KI is a novel polyomavirus identified in the respiratory secretions of children with acute respiratory symptoms. Whether this reflects a causal role of the virus in the human respiratory disease remains to be established. To investigate the presence of KIV in the respiratory tissue, we examined 20 fresh lung cancer specimens and surrounding normal tissue along with one paranasal and one lung biopsy from two transplanted children. KIV-VP1 gene was detected in 9/20 lung cancer patients and 2/2 transplanted patients. However, amplification of the sequence coding for the C-terminal part of the early region of KIV performed on the 11 positive cases was successful only in two malignant lung tissues, one surrounding normal tissue, and 1/2 biopsies tested. Phylogenetic analysis performed on the early region of KIV (including the four Italian isolates), BKV and JCV revealed the presence of three distinct clades. Within the KIV clade two sub-clades were observed. A sub-clade A containing the four Italian strains, and a sub-clade B comprising the Swedish and Australian isolates. Interestingly, the two Italian strains identified in normal tissue clustered together, whereas those detected in malignant tissue fell outside this cluster. In vitro studies are needed to investigate the transforming potential of KIV strains. J. Med. Virol. 81:558-561,2009. (C) 2009 Wiley-Liss, Inc

Aquaro, S

Babakir-Mina, M

Ciccozzi, M, Campitelli, L

Ciotti, M

Lo Coco, A

Perno, CF

Ciccozzi, M

Perno, C

Cineca Institutional Research Information System

Identification of the novel KI polyomavirus in paranasal and lung tissues

Archivio della Ricerca - Università di Roma Tor vergata

Journal of Medical Virology 81:558–561 (2009)
Identification of the Novel KI Polyomavirus in
Paranasal and Lung Tissues
Muhammed Babakir-Mina,1 Massimo Ciccozzi,2 Laura Campitelli,2 Stefano Aquaro,3
Achille Lo Coco,4,5 Carlo Federico Perno,1,5 and Marco Ciotti1*
1Laboratory of Molecular Virology, University Hospital Tor Vergata, Rome, Italy
2Department of Infectious, Parasitic and Immunomediated Disease, Istituto Superiore di Sanita’, Rome, Italy
3Department of Pharmaco-Biology, University of Calabria, Rende, Italy
4Thoracic Surgery, Santo Spirito Hospital, Pescara, Italy
5Department of Experimental Medicine and Biochemical Science, University of Rome Tor Vergata, Rome, Italy
KI is a novel polyomavirus identified in the
respiratory secretions of children with acute
respiratory symptoms. Whether this reflects a
causal role of the virus in the human respiratory
disease remains to be established. To investigate
the presence of KIV in the respiratory tissue, we
examined 20 fresh lung cancer specimens and
surrounding normal tissue along with one para-
nasal and one lung biopsy from two transplanted
children. KIV-VP1 gene was detected in 9/20 lung
cancer patients and 2/2 transplanted patients.
However, amplification of the sequence coding
for the C-terminal part of the early region of KIV
performed on the 11 positive cases was success-
ful only in two malignant lung tissues, one
surrounding normal tissue, and 1/2 biopsies
tested. Phylogenetic analysis performed on the
early region of KIV (including the four Italian
isolates), BKV and JCV revealed the presence of
threedistinct clades. Within the KIV clade two sub-
clades were observed. A sub-clade A containing
the four Italian strains, and a sub-clade
B comprising the SwedishandAustralian isolates.
Interestingly, the two Italian strains identified in
normal tissue clustered together, whereas those
detected in malignant tissue fell outside this
cluster. In vitro studies are needed to investigate
the transforming potential of KIV strains. J. Med.
Virol. 81:558–561, 2009.  2009 Wiley-Liss, Inc.
KEY WORDS: KIV polyomavirus; human
tumors; molecular epidemio-
logy
INTRODUCTION
Polyomaviruses are small non-enveloped double-
stranded DNA viruses that can infect mammals and
birds. Until recently, two human polyomaviruses BKV
[Gardner et al., 1971] and JCV [Padgett et al., 1971]
were known to infect human beings. Both viruses are
