus inneholder og frigjør smittsom XMRV. Laboratorier som arbeider med xenograft deriverte humane cellekulturer bør være seg bevisst over forurensingen/kontaminering med potensielt fare for biologisk smittefarlig materiale (biohazard) fra humantrofiske virus med opprinnelse fra mus og evnen varianter av MLVs har til å bli horisontalt spredt og infisere andre kulturer.

Konklusjonen fra denne studien slår fast at det er omfattende kontaminering og dertil også smittefare med hensyn til gammaretrovirus av ulike trofiske varianter av MLVs i laboratorier som har håndtert cellelinjer xenograft cellelinjer som brukes i kultur. Finnes disse er også risiko for at de infiserer andre humane cellelinjer som ikke er av denne typen.

Thus laboratories handling or culturing human xenografts should monitor for the presence of MLV, and should consider monitoring personnel for viral antigens or antibodies to them.

Laboratories working with xenograft cultures should have full knowledge and understanding of the potential biological and biohazardous risks and should not distribute or publish their findings without full disclosure of the virus status of their xenograft-derived materials.

I forskningssammenheng og cellekultur hvor en tester for XMRV og andre gammaretrovirus kan dette resultatet tolkes begge veier. Risiko for kontaminering er stor og høy nøyaktighet med prøvemateriale og håndtering av cellekulturer kan i verste fall medføre smitterisiko og spredning av MLVs. I testsammenheng er det fare for falsk positiver om ikke en er varsom og oppmerksom på hvor lett prøvemateriale i en kultur blir infisert av gammaretrovirus.

Om en anser/betrakter kroppen våres som en eneste stor cellekultur, kan en ikke utelukke at smitte fra laboratorier har spredt biologisk smitterisiko.

Så lenge vi snakker om at dette er retrovirus som har sitt opphav fra mus, kan en ei heller utelukke smitte i naturlig setting.

For WPIs med samarbeidspartneres orginalstudie Lombardi et al (2009) kan en anta at denne studien enten verifiserer dataene dersom alle cellelinjer og reagenser er fri for forurensning og hvis ikke er da et resultat av laboratorieforurensing.

For mer info om XMRV og andre gammarelaterte virus kan du lese disse innleggene:

Info om: Hva er ett retrovirus og hvorfor er de unike?

Info om: Mer om MLVs og XMRV


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Potensiell fare for biohazard av gammaretrovirus

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En kommentar om “XMRV nytt: Systematisk studie av Zhang et al juni 2011 på hyppighet av XMRV forekomst i humane cellelinjer og bevis for en horisontal virusspredning til andre cellekulturer/cellelinjer

  1. Tilbaketråkk: Vaksiner-Autisme-Neuroimmunologiske sykdommer-XMRV « ToTo NeuroImmunologisk Kurativ Behandling

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