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Scien i ic RepoR s | 6:39267 | DOI: 10.1038/s ep39267
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Epidemiology o wo human
p o opa o i uses, bu a i us
and usa i us
Elina Väisänen1, Minna Paloniemi2,3, Inka Kuisma1, Väinö Li ho ius1, A un Kuma 1,4,
Rauli F anssila1, Kam uddin Ahmed5, E ic Delwa 6,7, Timo Vesika i2, Klaus Hedman1,8 &
Ma ia Söde lund-Vene mo1
Two human pa o i uses we e ecen ly disco e ed by me agenomics in A ica, bu a i us (BuV) in 2012
and usa i us (TuV) in 2014. These i uses ha e been s udied exclusi ely by PCR in s ool and de ec ed
only in pa ien s wi h dia hoea, al hough a low p e alence. Th ee geno ypes o BuV ha e been
iden i ied. We de ec ed, by in-house EIA, BuV1-3 IgG an ibodies in 7/228 child en (3.1%) and 10/180
adul s (5.6%), whe eas TuV IgG was ound in one child (0.4%). All child en and 91% o he adul s we e
Finnish, ye in e es ingly 3/6 adul s o Indian o igin we e BuV-IgG posi i e. By compe i ion EIA, no
c oss- eac i i y be ween he BuVs was de ec ed, indica ing ha he BuV geno ypes ep esen dis inc
se o ypes. Fu he mo e, we analysed by BuV qPCR s ool and nasal swab samples om 955 child en wi h
gas oen e i is, espi a o y illness, o bo h, and ound BuV DNA in h ee s ools (0.3%) and o he i s
ime in a nasal swab (0.1%). This is he i s s udy documen ing he p esence o BuV and TuV an ibodies
in humans. Al hough he se op e alences o bo h i uses we e low in Finland, ou esul s indica e ha
BuV in ec ions migh be widesp ead in Asia. The BuV-speci ic humo al immune esponses appea ed o
be s ong and long-las ing, poin ing o sys emic in ec ion in humans.
The de elopmen o mode n deep sequencing and me agenomic echniques ha e acili a ed ecen disco e ies o
se e al no el pa o i uses in a ious animal species, including humans. O he no el pu a i e human pa o i-
uses, bu a i us (BuV) and usa i us (TuV) a e among he newes : bo h o hese i uses we e o iginally disco -
e ed in he s ools o dia heic child en in A ica - BuV in 2012 in Bu kina Faso and TuV in 2014 in Tunisia1,2.
Pa o i uses a e small, non-en eloped i uses wi h a single-s anded DNA genome o 4–6 kb, encoding only a
ew p o eins. The classi ica ion o pa o i uses is cu en ly based on he non-s uc u al p o ein (NS1) sequence3,
and bo h BuV and TuV a e classi ied in he P o opa o i us genus in he Pa o i idae amily. Human bu a i uses
a e conse ed in he NS1 p o ein (94–96% simila i y a he amino acid (aa) le el)4, and hus belong o one species,
P ima e p o opa o i us 13. Howe e , he BuV capsid p o eins VP1 and VP2 sha e only 71–78% and 64–73% sim-
ila i ies a he aa le el, espec i ely, gene a ing h ee geno ypes4,5. Tusa i us on he o he hand has been desc ibed
only in one child2, al hough pa ial TuV-like sequences ha e been de ec ed in u seals in B azil6.
To da e, bu a i us has been sea ched o exclusi ely in s ool samples, and i has been de ec ed in dia heal
s ools o child en and adul s in A ica, Eu ope and Asia, including Bu kina Faso, Tunisia, Bhu an, Finland, he
Ne he lands, Thailand, Tu key and China1,5,7–11. The p e alence in pa ien s has been low, 0.3% o 4%, and al hough
in h ee publica ions s ool samples om non-dia heic pa ien s o heal hy subjec s we e shown o be BuV-DNA
nega i e9–11, he e iological ole o he i us in dia hoea o in o he human diseases emains unce ain.
