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Research
Cite this article: Shioda S et al. 2018
Screening for 15 pathogenic viruses in humancell lines registered at the JCRB Cell Bank:
characterization of in vitro human cells by viral
infection. R. Soc. open sci. 5: 172472.
http://dx.doi.org/10.1098/rsos.172472

Screening for 15 pathogenic
viruses in human cell lines
registered at the JCRB Cell
Bank: characterization ofin vitro human cells by viral
infection
Setsuko Shioda1 , Fumio Kasai1 , Ken Watanabe2 , Kohei
Kawakami1 , Azusa Ohtani1 , Masashi Iemura1 , Midori

Received: 5 January 2018
Accepted: 23 March 2018

Ozawa1 , Akemi Arakawa1 , Noriko Hirayama1 , Eiko
Kawaguchi1 , Tomoko Tano1 , Sayaka Miyata1 ,Motonobu Satoh1 , Norio Shimizu2 and Arihiro Kohara1
1 Japanese Collection of Research Bioresources (JCRB) Cell Bank, Laboratory of Cell

Subject Category:
Cellular and molecular biology
Subject Areas:
cellular biology
Keywords:
virus test, cell culture, in vitro virus infection,
viral tropism, cellular characteristics

Author for correspondence:
Fumio Kasai
e-mail : k-230@umin.ac.jp

Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.
figshare.c.4068770.

Cultures, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka,Japan
2 Department of Virology, Medical Research Institute, Tokyo Medical and Dental
University, Tokyo, JapanFK, 0000-0001-5933-7714
Human cell lines have been used in a variety of research fields
as an in vitro model. These cells are all derived from humantissue samples, thus there is a possibility of virus infection.
Virus tests are routinely performed in clinical practice, but
are limited in cell lines. In this study, we investigated 15
kinds of viruses in 844 human cell lines registered at theJapanese Collection of Research Bioresources (JCRB) Cell Bank.
Our real-time PCR analysis revealed that six viruses, EBV,HTLV-1, HBV, B19V, HHV-6 and HHV-7, were detected in
43 cell lines. Of them, 20 cell lines were transformed byintentional infection in vitro with EBV or HTLV-1. Virusesin the other 23 cell lines and one EBV transformed cell line
are derived from an in vivo infection, including five de novo
identifications of EBV, B19V or HHV-7 carriers. Among them,
17 cell lines were established from patients diagnosed withvirus-associated diseases. However, the other seven cell lines
originated from in vivo cells unrelated to disease or cellulartropism. Our approach to screen for a set of 15 viruses in eachcell line has worked efficiently to identify these rare cases. Virus

2018 The Authors. Published by the Royal Society under the terms of the Creative Commons
Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted
use, provided the original author and source are credited.

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tests in cell lines contribute not only to safety assessments but also to investigation of in vivo viral
infection which can be a characteristic feature of cell lines.

2. Material and methods
2.1. Cell lines
Cell lines examined in this study are registered with the JCRB Cell Bank and are listed in the electronic
supplementary material, table S1. These cells had been authenticated by short tandem repeat (STR)
analysis and had been confirmed as mycoplasma-negative in our routine quality control. Certain cell................................................