widespread in the human population, establish a latent
infection in the kidney [Chesters et al., 1983], and can be
detected in the urine of healthy individuals, where
infection is asymptomatic [Brown et al., 1975].
In immunocompromised patients, reactivation of
BKV and JCV may cause severe disease. JCV can
cause a demyelinating disease known as progressive
multiphocal leukoencephalopathy (PML) which occurs
mainly in AIDS patients [Gordon and Khalili, 1998].
BKV can be responsible of a tubular nephritis, which can
lead to allograft failure in renal transplant recipients
and hemorrhagic cystitis in hematopoietic stem cell
transplant recipients [Nickeleit et al., 2002; Hirsch and
Steiger, 2003; Chen et al., 2004].
Polyomaviruses BKV, JCV, and SV40 have been
detected in human tumors [Sariyer et al., 2004; Giuliani
et al., 2007, 2008] and induce numerous tumors in
experimental animals [Sariyer et al., 2004]. The mech-
anism by which the oncogenic transformation is
achieved is through the action of the early proteins
large T antigen (T-Ag) and small t antigen (t-Ag). The T-
Ag binds the oncosuppressor proteins p53 and pRb, two
key regulators of cell cycle progression [Bollag et al.,
1989; Cress and Nevins, 1996] inhibiting apoptosis
and driving the cell into S phase. The small t antigen
interacting with the protein phosphatase 2A stimulates
the map kinase pathway and induces cell proliferation
[Sontag et al., 1993]. This aberrant stimulation of the
cell cycle is responsible for oncogenic transformation.
Recently, a new human polyomavirus, KI polyoma-
virus (KIV) has been identified in the respiratory
secretions of children with acute respiratory symptoms
*Correspondence to: Marco Ciotti, Laboratory of Molecular
Virology, University Hospital Tor Vergata, Viale Oxford, 81-00133
Rome, Italy. E-mail: marco.ciotti@ptvonline.it
Accepted 18 November 2008
DOI 10.1002/jmv.21417
Published online in Wiley InterScience
(www.interscience.wiley.com)
 2009 WILEY-LISS, INC.
[Allander et al., 2007]. Debated is its causal role in the
respiratory disease [Bialasiewicz et al., 2007; Norja
et al., 2007; Babakir-Mina et al., 2008] as completely
unknown is its oncogenic potential. The objective of the
study was to determine the presence of KIV in normal
and malignant lung tissue as well as in a normal
paranasal tissue and to compare the early region of KIV
genome, comprising the C-terminal region of the T-Ag
gene and almost the entire t-Ag gene [Allander et al.,
2007] with those of known KIV, JCV, and BKV strains.
MATERIALS AND METHODS
Study Group
A series of 20 fresh lung cancer specimens and
surrounding normal tissue provided by the Department
of Surgery of the Santo Spirito Hospital, Pescara, Italy,
between 2005 and 2006 years were tested for the
presence of the novel polyomavirus KI. These specimens
were already tested for the presence of HPV, SV40, BKV,
JCV, and CMV [Giuliani et al., 2007]. The patients
included 13 men and 7 women and the age ranged from
40 to 85 years, mean age 68. In addition, one paranasal
and one lung biopsy obtained from two thalassemic
transplanted children (13 and 3 years old, respectively)
were included in the study. Informed consent was
obtained from all patients enrolled in the study.
DNA Extraction and Viral Detection
Twenty-five milligrams of normal and malignant
tissue from each patient was digested overnight at
568C with proteinase K and then total DNA extracted
with the QIAamp DNA mini kit according to the
manufacturer’s instruction (Qiagen S.P.A., Milan,
Italy). The DNA was eluted in a volume of 50 ml and
stored at 808C until analysis.
Amplification of the VP1 Gene and
Early Region of KIV Genome
The KIV was detected amplifying a fragment of the
VP1 geneasdescribed [Allander etal., 2007]. The positive
cases were then amplified for the early region of KIV
genome comprising the C-terminal part of T-Ag gene
and the almost entire t-Ag gene [Allander et al., 2007].
The PCR primers were as follows: FT-Ag 50-CCACA-
CAGGTGGTTTTCTATAA-30 (position 4521–4542) and
RT-Ag 50-GCCCCAGCTTTTTCTAGAAGAA-30 (position