BuV-like i uses ha e been ound in wild and cap i e non-human p ima es as well as in swine, sh ews, a s,
ba s and u seals4,6,12–17. The de ec ion o hese i uses in se a o hesus monkeys in he USA, and in he spleen o
wild baboons and sh ews in Zambia, sugges s ha BuV-like i uses may cause sys emic in ec ions12,13.
1Depa men o Vi ology, Uni e si y o Helsinki, Helsinki 00290, Finland. 2Vaccine Resea ch Cen e , Uni e si y o
Tampe e, Tampe e 33520, Finland. 3Fimlab labo a o ies l d, Tampe e 33520, Finland. 4Heal h Sciences No h
Resea ch Ins i u e, Sudbu y, ON P3E 5J1, Canada. 5Depa men o Pa hobiology and Medical Diagnos ics, Facul y
o Medicine, Uni e si i Malaysia Sabah, Ko a Kinabalu 88400, Sabah, Malaysia. 6Blood Sys ems Resea ch Ins i u e,
San F ancisco, CA 94118, USA. 7Depa men o Labo a o y Medicine, Uni e si y o Cali o nia a San F ancisco, San
F ancisco, CA 94118, USA. 8Helsinki Uni e si y Hospi al, HUSLAB, Helsinki 00290, Finland. Co espondence and
eques s o ma e ials should be add essed o E.V. (email: elina.[email p o ec ed])
Recei ed: 26 Sep embe 2016
Accep ed: 21 No embe 2016
Published: 14 Decembe 2016
OPEN
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Scien i ic RepoR s | 6:39267 | DOI: 10.1038/s ep39267
We i s analysed by quan i a i e PCR he p esence o BuV DNA in s ool and, o he i s ime, also in nasal
swab samples om 955 child en wi h symp oms o acu e gas oen e i is, acu e espi a o y in ec ion o bo h. The
me e p esence o i al DNA in s ool o in o he sample ypes does, howe e , no necessa ily indica e a causal ole
o a disease, nei he does he ansien PCR posi i i y gi e a ue pic u e o how common he i us is in he popu-
la ion. In his s udy we he e o e in es iga ed he p e alence o BuV- and TuV-speci ic IgG an ibodies, which can
e eal p io i us encoun e s. We c ea ed i us-like pa icles (VLP) o he majo VP2 capsid p o eins o all h ee
BuV geno ypes and o TuV, applied hem as an igens in IgG EIAs, and analysed 180 se um samples om heal hy
adul s and 228 se um samples om he same paedia ic coho o assess he epidemiology o hese i uses.
Resul s
Bu a i us DNA in nasal swab and s ool samples in he paedia ic coho . BuV DNA was de ec ed
in 1/955 (0.1%) nasal swab and in 3/955 (0.3%) s ools (Table1). The esul s we e con i med by sequencing he
qPCR amplicons. We we e no able o ampli y he VP egion o he i al genome om hese samples, possibly
due o low quan i y and/o limi ed sample olume; he geno ypes he e o e emained unknown. The i al loads
o he samples we e 2.1 × 102–1.6 × 103 copies/ml s ool suspension and 4.6 × 103 copies/ml nasal swab medium
(Table1). All ou BuV DNA-posi i e child en had gas oin es inal symp oms, while all o he 545 non-dia heic
child en we e BuV-DNA nega i e. Howe e , all ou child en had in s ool also ano he i us known o cause
gas oen e i is (Table1).
Bu a i us and usa i us VP2 VLPs. The VP2 genes we e cloned om he o iginal BuV and TuV
DNA-posi i e s ool samples and used o c ea e ecombinan VP2 VLPs o BuV1, 2 and 3 as well as TuV. The
ou ypes o VLPs we e exp essed in insec cell cul u es and pu i ied by CsCl ul acen i uga ion. SDS-gel elec-
opho esis showed p o eins o expec ed sizes, app oxima ely 64 kDa o BuVs and 62 kDa o TuV, and elec on
mic oscopy disclosed pa o i us-like VLPs o ~25 nm in diame e (Fig.1).