A number of human cell lines have been established from the various tissues of normal individuals,
as well as patients with a range of cancers. These resources have been used in a broad research area,
including as an experimental model for drug development and to gain a deeper understanding ofmolecular pathogenesis. General guidelines for the use of cell lines are provided to obtain reliable datafrom experiments using cultured cells [1,2]. Authentication failures caused by cross-contamination or
misidentification and mycoplasma contamination are fundamental issues, which affect the quality ofmaterials in cell culture [3–6]. However, these concerns can be avoided by following the guidelines inaccordance with best practices [7,8].
A diverse range of viruses have been identified within specific tissues and reported in relation todisease pathogenesis [9]. Human tissue samples are often subjected to viral infection in diagnostic
laboratories and certain viral tests are performed in routine clinical practice. For example, the humanherpesvirus family is known as a common pathogen that causes disease in humans, and has been
investigated in clinical samples [10]. A method of detecting the herpesviruses has been establishedbased on multiplex real-time PCR, and this assay has been applied in clinical samples, demonstrating
a robust screening method [11]. Although human cell lines could potentially carry a virus through anestablishment, little attention has been paid to the possibility of viral infections in cell samples.
When viruses enter the human body, they proliferate in susceptible cells and establish an infection.
This can cause illness with clinical symptoms such as fever, rash or headache, leading to a certain typeof infectious disease unique to infected tissues. Alternatively, viruses are often observed in a latent state,
which is sometimes related to reactivation [12]. Viruses are structurally classified by genome types, DNA
and RNA, which are represented by herpesviruses and retroviruses, respectively (table 1). DNA viruses
retain the ability to repair mismatched base pairs, exhibiting low diversity. RNA viruses are sorted
into two groups distinguished by the presence or absence of reverse transcriptase. Because of reverse
transcription of the viral RNA into DNA, retroviruses, such as human T-cell leukaemia virus (HTLV)and human immunodeficiency virus (HIV), can be detected in cellular DNA. These RNA viruses are
known to be integrated in host cell genome DNA after infection.
Certain types of viruses are employed to induce cellular oncogenesis and immortalization [13],represented by Epstein–Barr virus (EBV), discovered as the first human oncogenic virus. EBVtransformed lymphoblastoid cell lines are conventionally established from patients with congenital
anomalies to provide continuous supplies of genomic DNA as a valuable resource [14]. In contrast to
viruses experimentally used for transformation in vitro, some viruses originated from in vivo infection
are detected in certain cell lines. It is reported that HHV-8 was detected in human cell lines specificallyderived from primary effusion lymphomas [15]. Screening for seven kinds of viruses has been performedin 460 primate cell lines, revealing that some samples were positive for EBV by PCR but negative by
southern blot [16]. This implies that the presence of viruses in cell lines can be more accurately detectedby PCR.
Bacterial contamination is visually found in cell cultures through a conventional light microscope,and can be prevented using antibiotics. However, virus detection requires molecular analysis, and,once cells have become infected the viruses cannot be easily removed from the cells. Datasets fromvirus examination in cell lines contribute to the safe management of cell culture and characterizationof cell lines. In this report, we have screened for 15 viruses in 844 human cell lines registered with theJapanese Collection of Research Bioresources (JCRB) Cell Bank and have detected six different viruses in
43 cell lines.

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1. Introduction

2

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genometype
DNAviral DNA
test◦

viral tropism
monocyte, epithelial cells,
endothelial cells

related diseasebirth defects, cytomegalic inclusion disease

B-cell, epithelial cells

infectious mononucleosis, Burkitt lymphoma,
nasopharyngeal cancer, CNS lymphoma,
leukaemia◦

viral RNA
test.........................................................................................................................................................................................................................

EBV

DNA

.........................................................................................................................................................................................................................

HHV-6

DNA

T-cell, NK cell

exanthem subitum (HHV-6B)

◦

HHV-7

DNA

T-cellexanthem subitum, febrile convulsions

◦

BKV

DNA

reno-urinary tract

haemorrhagic cystitis after allogeneic
haematopoietic stem cell transplantation◦

progressive multifocal leucoencephalopathy in
HIV-AIDS, haemorrhagic cystitis after allogeneic
haematopoietic stem cell transplantation◦

haemorrhagic cystitis after allogeneic
haematopoietic stem cell transplantation◦

erythema infectiosum, arthritis, chronic anaemia
etc.

◦.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

.........................................................................................................................................................................................................................

JCV

DNA

reno-urinary tract, brain.........................................................................................................................................................................................................................

ADV

DNA

genitourinary tract.........................................................................................................................................................................................................................

B19V

DNA

erythroid cells, skin, liver.........................................................................................................................................................................................................................

HBV

DNA

hepatocyte

hepatitis, cirrhosis and hepatocellular carcinoma◦

HTLV-1RNA

T-cellhuman T-cell leukaemia◦

HTLV-2RNA

T-cellhuman T-cell leukaemia

◦

◦

HIV-1

RNA

CD4 (+) T-cell

AIDS

◦

◦

HIV-2

RNA

CD4 (+) T-cell

AIDS

◦

◦

HAV

RNA

hepatocyte

hepatitis

◦

HCV

RNA

hepatocyte

hepatitis, cirrhosis and hepatocellular carcinoma◦.........................................................................................................................................................................................................................