5032–5011). The cycling conditions were: 10 min at 948C,
1 cycle; followed by 35 cycles of amplification at 948C for
1 min, 568C for 1 min, and 728C for 2 min. The PCR
products were analyzed on a 2% agarose gel stained with
ethidium bromide and visualized under ultraviolet
light. All the necessary precautions were taken to avoid
contamination.
Sequencing of the Early Region and
Phylogenetic Analysis
The amplified fragments of the early region of KIV were
sequenced using the Genome Lab DTCS Quick Start Kit
(Beckman Coulter, Fullerton, CA) and run on a Beckman
Coulter CEQ2000XL sequence analyzer after column
purification. The obtained sequences were submitted to
the Genebank and matched against all deposited sequen-
ces (http://www.ncbi.nlm.nih.gov/BLAST). An alignment
with a set of reference sequences was obtained using
CLUSTAL X [Thompson et al., 1994] and manually edited
with the Bioedit software [Hall, 1999]. Positions contain-
ing gaps were removed from the final alignment. For our
data set, the best fitting nucleotide substitution model was
tested with a hierarchical likelihood ratio test following
the strategy described by Swofford and Sullivan [2003],
using a neighbor-joining (NJ) base-tree with LogDet
corrected distances. Maximum likelihood (ML) trees were
then inferred with the selected model and ML-estimated
substitution parameters. The heuristic search for the ML
tree was performed using an NJ tree as starting tree and
the TBR branch-swapping algorithm. NJ trees were also
estimated using pair-wise distances inferred by ML with
the best fitting nucleotide substitution model. Calcula-
tions were performed with PAUP* 4.0b10 according to
Swofford and Sullivan [2003]. Statistical support for
internal branches in the NJ trees was obtained by
bootstrapping (1,000 replicates) and with the ML-based
zero-branch-length test for the ML trees [Swofford and
Sullivan, 2003]. Trees were rooted by outgroup rooting
using BKV-ETH-3 strain as outgroup. The accession
numbers of the sequences utilized for the phylogenetic
analysis including those referred to KIV strains identified
in our laboratory are the following: AB263916; AB269841;
AB269826; AB211387; AB269851; AB269822; AB211370;
AY628225; AY628234; AY628232; AY628233; AY628231;
AY628230; AY628229; AY628227; AY628226; AB127343;
AB127018; AY536242; AB372037; AB113124; AY382184;
AB038255; AB212954; AB212952; AF004349; AF030085;
AY536239; EF520287; EF520288; EF520289; EF127906;
EF127907; EF127908; FJ389513; FJ389514; FJ389515;
FJ389516.
RESULTS
Detection of KIV-DNA
KIV-VP1 gene was detected in 9 out of 20 lung cancer
specimens tested (45%) and in 1 case the VP1 gene was
detected either in normal as well as in malignant tissue.
Following this primary screening, a second PCR was
performed on the nine positive cases targeting the early
region of KIV. A positive result was obtained only in
three cases: one surrounding normal tissue (KIV-RM1)
and two malignant tissues (KIV-RM3 and KIV-RM4).
In the two transplanted thalassemic children, the VP1
gene was detected in both cases while amplification of
the early region of the viral genome was positive only in
one case (KIV-RM2).
Phylogenetic Analysis
Phylogenetic analysis revealed the presence of three
main clades, supported by highly significant bootstrap
and P values in the zero-branch-length test. Two
J. Med. Virol. DOI 10.1002/jmv
Presence of KIV in the Respiratory Tissue 559
represented BK and JC polyomaviruses (Fig. 1) while
the third one contained the novel KIV strains. This third
clade can be subdivided into two statistically supported
sub-clades, named A and B, containing the four Italian
isolates and the Swedish and Australian isolates,
respectively (Fig. 1; P< 0.001 in the zero-branch-length
test, Bayesian posterior probability >90%, and boot-
strap support >75%.). Within sub-clade A, KIV-RM1
and KIV-RM2 resulted more closely related compared to
KIV-RM3 and KIV-RM4 isolates which were placed
outside this cluster.
The mean genetic distance between groups (BKV,
JCV, and KIV) was 0.26, whereas within clades A and B