Bu a i us and usa i us IgG in adul s. Ten subjec s (10/180, 5.6%) we e BuV IgG se oposi i e: ou sub-
jec s o BuV1, i e o BuV2 and one o all h ee bu a i uses ( iple posi i e) (Tables2 and 3, and Fig.2). Th ee
o he BuV1 se oposi i es and he iple se oposi i e showed e y high OD alues (OD 1.9–4.0, Table2 and
Fig.2). None o he 180 adul se a we e posi i e o TuV IgG. Al hough mos o he adul s we e o Finnish descen
(163/180, 90.6%), o he en BuV IgG-posi i e subjec s i e we e om he Middle-Eas , India o China, including
all ou wi h high OD alues. They had mo ed o Finland as adul s, and we e 26 o 34 yea s o age a he ime o
No. Sample ype
BuV DNA quan i y (copies
pe ml supe na an ) Age, gende Sampling da e Symp oms O he pa hogens ound
1Swab 4.6 × 10320 mon hs, M 27.3.2011 AGE + ARTI swab: neg* (s ool: no o i us,
HBoV2# se um: HBoV2#)
2S ool 2.1 × 1024 y s 5 mon hs, F 23.2.2010 AGE no o i us
3S ool 8.5 × 10223 mon hs, F 6.3.2011 AGE o a i us, HBoV2
4S ool 1.6 × 10310 mon hs, F 9.1.2011 AGE + ARTI no o i us
Table 1. Bu a i us DNA-posi i e child en. *Only boca- and co ona i uses we e es ed om nasal swabs. #An
acu e boca i us 2 (HBoV2) in ec ion was diagnosed based on se ology in his child18. The co esponding s ool
and se um samples o his child we e BuV-DNA nega i e.
Figu e 1. SDS-PAGE (a) and elec on mic oscopy pic u es (b) o VP2-VLPs o BuV1-3 and TuV.
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sampling. Consequen ly, he se op e alence among he na i e Finnish subjec s was 3.1% (5/163), whe eas 5/12
(41.7%) Asian subjec s and 3/6 (50%) Indian subjec s we e se oposi i e. All BuV IgG-posi i e adul s o Finnish
backg ound had low OD alues.
Follow-up samples we e a ailable om i e BuV IgG-posi i e adul s, including all ou wi h high OD alues.
The samples we e analysed wi h BuV-IgG EIA, and he OD alues, which e lec he an ibody i es in he es ed
subjec s, we e shown o emain s able o up o six yea s (Table2).
Bu a i us and usa i us IgG in child en. Among he 228 child en s udied o BuV and TuV IgG an i-
bodies, se en (3.1%) had BuV IgG and one (0.4%) had TuV IgG. O he BuV IgG-posi i e cases, one had IgG o
BuV1, h ee o BuV2, wo o BuV3 and one o bo h BuV1 and 2 (Fig.2). The BuV-se oposi i e child en we e
be ween 7 mon hs and 5 yea s o age a he ime o sampling, whe eas he TuV-se oposi i e child was 9 yea s o
age.
In addi ion o he ue BuV o TuV se oposi i e samples, we encoun e ed ou child en wi h low BuV IgG
OD alues wi h inde e mina e blocking esul s (one BuV1 and h ee BuV2). These esul s we e in e p e ed as
nega i e.
Among he BuV DNA-posi i e cases, a se um sample was a ailable only om he nasal swab-posi i e child.