◦.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

lines carrying the same name appear with different registration numbers. These cell lines were depositedby various depositors through different routes, and would normally have the same characteristics, butthere is the possibility of subtle strain differences.

2.2. Microbiological tests
The residual cells from every freezing lot were inoculated into two tubes and one agar plate (NBmedium—nutrient broth with 2% yeast extract, thioglycollate medium, 1-blood agar plate) for a sterility
test. These tubes and plates were incubated at 35°C for 2 weeks to examine bacterial and fungal
contamination.

2.3. DNA and RNA extraction
Total DNA and RNA samples were extracted from each cell line with the AllPrep DNA/RNA Minikit (QIAGEN, Germany, 80204). Some DNA was prepared by a conventional protocol using phenol–
chloroform. When extracting RNA for copy number titration, DNA contamination was eliminated bydigestion with RNase-free DNase I (QIAGEN, Germany, 79254) at room temperature.

2.4. Detection of viral DNA with real-time PCR
The viral DNA test was targeted to nine and four kinds of DNA and RNA viruses, respectively(table 1), CMV, EBV, BKV, JCV, HHV-6, HHV-7, ADV, B19V, HBV, HTLV-1, HTLV-2, HIV-1 and HIV-2,................................................

name
CMV

3

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Table 1. List of pathogenic viruses examined in human cell lines. CMV, cytomegalovirus; EBV, Epstein-Barr virus; HHV-6, humanherpesvirus 6; HHV-7, human herpesvirus 7; BKV, human polyomavirus BK; JCV, human polyomavirus JC; ADV, human adenovirus; B19V,
human parvovirus B19; HBV, hepatitis B virus; HTLV-1, human T-cell leukaemia virus type 1; HTLV-2, human T-cell leukaemia virus type 2;
HIV-1, human immunodeficiency virus 1; HIV-2, human immunodeficiency virus 2; HAV, hepatitis A virus; HCV, hepatitis C virus.

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Viral RNA was examined for six kinds of RNA viruses (table 1), HAV, HCV, HTLV-1, HTLV-2, HIV-1
and HIV-2. The reaction mixture using the One-Step PrimeScript RT-PCR Kit (TaKaRa, Japan, RRO64A)
was prepared by adding 100 ng of total RNA following the manufacture’s protocol. Primers and TaqMan
probes are shown in the electronic supplementary material, table S3. PCR was carried out using theApplied Biosystems 7300 Real-Time PCR System under conditions consisting of 42°C for 5 min and 95°C
for 10 s followed by 50 cycles of 95°C for 5 s and 60°C for 34 s.

2.6. Reverse Transcriptase Assay
Some cell lines were transfected with the luciferase expressing gene using retrovirus or lentivirus vectors.
To examine lentivirus replication, the cultured medium of these cell lines was centrifuged at 250g for
10 min at 4°C to remove cell debris. The supernatant was concentrated by centrifugation (22 000g) for 2 h
at 4°C, then the pellets were analysed by the Reverse Transcriptase Assay (Roche, Germany, 11468120910)
as described by the manufacturer.

3. Results
Our virus test detected six viruses, EBV, HTLV-1, HBV, B19V, HHV-6 and HHV-7, in 43 cell lines (table 2).
One of them, AT(L)7KY, was positive for both EBV and HHV-7. Six cell lines are sublines, corresponding
to three individuals, owing to duplicated registrations, different names or misidentification. The othernine viruses were not found in any of the 844 cell lines. Whole results are listed in the electronic
supplementary material, table S1 in which positive data are shown by Ct value or DNA copy number.
All cell lines were negative for bacterial microorganisms.