was 0.19 and 0.137, respectively. These values were not
statistically significant. Conserved mutations/motifs
were not observed in the virus infecting the neoplastic
tissue.
DISCUSSION
The novel polyomavirus KIV was first described by
Allander et al. [2007] in the respiratory secretions
of children with acute respiratory symptoms and
later confirmed by other groups around the world
[Bialasiewicz et al., 2007; Babakir-Mina et al., 2008].
Whether this reflects a causal role of the virus in the
respiratory illness is still debated and molecular
epidemiological studies are needed to establish a firm
link with the respiratory disease. Up to now, completely
unexplored is the oncogenic potential of KIV.
The human polyomaviruses BKV and JCV have been
detected in several human tumors [White and Khalili,
2004; Giuliani et al., 2008] including the respiratory
tract [Giuliani et al., 2007; Zheng et al., 2007], and
induce tumors in animal models [Sariyer et al., 2004].
This prompted us to investigate the presence of KIV in
normal and malignant respiratory tissue. PCR analysis
revealed the presence of KIV-VP1 gene in some of the
lung cancer tissues examined and in both normal
biopsies (paranasal and lung) from the transplanted
patients. However, when the same positive samples
were tested for the presence of the early region of the
viral genome, an amplified product was obtained only
in four of the KIV-VP1 positive cases. This apparent
discrepant result might be explained by the nucleotide
variability observed in the C-terminal part of T-Ag gene
of polyomaviruses [Lednicky et al., 1997; White and
Khalili, 2004].
Phylogenetic analysis performed on the early region of
polyomaviruses included in this study showed the
presence of three main clades comprising the BKV,
JCV, and KIV strains, respectively. Interestingly,
within the KIV clade two sub-clades named A and B
were observed. The sub-clade A contained all four
Italian isolates, while sub-clade B included all Swedish
and Australian strains. Among the Italian isolates, both
KIV-RM1 and -RM2 identified in normal tissue (lung
tissue surrounding the tumor and paranasal tissue)
appeared closely related and clustered together,
whereas both the KIV-RM3 and -RM4 strains detected
J. Med. Virol. DOI 10.1002/jmv
Fig. 1. Phlylogenetic analysis of the early region (512 nt) of human
polyomaviruses was carried out using HKYþ IþC (alpha-parame-
ter¼ 0.0763) as the best evolutionary model. Branch lengths were
estimated with the best fitting nucleotide substitution model according
to a hierarchical likelihood ratio test [Posada and Crandall, 1998], and
were drawn in scale with the bar at the bottom indicating 0.3 nucleotide
substitutions per site. One * along a branch represents significant
statistical support for the clade subtending that branch (P<0.001 in
the zero-branch-length test) and bootstrap support>75%. The tree was
rooted using the strain BKV-ETH-3 as outgroup. Sub-clades within the
main KIV clade are indicated with the letters A and B.
560 Babakir-Mina et al.
in lung cancer tissue fell outside this cluster, being more
distantly related to KIV-RM1 and -RM2.
Regarding the genetic distance observed between
sub-clades A and B, it might be related to the
different source of isolation (malignant lung tissue vs.
respiratory secretions), and perhaps reflects a different
biological behavior of the virus in vivo. To test this
hypothesis would be interesting to study the trans-
forming potential of KIV strains in vitro as it is known
that the C-terminal part of the T-Ag of polyomaviruses
retains binding activity for p53 and Rb proteins.
The inactivation of these oncosuppressor proteins by
the T-Ag leads to cell cycle deregulation, inhibition of
apoptosis, and cell transformation. Therefore, in vitro
experiments investigating the transforming potential of
KIV isolates are warranted.
ACKNOWLEDGMENTS
We thank Paola Galati and Amalia Mastrofrancesco
for their technical assistance.
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J. Med. Virol. DOI 10.1002/jmv
Presence of KIV in the Respiratory Tissue 561