The se um was bo h BuV-DNA and BuV-IgG nega i e o all BuV ypes poin ing ei he o a supe icial o local
espi a o y ac in ec ion wi hou i emia o an ibody esponses o o a e y ea ly acu e sys emic in ec ion s ill
Geog aphical
backg ound Sampling da e
Abso bance alue (OD) wi hou compe i ion
BuV1 IgG BuV2 IgG BuV3 IgG TuV IgG H5 IgG
Case 1 Middle-Eas 4.3.2010 2.742 2.793 2.827 0.030 0.040
24.9.2010 2.657 2.958 2.958 0.028 0.042
22.12.2010 2.880 3.209 3.482 0.026 0.035
19.7.2011 2.437 2.666 2.576 0.027 0.032
18.2.2016 2.683 2.419 2.675 0.033 0.046
Case 2 India 6.1.2011 2.977 0.032 0.054 0.026 0.036
19.7.2011 2.900 0.039 0.056 0.028 0.038
Case 3 India 10.3.2010 3.642 0.040 0.061 0.041 0.037
6.1.2011 4.071 0.025 0.046 0.029 0.024
Case 4 India 11.3.2010 1.924* 0.454*0.164*0.079 0.496*
9.4.2010 2.330* 0.534*0.228*0.096 0.616*
Case 5 Finland 19.10.2009 0.248 0.069 0.022 0.020 0.050
1.11.2011 0.258 0.084 0.028 0.020 0.055
21.1.2014 0.271 0.091 0.027 0.024 0.057
17.2.2016 0.295 0.126#0.034 0.029 0.076
Table 2. Bu a i us IgG-posi i e adul s wi h ollow-up samples. The posi i es a e indica ed wi h bolded
numbe s. H5 = High5 insec cells. *BuV2, BuV3 and H5 eac i i y was comple ely blocked in compe i ion assay
by insec cell suspension, bu no wi h he speci ic BuV VLP. The BuV1 esul was blocked only wi h BuV1. See
also Table3. #BuV2 eac i i y was blocked wi h insec cell suspension, no by BuV2 VLP.
Blocking VLP/
suspension
Case 1, sample aken
4.3.2010
Case 4*, sample aken
11.3.2010 Case 13 Case 16 Case 18
An igen in he well An igen in he well
An igen in
he well
An igen in
he well
An igen in
he well
BuV1 BuV2 BuV3 BuV1 BuV2 BuV3 BuV2 BuV3 TuV
none 2.588 2.761 2.896 2.011 0.644 0.174 0.156 0.142 0.314
BuV1 0.030 2.587 2.661 0.127 0.458 0.132 0.177 0.107 0.299
BuV2 2.579 0.038 2.722 1.949 0.167 0.143 0.023 0.111 0.191
BuV3 2.400 2.614 0.080 1.904 0.472 0.151 0.191 0.024 0.377
TuV 2.513 2.720 2.819 1.997 0.525 0.172 0.164 0.116 0.101§
High5 insec
cells 2.638 2.845 2.954 2.016 0.074#0.087#0.211 0.116 0.401
Table 3. Examples o compe i ion assay esul s. Speci ic blocking is demons a ed wi h bolded numbe s.
*Case 4 is s ongly IgG posi i e o bo h BuV1 and insec cells (also seen in Table2). BuV2 and BuV3 VLPs ha e
been shown o ha e insec cells impu i ies (da a no shown), which causes mo e eac i i y owa ds BuV2 and
BuV3 an igens. This eac i i y is comple ely blocked by insec cell suspension (ma ked wi h#), and he sample
is he e o e conside ed o be IgG posi i e only o BuV1 among he es ed i al an igens. The blocking e ec o
BuV2 VLP owa ds BuV2 is in e p e ed o be caused by he insec cell impu i ies a he ha he VLP i sel . §5x
mo e blocking an igen was used o block he IgG eac i i y.
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awai ing an immune esponse. Al hough his 20-mon h-old child had been hospi alized o ou days and had
unde lying heal h issues including ope a ed cle pala e, hea p oblems and as hma, a se ologically con i med
acu e HBoV2 in ec ion sugges s ha his child was B-cell immunocompe en 18.
Speci ici y o bu a i us and usa i us B-cell immuni y. In o de o ensu e ha he EIA abso bance
alues we e speci ic o he pa icula i us, i.e. no due o c oss- eac i i y wi h known simila i uses o unspe-
ci ic eac ions, all samples wi h a measu able OD alue o ≥0.1 unde wen con i ma o y compe i ion assays by
blocking wi h soluble VLPs and insec cell lysa e. Samples wi h OD alues below 0.1 we e oo di icul o unam-
biguously con i m by blocking and we e hus conside ed nega i e. O no e, he de e mina ion o an exac cu o
alue was no c ucial o he in e p e a ion o he esul s as all samples wi h OD alues o ≥ 0.1 we e con i med by
compe i ion assay and only he con i med esul s we e conside ed genuinely posi i e.