3.1. EBVThe most common virus we detected in human cell lines was EBV, found in 30 cell lines, all of which
are derived from blood cells (table 3.1). Two of them, P32/ISH and ITSM, have not been reported to
be EBV positive and have been identified in this study. EBV is closely associated with endemic Burkitt
lymphoma, but this is low in Japanese cases [18]. Six of nine Burkitt lymphoma cell lines, Daudi, HSSultan, Namalwa, NC-37, P32/ISH and RAJI, were positive for EBV in contrast to three negative celllines, KHM-10B, Minami-2 and Ramos (RA1). NC-37 is a misidentified cell line and has been revealed
as the derivative of RAJI. Except for P32/ISH, the other five positive cell lines different from Japaneseorigin showed a high copy number of EBV. P32/ISH, as described previously [19], has an immature
B-cell phenotype on the basis of immunological surface marker analysis. EBV nuclear antigen andterminal deoxynucleotidyl transferase activity were negative, which would be related to low copy
number of EBV. The other eight EBV-positive cell lines were established from different haematologicaldiseases, leukaemia, multiple myeloma, lymphoma, pseudomyxoma peritonei or severe chronic active
EBV infection. One of them, KAI3, is non-B-cell and has been reported as an NK-like cell line [20]. RPMI
1788 was not associated with any disease and was established from normal cells, which would be EBV
positive in vivo.
Compared with these 14 EBV-positive cell lines originated from in vivo infection, 16 lymphoblastoid
cell lines were established from in vitro transformation of peripheral blood mononuclear cells using EBV.
They were obtained from patients with retinoblastoma and their parents, or patients with a rare diseasesuch as Bloom syndrome, ataxia telangiectasia or xeroderma pigmentosum. Different from these cell lines................................................

2.5. Detection of viral RNA with One-Step RT-PCR

4

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and performed using customized 96-well plates inoculated with primers and probes in trehalosesolution [17]. This format included two sets of multiplex PCR amplification: one for BKV and JCV,the other for B19V and HBV. Primers and TaqMan probes designed for each virus are shown in theelectronic supplementary material, table S2. An aliquot of 50 µl of reaction mixture consisting of 500 ng
of the cellular DNA with AmpliTaq Gold DNA Polymerase (Applied BioSystems, USA, N8080245) orTaq DNA Polymerase (Thermo Fisher Scientific, USA, EP0406) was added to each well. The assay was
conducted using the Applied Biosystems 7300 Real-Time PCR System, with an initial denaturation at
95°C for 10 min, followed using 50 cycles of 95°C for 15 s and 60°C for 1 min for the AmpliTaq Gold DNA
Polymerase or with an initial denaturation at 95°C for 1 min followed by 45 cycles of 95°C for 10 s and
60°C for 30 s for Taq DNA Polymerase.

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Table 2. Summary of virus screening in human cell lines.

5

EBV

30*

12

2

16

HTLV-1

6

1

1

4

HBV

4

4

disease unrelatedtransformed in vitro.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

B19V

2

2

HHV-6

1

1

HHV-7

1*

1

*double positive−1

subtotal

43

negative

801

total

844

.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.................................................................................
.................................................................................
.........................................................................................................................................................................................................................

17

7

20

.........................................................................................................................................................................................................................

related to inherited diseases, TK6(IVGT) is characterized by being heterozygous for thymidine kinaseand has been used for in vitro mutagenesis assays [21]. EBV-transformed cell lines generally proliferate
well, and high copy number of EBV was detected.

3.2. HTLV-1
HTLV-1 was detected in six cell lines, one lung cancer and four leucocytes (table 3.2). KHM-3S wasestablished from pleural effusion of a patient with small cell lung cancer and showed negative forleucocyte common antigen [22], indicating its unique tropism. MT-1 was derived from a patient withadult T-cell leukaemia (ATL), which shows strong association with HTLV-1 [23]. These two cell lines
originated from an in vivo infection. MT-2, MT-3 and MT-4 were established by co-culture with lymphoidcells from ATL patients and were infected with HTLV-1 in vitro [24]. The viral RNA was detected fromthese six cell lines and the DNA showed a high copy number. Five types of HTLV-1 proviral sequenceswere detected in 11 different sites of the MT-2 genome, indicating the integration [25]. It is reported thatthese MT-1, MT-2, MT-3 and MT-4 cell lines are virus-producing cell lines [26].

3.3. HBVHBV was detected from three hepatoma cell lines, huH-1, JHH-7 and PLC/PRF/5 (table 3.3). PLC/PRF/5
is also known as Alexander cells, registered as IFO50069. The viral DNA copy number varied between
these cell lines. It is reported that huH-1 and PLC/PRF/5 carry hepatitis B surface antigen (HBsAg) [27].
Although the target region of our real-time PCR is the S gene coding for HBsAg of the virus envelope,
the antigen was not detected in JHH-7 [28]. Two integration sites composed of incomplete HBV genome
are identified in the JHH-7 genome [28]. Other hepatoma cell lines without HBsAg, JHH-1, -2, -4, -5
and -6, were negative for HBV [28], indicating that JHH-7 exhibits a distinctive feature in the HBVpositive cells.