Babakir Mina, M

Campitelli, L

Ciotti, M.

AQUARO, STEFANO

PERNO, CARLO FEDERICO

Journal of Medical Virology 81:558–561 (2009)Identification of the Novel KI Polyomavirus inParanasal and Lung TissuesMuhammed Babakir-Mina,1 Massimo Ciccozzi,2 Laura Campitelli,2 Stefano Aquaro,3Achille Lo Coco,4,5 Carlo Federico Perno,1,5 and Marco Ciotti1*1Laboratory of Molecular Virology, University Hospital Tor Vergata, Rome, Italy2Department of Infectious, Parasitic and Immunomediated Disease, Istituto Superiore di Sanita’, Rome, Italy3Department of Pharmaco-Biology, University of Calabria, Rende, Italy4Thoracic Surgery, Santo Spirito Hospital, Pescara, Italy5Department of Experimental Medicine and Biochemical Science, University of Rome Tor Vergata, Rome, ItalyKI is a novel polyomavirus identified in therespiratory secretions of children with acuterespiratory symptoms. Whether this reflects acausal role of the virus in the human respiratorydisease remains to be established. To investigatethe presence of KIV in the respiratory tissue, weexamined 20 fresh lung cancer specimens andsurrounding normal tissue along with one para-nasal and one lung biopsy from two transplantedchildren. KIV-VP1 gene was detected in 9/20 lungcancer patients and 2/2 transplanted patients.However, amplification of the sequence codingfor the C-terminal part of the early region of KIVperformed on the 11 positive cases was success-ful only in two malignant lung tissues, onesurrounding normal tissue, and 1/2 biopsiestested. Phylogenetic analysis performed on theearly region of KIV (including the four Italianisolates), BKV and JCV revealed the presence ofthreedistinct clades. Within the KIV clade two sub-clades were observed. A sub-clade A containingthe four Italian strains, and a sub-cladeB comprising the SwedishandAustralian isolates.Interestingly, the two Italian strains identified innormal tissue clustered together, whereas thosedetected in malignant tissue fell outside thiscluster. In vitro studies are needed to investigatethe transforming potential of KIV strains. J. Med.Virol. 81:558–561, 2009.  2009 Wiley-Liss, Inc.KEY WORDS: KIV polyomavirus; humantumors; molecular epidemio-logyINTRODUCTIONPolyomaviruses are small non-enveloped double-stranded DNA viruses that can infect mammals andbirds. Until recently, two human polyomaviruses BKV[Gardner et al., 1971] and JCV [Padgett et al., 1971]were known to infect human beings. Both viruses arewidespread in the human population, establish a latentinfection in the kidney [Chesters et al., 1983], and can bedetected in the urine of healthy individuals, whereinfection is asymptomatic [Brown et al., 1975].In immunocompromised patients, reactivation ofBKV and JCV may cause severe disease. JCV cancause a demyelinating disease known as progressivemultiphocal leukoencephalopathy (PML) which occursmainly in AIDS patients [Gordon and Khalili, 1998].BKV can be responsible of a tubular nephritis, which canlead to allograft failure in renal transplant recipientsand hemorrhagic cystitis in hematopoietic stem celltransplant recipients [Nickeleit et al., 2002; Hirsch andSteiger, 2003; Chen et al., 2004].Polyomaviruses BKV, JCV, and SV40 have beendetected in human tumors [Sariyer et al., 2004; Giulianiet al., 2007, 2008] and induce numerous tumors inexperimental animals [Sariyer et al., 2004]. The mech-anism by which the oncogenic transformation isachieved is through the action of the early proteinslarge T antigen (T-Ag) and small t antigen (t-Ag). The T-Ag binds the oncosuppressor proteins p53 and pRb, twokey regulators of cell cycle progression [Bollag et al.,1989; Cress and Nevins, 1996] inhibiting apoptosisand driving the cell into S phase. The small t antigeninteracting with the protein phosphatase 2A stimulatesthe map kinase pathway and induces cell proliferation[Sontag et al., 1993]. This aberrant stimulation of thecell cycle is responsible for oncogenic transformation.Recently, a new human polyomavirus, KI polyoma-virus (KIV) has been identified in the respiratorysecretions of children with acute respiratory symptoms*Correspondence to: Marco Ciotti, Laboratory of MolecularVirology, University Hospital Tor Vergata, Viale Oxford, 81-00133Rome, Italy. E-mail: marco.ciotti@ptvonline.itAccepted 18 November 2008DOI 10.1002/jmv.21417Published online in Wiley InterScience(www.interscience.wiley.com) 2009 WILEY-LISS, INC.