The an ibodies in all BuV- o TuV-se oposi i e samples showed no c oss- eac i i y be ween he BuV geno-
ypes o TuV, including he iple-BuV se oposi i e plasma (Case 1; Table3). Bo h in a high-posi i e adul (Case 1)
and in a low-posi i e adul (Case 5), we could show ha he BuV an ibody le els and speci ici y emained con-
s an o a leas six yea s (Table2). Fu he mo e, among he 84 medical s uden s, he BuV1-3 and TuV IgG EIA
esul s co ela ed nei he wi h each o he no wi h he p e iously analysed IgG EIA esul s o six o he human
pa o i uses: HBoV1-4, PARV4, o pa o i us B19 (da a no shown)19–21.
No ably, some o he se a did con ain an ibodies owa d High5 insec cells in which ou an igens we e
exp essed: 9/180 (5.0%) in he adul coho wi h ODs o 0.104–0.616, and 13/228 (5.7%) in he paedia ic coho
wi h ODs o 0.096–0.404. Some o hese High5 backg ounds would ha e yielded inco ec posi i i y in he
unblocked BuV IgG EIAs, mo e wi h BuV2 and 3 han BuV1 o TuV an igens. Howe e , we we e able o dis-
inguish alse om genuine eac i i y by compe ing wi h insec cell lysa e, as can be seen in pa ien numbe 4
(Tables2 and 3): he BuV2 and BuV3 eac i i ies we e comple ely blocked in he compe i ion assay by insec
cells, bu less o no a all by VLPs o ei he o hese wo BuVs, whe eas he BuV1 esul was blocked wi h he
homologous BuV1 (Table3). In ou p e ious s udies we ha e seen he same phenomenon wi h S 9 insec cells.
Human an ibodies owa ds insec cell componen s should he e o e be conside ed when applying in EIA insec
cell-de i ed an igens.
Discussion
Bu a i us and usa i us a e among he newes pa o i uses disco e ed in humans by deep sequencing1,2. Un il
now, human bu a i uses, mos ly BuV1 o BuV3, has been de ec ed by PCR in se e al geog aphical loca ions in
A ica, Eu ope and Asia; bu exclusi ely in dia heal s ools1,5,7–11. S udies ha e no included o he sample ypes
o o he diagnos ic means, ye he closely ela ed BuV-like i uses ha e been ound in spleens o se a o in ec ed
animals, indica ing sys emic in ec ions12,13. He e we analysed o BuV DNA, s ool samples and nasal swabs; as
Figu e 2. BuV and TuV IgG-posi i e adul s and child en. The igu e p esen s he EIA OD alues om he
samples. Blue x ma ks he BuV1 IgG le el in he sample, ed ci cle he BuV2 IgG le el, g een diamond he BuV3
IgG le el and he black squa e he TuV IgG le el. Below each esul he ollowing in o ma ion is p esen ed:
Age, gende , geog aphical backg ound (M-E = Middle-Eas , IND = India, CHN = China, FIN = Finland), case
numbe and coho .
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Scien i ic RepoR s | 6:39267 | DOI: 10.1038/s ep39267
well as o BuV and TuV an ibodies, se a o child en wi h espi a o y in ec ion, gas oen e i is o bo h. We also
de e mined he co esponding se op e alences among asymp oma ic adul s.
In he s ool samples, we ound a low BuV-DNA p e alence in Finnish child en p esen ing wi h acu e gas-
oen e i is wi h o wi hou espi a o y ac in ec ion, in line wi h p io epo s1,5,9–11. We obse ed o he i s
ime BuV DNA in a nasal swab, albei a a e y low quan i y, and wi h no BuV IgG no DNA de ec able in a co -
esponding se um. In each child wi h BuV DNA in s ool, a known gas oin es inal i us was iden i ied, possibly
accoun ing o he illness. Howe e , none o he 545 child en wi hou gas oen e i is had BuV DNA in s ool,
sugges ing BuV could be a con ibu ing ac o o he disease.