3.4. B19V
B19V was detected in two cell lines, FPC5JTO and XP2SA (table 3.4), which could be the first cell lines
positive for B19V, originated from in vivo infection. These cell lines were fibroblast derived from theskin of patients with hereditary diseases, carrying a high cancer risk. Different from the EBV, HTLV-1
and HBV detected mostly in cancer cell lines, the two B19V-positive cell lines have no association withdiseases. Their copy numbers were low, which would be related to their primary cells. Although B19V
can propagate in a leukaemia cell line, UT-7 [29], the positive cell lines have not been registered with any
major public cell banks.

................................................

number of cell lines

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Table 3. List of virus-positive cell lines. Bold italic names indicate cell lines first identified as virus positive in this report. Bold italic origins
show different tropism compared to available data.

.........................................................................................................................................................................................................................

1.1. Burkitt lymphoma.........................................................................................................................................................................................................................

Daudi

B lymphocyte

JCRB9071

HS-Sultan

B lymphocyte

JCRB9062

Namalwa

B lymphocyte

IFO50040

NC-37

Identical STR profile with the RAJI cell line

IFO50039

P32/ISH

B lymphocyte

JCRB0095a

B lymphocyte

JCRB9012.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

a.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

RAJI.........................................................................................................................................................................................................................

1.2. leukaemia, myeloma, lymphoma.........................................................................................................................................................................................................................

CCRF-SB

B lymphoblastoid leukaemia

JCRB0032

IM-9

B lymphoblastoid multiple myeloma

JCRB0024

KAI3

NK-like cell line from a patient with severe chronic active EBV infection

IFO50518

RM-P1

B cell primary effusion lymphoma

JCRB1650

SLVL

B cell lymphoma

JCRB0159

WIL2-NS

B lymphocyte

JCRB9063.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

1.3. disease-unrelated in vivo infection.........................................................................................................................................................................................................................

ITSM

pseudomyxoma peritonei, cecum

JCRB1635

RPMI 1788

peripheral blood from normal individual

JCRB0035.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

1.4. in vitro EBV transformed.........................................................................................................................................................................................................................

AT(L)5KY

peripheral blood, ataxia telangiectasia patient

JCRB0331

AT(L)6KY

peripheral blood, ataxia telangiectasia patient

JCRB0332

AT(L)7KY

peripheral blood, ataxia telangiectasia patient, HHV-7 positive

JCRB0333

BSL2KA

peripheral blood, Bloom syndrome patient

JCRB1042

GM1526

peripheral blood, ataxia telangiectasia patient

JCRB3008

HLCL-1

peripheral lymphocyte, normal

JCRB0041.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

KCMC-1528

peripheral blood, Rubinstein–Taybi syndrome patient

JCRB1541

NCC-CoC-K115B

peripheral blood, colon cancer patient

JCRB1339

RB182F(L)peripheral blood, father of retinoblastoma patient

JCRB3038

RB257F(L)peripheral blood, father of retinoblastoma patient

JCRB3037

RBL162T

peripheral blood, retinoblastoma patient

JCRB0328

RBL182T

peripheral blood, retinoblastoma patient

JCRB0330

RBL221T

peripheral blood, retinoblastoma patient

JCRB0329

TK6(IVGT)

lymphoblast

JCRB1435

XPL15OSperipheral blood, xeroderma pigmentosum patient

JCRB0307

XPL3KA

peripheral blood, xeroderma pigmentosum patient

JCRB0306.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

2. HTLV-1-positive cell lines.........................................................................................................................................................................................................................

KHM-3S

small cell lung cancer

JCRB0138

MT-1

lymphoid cell line from adult T-cell leukaemia, low level HTLV-1 producer

JCRB1209

MT-2

human cord leucocyte cell line established by co-cultivation with human ATL cells

JCRB1210

MT-3

human cord leucocyte cell line established by co-cultivation with human ATL cells

JCRB1217.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

(Continued.)................................................

JCRB registration no.