[Allander et al., 2007]. Debated is its causal role in therespiratory disease [Bialasiewicz et al., 2007; Norjaet al., 2007; Babakir-Mina et al., 2008] as completelyunknown is its oncogenic potential. The objective of thestudy was to determine the presence of KIV in normaland malignant lung tissue as well as in a normalparanasal tissue and to compare the early region of KIVgenome, comprising the C-terminal region of the T-Aggene and almost the entire t-Ag gene [Allander et al.,2007] with those of known KIV, JCV, and BKV strains.MATERIALS AND METHODSStudy GroupA series of 20 fresh lung cancer specimens andsurrounding normal tissue provided by the Departmentof Surgery of the Santo Spirito Hospital, Pescara, Italy,between 2005 and 2006 years were tested for thepresence of the novel polyomavirus KI. These specimenswere already tested for the presence of HPV, SV40, BKV,JCV, and CMV [Giuliani et al., 2007]. The patientsincluded 13 men and 7 women and the age ranged from40 to 85 years, mean age 68. In addition, one paranasaland one lung biopsy obtained from two thalassemictransplanted children (13 and 3 years old, respectively)were included in the study. Informed consent wasobtained from all patients enrolled in the study.DNA Extraction and Viral DetectionTwenty-five milligrams of normal and malignanttissue from each patient was digested overnight at568C with proteinase K and then total DNA extractedwith the QIAamp DNA mini kit according to themanufacturer’s instruction (Qiagen S.P.A., Milan,Italy). The DNA was eluted in a volume of 50 ml andstored at 808C until analysis.Amplification of the VP1 Gene andEarly Region of KIV GenomeThe KIV was detected amplifying a fragment of theVP1 geneasdescribed [Allander etal., 2007]. The positivecases were then amplified for the early region of KIVgenome comprising the C-terminal part of T-Ag geneand the almost entire t-Ag gene [Allander et al., 2007].The PCR primers were as follows: FT-Ag 50-CCACA-CAGGTGGTTTTCTATAA-30 (position 4521–4542) andRT-Ag 50-GCCCCAGCTTTTTCTAGAAGAA-30 (position5032–5011). The cycling conditions were: 10 min at 948C,1 cycle; followed by 35 cycles of amplification at 948C for1 min, 568C for 1 min, and 728C for 2 min. The PCRproducts were analyzed on a 2% agarose gel stained withethidium bromide and visualized under ultravioletlight. All the necessary precautions were taken to avoidcontamination.Sequencing of the Early Region andPhylogenetic AnalysisThe amplified fragments of the early region of KIV weresequenced using the Genome Lab DTCS Quick Start Kit(Beckman Coulter, Fullerton, CA) and run on a BeckmanCoulter CEQ2000XL sequence analyzer after columnpurification. The obtained sequences were submitted tothe Genebank and matched against all deposited sequen-ces (http://www.ncbi.nlm.nih.gov/BLAST). An alignmentwith a set of reference sequences was obtained usingCLUSTAL X [Thompson et al., 1994] and manually editedwith the Bioedit software [Hall, 1999]. Positions contain-ing gaps were removed from the final alignment. For ourdata set, the best fitting nucleotide substitution model wastested with a hierarchical likelihood ratio test followingthe strategy described by Swofford and Sullivan [2003],using a neighbor-joining (NJ) base-tree with LogDetcorrected distances. Maximum likelihood (ML) trees werethen inferred with the selected model and ML-estimatedsubstitution parameters. The heuristic search for the MLtree was performed using an NJ tree as starting tree andthe TBR branch-swapping algorithm. NJ trees were alsoestimated using pair-wise distances inferred by ML withthe best fitting nucleotide substitution model. Calcula-tions were performed with PAUP* 4.0b10 according toSwofford and Sullivan [2003]. Statistical support forinternal branches in the NJ trees was obtained bybootstrapping (1,000 replicates) and with the ML-basedzero-branch-length test for the ML trees [Swofford andSullivan, 2003]. Trees were rooted by outgroup rootingusing BKV-ETH-3 strain as outgroup. The accessionnumbers of the sequences utilized for the phylogeneticanalysis including those referred to KIV strains identifiedin our laboratory are the following: AB263916; AB269841;AB269826; AB211387; AB269851; AB269822; AB211370;AY628225; AY628234; AY628232; AY628233; AY628231;AY628230; AY628229; AY628227; AY628226; AB127343;AB127018; AY536242; AB372037; AB113124; AY382184;AB038255; AB212954; AB212952; AF004349; AF030085;AY536239; EF520287; EF520288; EF520289; EF127906;EF127907; EF127908; FJ389513; FJ389514; FJ389515;FJ389516.RESULTSDetection of KIV-DNAKIV-VP1 gene was detected in 9 out of 20 lung cancerspecimens tested (45%) and in 1 case the VP1 gene wasdetected either in normal as well as in malignant tissue.Following this primary screening, a second PCR wasperformed on the nine positive cases targeting the earlyregion of KIV. A positive result was obtained only inthree cases: one surrounding normal tissue (KIV-RM1)and two malignant tissues (KIV-RM3 and KIV-RM4).In the two transplanted thalassemic children, the VP1gene was detected in both cases while amplification ofthe early region of the viral genome was positive only inone case (KIV-RM2).Phylogenetic AnalysisPhylogenetic analysis revealed the presence of threemain clades, supported by highly significant bootstrapand P values in the zero-branch-length test. TwoJ. Med. Virol. DOI 10.1002/jmvPresence of KIV in the Respiratory Tissue 559represented BK and JC polyomaviruses (Fig. 1) whilethe third one contained the novel KIV strains. This thirdclade can be subdivided into two statistically supportedsub-clades, named A and B, containing the four Italianisolates and the Swedish and Australian isolates,respectively (Fig. 1; P< 0.001 in the zero-branch-lengthtest, Bayesian posterior probability >90%, and boot-strap support >75%.). Within sub-clade A, KIV-RM1and KIV-RM2 resulted more closely related compared toKIV-RM3 and KIV-RM4 isolates which were placedoutside this cluster.The mean genetic distance between groups (BKV,JCV, and KIV) was 0.26, whereas within clades A and Bwas 0.19 and 0.137, respectively. These values were notstatistically significant. Conserved mutations/motifswere not observed in the virus infecting the neoplastictissue.DISCUSSIONThe novel polyomavirus KIV was first described byAllander et al. [2007] in the respiratory secretionsof children with acute respiratory symptoms andlater confirmed by other groups around the world[Bialasiewicz et al., 2007; Babakir-Mina et al., 2008].Whether this reflects a causal role of the virus in therespiratory illness is still debated and molecularepidemiological studies are needed to establish a firmlink with the respiratory disease. Up to now, completelyunexplored is the oncogenic potential of KIV.The human polyomaviruses BKV and JCV have beendetected in several human tumors [White and Khalili,2004; Giuliani et al., 2008] including the respiratorytract [Giuliani et al., 2007; Zheng et al., 2007], andinduce tumors in animal models [Sariyer et al., 2004].This prompted us to investigate the presence of KIV innormal and malignant respiratory tissue. PCR analysisrevealed the presence of KIV-VP1 gene in some of thelung cancer tissues examined and in both normalbiopsies (paranasal and lung) from the transplantedpatients. However, when the same positive sampleswere tested for the presence of the early region of theviral genome, an amplified product was obtained onlyin four of the KIV-VP1 positive cases. This apparentdiscrepant result might be explained by the nucleotidevariability observed in the C-terminal part of T-Ag geneof polyomaviruses [Lednicky et al., 1997; White andKhalili, 2004].Phylogenetic analysis performed on the early region ofpolyomaviruses included in this study showed thepresence of three main clades comprising the BKV,JCV, and KIV strains, respectively. Interestingly,within the KIV clade two sub-clades named A and Bwere observed. The sub-clade A contained all fourItalian isolates, while sub-clade B included all Swedishand Australian strains. Among the Italian isolates, bothKIV-RM1 and -RM2 identified in normal tissue (lungtissue surrounding the tumor and paranasal tissue)appeared closely related and clustered together,whereas both the KIV-RM3 and -RM4 strains detectedJ. Med. Virol. DOI 10.1002/jmvFig. 1. Phlylogenetic analysis of the early region (512 nt) of humanpolyomaviruses was carried out using HKYþ IþC (alpha-parame-ter¼ 0.0763) as the best evolutionary model. Branch lengths wereestimated with the best fitting nucleotide substitution model accordingto a hierarchical likelihood ratio test [Posada and Crandall, 1998], andwere drawn in scale with the bar at the bottom indicating 0.3 nucleotidesubstitutions per site. One * along a branch represents significantstatistical support for the clade subtending that branch (P<0.001 inthe zero-branch-length test) and bootstrap support>75%. The tree wasrooted using the strain BKV-ETH-3 as outgroup. Sub-clades within themain KIV clade are indicated with the letters A and B.560 Babakir-Mina et al.in lung cancer tissue fell outside this cluster, being moredistantly related to KIV-RM1 and -RM2.Regarding the genetic distance observed betweensub-clades A and B, it might be related to thedifferent source of isolation (malignant lung tissue vs.respiratory secretions), and perhaps reflects a differentbiological behavior of the virus in vivo. To test thishypothesis would be interesting to study the trans-forming potential of KIV strains in vitro as it is knownthat the C-terminal part of the T-Ag of polyomavirusesretains binding activity for p53 and Rb proteins.The inactivation of these oncosuppressor proteins bythe T-Ag leads to cell cycle deregulation, inhibition ofapoptosis, and cell transformation. 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Identification of the novel KI polyomavirus in paranasal and lung tissues

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