Human pa o i us VLPs can be cons uc ed using solely he majo capsid p o ein, and such VLPs a e o en
he an igens o choice in se odiagnosis19,22–27. In his s udy we showed ha he VP2 p o eins o all h ee BuV gen-
o ypes and TuV a e capable o assembling in o VLPs, as wi h o he human pa o i uses, and used hese VLPs in
EIAs o BuV and TuV se ology.
The highes BuV IgG p e alence was de ec ed among Asian asymp oma ic adul s; howe e , wi hou s a is ical
powe due o low sample numbe . O he Asian subjec s 5/12 we e posi i e, and he highes numbe was obse ed
among Indian pe sons o whom 3/6 we e BuV-IgG posi i e. These indi iduals had also he highes BuV IgG OD
alues, unlike he child en o adul s o Finnish descen . In addi ion, he p e alence o BuV an ibodies in Finnish
child en (3.1%, 7/228) was iden ical o ha in he na i e Finnish adul s (3.1%, 5/163) aising he ques ion why he
BuV IgG p e alence did no inc ease wi h age. Al hough we did show ha he an ibody le els among adul s can
emain cons an o a leas six yea s, waning o he an ibodies o e ime migh play a ole. Fu he mo e, i he
p ima y in ec ion o he hos is local and supe icial, i migh no induce a s ong long-las ing an ibody esponse.
Wi h HBoVs i has been shown ha exposu e o homo- o he e ologous HBoV species may boos he an ibody
esponse28,29. Thus, he simila BuV se op e alence among Finnish adul s and child en migh be a consequence
o he gene al a i y o BuV in ec ions in he No dic coun ies, leading o inadequa e main enance o an ibody
esponses.
In ou p e ious s udy o s ools o Finnish child en and adul s du ing 2012–2013, we de ec ed BuV DNA
exclusi ely o geno ype 17. In he p esen s udy we ound six child en wi h an ibodies o geno ypes 2 o 3, while
only wo had an ibodies o geno ype 1 (o no e, one child had bo h BuV1 IgG and BuV2 IgG). These BuV2 o
3 IgG-posi i e child en we e 7 mon hs o 5 yea s o age a he ime o sampling (2009 o 2011), sugges ing ha
BuV2 and maybe BuV3 migh ha e ci cula ed in Finland be ween 2006 and 2011. Un o una ely, we do no
ha e a el in o ma ion o hese child en. The BuV3 an ibodies in he wo 7- o 8-mon h-old in an s migh be
ma e nal. I is in e es ing ha in bo h o ou coho s BuV2 IgG was he mos equen , while BuV2 DNA has been
de ec ed in only a single pa ien , in he o iginal publica ion1.
Mos o he human pa o i uses ha e se e al a ian s o geno ypes. This is he case also o BuV. The a i-
an s o human pa o i us B19 (B19V) di e in VP2 aa sequence by only 1–2%30,31, and c oss- eac ully in EIA.
The e o e, B19V se odiagnosis can be done eliably using as an igen VP2 VLPs o any o he a ian s32–34. On he
o he hand, he mo e di e gen ou HBoVs (wi h a 11–23% di e ence in he majo capsid p o ein aa sequence)
a e pa ly c oss- eac i e in EIA19. The c oss- eac i i y can be isualized by blocking he se um wi h he e ologous
HBoVs p io o he EIA. To iden i y such c oss- eac i i y is a p e equisi e o co ec in e p e a ion o he HBoV
EIA esul s19,29. The human bu a i us geno ypes di e by 27–36% in VP24, and in ou EIA analysis he BuV
geno ypes we e no c oss- eac i e. Nei he he BuV geno ype, he an ibody le el in he sample, no he age o he
subjec a ec ed assay speci ici y. In e es ingly, we ound one adul and one child wi h speci ic an ibodies owa ds
mo e han one BuV ype (case 1 wi h a iple and case 7 wi h a double posi i i y, shown in Fig.2). In conclusion,
we showed he h ee BuV geno ypes also o ep esen h ee di e se se o ypes.