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cell nameorigin of tissue or cell
1. EBV-positive cell lines

6

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Table 3. (Continued.)
JCRB registration no.

human cord leucocyte cell line established by co-cultivation with human ATL cells

JCRB0135bhuman cord leucocyte cell line established by co-cultivation with human ATL cells

JCRB1216.........................................................................................................................................................................................................................

MT-4.........................................................................................................................................................................................................................

3. HBV-positive cell lines.........................................................................................................................................................................................................................

Alexander cellsc

hepatoma, HBs antigen-positive

IFO50069

huH-1

hepatoma, HBs antigen-positive

JCRB0199

hepatoma

JCRB1031

hepatoma, HBs antigen-positive

JCRB0406.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

JHH-7.........................................................................................................................................................................................................................

c

PLC/PRF/5.........................................................................................................................................................................................................................

4. B19V-positive cell lines.........................................................................................................................................................................................................................

FPC5JTOskin fibroblast of familial polyposis coli patient

JCRB0318

XP2SA

skin fibroblast of xeroderma pigmentosum (variant type) patient

JCRB0305.........................................................................................................................................................................................................................
.........................................................................................................................................................................................................................

5. HHV-6-positive cell line.........................................................................................................................................................................................................................

HUV-EC-C

human vascular endothelial cell

IFO50271.........................................................................................................................................................................................................................

6. HHV-7-positive cell line.........................................................................................................................................................................................................................

AT(L)7KY

peripheral blood, ataxia telangiectasia patient, EBV transformed

JCRB0333.........................................................................................................................................................................................................................

Six cell lines are sublines consisting of three different in vivo origins.
a Identical origin because of misidentification in NC-37.
b Different registration numbers due to independent depositions.
c PLC/PRF/5 has also been used as Alexander cells.

3.5. HHV-6
HHV-6 was detected in a human vascular endothelial cell line, HUV-EC-C, derived from normal
umbilical cord tissue (table 3.5). We have previously reported that chromosomally integrated HHV-6B
has been identified at the distal end of chromosome 9q in this particular cell line [30]. The copy number
is calculated to be one copy per cell, indicating that the integration could have occurred in the germ line.
The viral integration in the HUV-EC-C cell line would be caused by in vivo infection. This is distinct fromother HHV-6-positive cell lines generated by in vitro transfection [31].

3.6. HHV-7
HHV-7 was detected in one cell line, AT(L)7KY, which was not previously reported to be positive. This
cell line is a B-cell line transformed by EBV, indicating that both EBV and HHV-7 can be detected in thiscell line (table 3.6). It was established from peripheral blood mononuclear cells of a patient with ataxia
telangiectasia, which is not associated with HHV-7 infection. Different from HHV-7-infected cell linestransduced with an adenovirus vector [32], AT(L)7KY is, to the best of our knowledge, the first report ofan HHV-7-positive cell line likely to be originated from in vivo infection which remains detectable in cell
culture.

3.7. HIV-1 and HIV-2
A very small copy number of DNA fragments targeting the gag gene of HIV-1 and LTR of HIV-2 weredetected in some of the luciferase-expressing cell lines (electronic supplementary material, table S4).
However, these viral RNAs were not detected in these cell lines, and reverse transcriptase activity
was below the detection limit. The absence of the RNA and reverse transcriptase in these cell lines
confirmed that the cell lines are negative for HIV-1 and HIV-2. The luciferase gene was introduced
into these cell lines by the lentiviral vector [33], which may be associated with the detection by PCR.
Because HIV-1 and HIV-2 are highly pathogenic and must be handled under biosafety level 3 (BSL3),
such cell lines are not received and distributed from our JCRB Cell Bank. All cell lines examinedin this study are free of HIV-1 and HIV-2, which minimizes safety concerns over the use of ourcell lines.

................................................

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4. Discussion

This work did not involve direct access to any human samples and is not subject to human ethics review.
Data accessibility. The datasets supporting this article have been uploaded as the electronic supplementary tables.

................................................

Ethics. This work is based on the analysis of publicly available materials, which are registered with the JCRB Cell Bank.

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Virus infection is one of the major public health concerns and is related to disease pathogenesis [34].
Virus tests have been established for clinical samples in diagnostic laboratories [9–11]. These tests arealso routinely performed on donated blood to avoid infections through blood products. Blood samples
are examined mainly by targeting antigens or antibodies detected in plasma. Positive results from theseindirect methods identify individuals carrying a viral infection, but do not specify which cells carrythe virus. Different from those on in vivo samples, our virus tests for cell lines detect viral DNA or
RNA in the cytoplasm or the nuclei. The presence of these viral components in cells can be a molecularcharacterization of the cell line.
Our previous study demonstrated that chromosomally integrated HHV-6B has been identified in ahuman vascular endothelial cell line, HUV-EC-C (IFO50271) [15]. The analysis of the HUV-EC-C cells
reveals that integration of HHV-6B is stable during long-term culture of HUV-EC-C, despite genomic
instability in the host cell. A complete HHV-6 genome can be present as the form of integration into
the host germ line genome [35], leading to the unexpected detection in different cell types of tropism
observed as the HUV-EC-C cell line. It was reported that a human B cell line, RPMI 1788, was establishedfrom the peripheral blood of a normal donor and labelled as normal cells [36], however this cell line wasfound to be EBV positive in another study [37]. This suggests that lymphocytes had been infected with
EBV in vivo, leading to establishment of the immortalized cell line. These examples show that further
investigation of virus-positive cell lines could lead to an understanding of viral dynamics and that suchcell lines can serve as invaluable research materials. Virus tests contribute to the characterization ofcell lines and elucidation of cellular origins. Because each cell line is derived from a specific tissue andestablished from a certain cell population in the sample, cell lines that show negative on virus tests are
not necessarily derived from uninfected individuals.
Cells in vitro always have a risk of viral infection in the culture environment because, by themselves,
they lack a defence system against infection. Assessment of virus contamination in cells is undertaken forthe purpose of risk management [38]. When cell lines are used as biological products in the biotechnology
industry, quality control is strictly maintained during production [39]. Examination of the parainfluenza
virus has been performed in 123 human cell lines without any such infection being detected, assuringthat those lines are free from the infection [40]. Another study of a screen for murine leukaemia viruses in
577 human cell lines reported that 17 of these cell lines produced active retroviruses [41]. This implies thathuman cell lines have a risk of viral contamination through transplantation of cells into mice, which isdistinguished from in vivo infection characterizing cell lines. The use of human cell lines is accompanied
by a potential biohazard, because virus tests are limited to certain species and do not cover all pathogens.
It is recommended that human cell lines are primarily handled at a biosafety level 2 laboratory, regardlessof results from virus tests. Especially, cell lines which produce virus particles need careful handling andtheir application should be restricted. Each virus has distinct host cell tropisms, implying that negative
results would be expected from the majority of cell lines. Because it is not clear that viral tropism isidentical between in vivo and in vitro environments, we applied the whole set of virus tests to all cell
types. Apart from the cell lines transformed using EBV or HTLV-1 by in vitro infection, 22 cell lines had
been infected with virus in vivo before their establishment. Of them, two cell lines are inconsistent withthe cellular tropism, showing unexpected results.
It is reported that induced pluripotent stem cells can be established from EBV-transformed
lymphoblastoid cell lines, and that the reprogrammed cells have been shown to be EBV free [42,43]. Thissuggests that a change in cell type could lead to elimination of viruses infecting cell lines. These viruses
might present in the form of episomes in cells, which are different from integration into host genomes.
Differences in the form of viruses in cells would be related to the amount of virus present during cell
culture. In this report, human papillomavirus (HPV) is excluded because it has diverse genotypes related
to malignant potential, which we will analyse in a future study. Analyses of RNA sequencing data led
to identification of EBV, HHV-8 and HTLV-1 in lymphoma cell lines [44]. DNA sequence data from alarge number of human cancer cell lines have been accumulated in the Cancer Cell Line Encyclopedia(CCLE) [45] and the Catalogue of Somatic Mutations in Cancer (COSMIC) [46]. Virus-positive cell lines
will be identified from these databases, and bioinformatics approaches will help to investigate viral
integration sites and genotypes.

8

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Authors’ contributions. S.S., F.K., N.S. and A.K. designed the study; S.S., K.W., K.K., A.O., M.I., M.O., A.A., N.H., T.T., S.M.,

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