We analysed he se um IgG eac i i ies agains a numbe o ac o s known o cause backg ound in EIA,
including insec cell lysa e, non-an igen-coa ed mic owells and s ep a idin. O bo h adul s and child en 5–6%
had an ibodies owa ds insec cell cons i uen s, which would cause alse posi i i y wi hou compe i ion assays.
Such alse eac ions should be conside ed when applying insec -cell p oduced an igens in EIA.
This is he i s published human s udy o TuV since he ini ial a icle. While we ound a single child o exhibi
TuV IgG, in con i ma o y blocking a 5- old highe concen a ion o he homologous an igen was equi ed han
in he co esponding BuV EIAs, o block he IgG binding. As none o he o he an igens (BuV1-3) o insec
cell lysa e blocked he TuV IgG eac i i y, he esul was conside ed TuV speci ic. Mo e s udies a e ne e heless
needed o con i m whe he TuV ac ually is a human i us.
Taken oge he , we obse ed o he i s ime BuV DNA, aside om pedia ic s ools, in a nasal swab. Mo e
impo an ly, his is he i s s udy documen ing he p esence o BuV and TuV IgG an ibodies in humans. We
u he mo e showed ha he h ee bu a i uses ep esen di e en se o ypes: none o he BuVs no TuV we e
mu ually c oss- eac i e in he coho s s udied. While in Finland he se op e alences o bo h o hese p o opa o-
i uses we e low, we obse ed bo h highe OD alues and a highe se op e alence o BuV among pe sons wi h
an Asian o igin, indica ing ha BuV in ec ions migh be mo e common in o he coun ies, pa icula ly in India.
The BuV-speci ic immune esponses o all h ee se o ypes appea ed o be s ong and las ing, poin ing o sys emic
in ec ions. O e all, ou esul s oge he wi h hose o o he s indica e ha BuV is a human i us, whe eas addi-
ional s udies a e needed o assess he hos opism o TuV and he clinical co ela es and global epidemiology o
hese i uses as well as hei ansmission ou e(s).
Ma e ials and Me hods
Pa ien s and Samples. The adul coho consis ed o se um o plasma samples om 180 heal hy adul ol-
un ee s: 84 medical s uden s om he Uni e si y o Helsinki and 96 s a membe s om he Helsinki Uni e si y
Hospi al o om he Facul y o Medicine, Uni e si y o Helsinki (median age 30 yea s, ange 19–65). A w i en
in o med consen was ob ained om all subjec s, and he samples we e collec ed be ween 2007 and 2013. All
medical s uden s and 79/96 o he s a we e o Finnish descen , he emaining 17 s a membe s we e o iginally

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Scien i ic RepoR s | 6:39267 | DOI: 10.1038/s ep39267
om Asia (12), he Ame icas (1) o o he Eu opean coun ies (4). Follow-up samples we e collec ed om BuV
IgG-posi i e pa icipan s, o ob ained ( ozen-s o ed) om ou a chi e. The E hics Commi ee o Helsinki and
Uusimaa Hospi al Dis ic app o ed he s udy p o ocol, and he s udy was conduc ed in acco dance wi h he
ele an guidelines and egula ions.
The samples and clinical in o ma ion o he paedia ic coho ha e been desc ibed p e iously18. Sho ly, he
pa ien s (n = 955) we e ec ui ed be ween Sep embe 2009 and Augus 2011 in Tampe e, Finland, and he sam-
ples we e collec ed h oughou he yea . The child en we e di ided in h ee g oups based on signs and symp-
oms: acu e gas oen e i is (AGE, n = 172), acu e espi a o y ac in ec ion (ARTI, n = 545) o symp oms o bo h
(n = 238). The median age was 14 mon hs ( ange 6 days – 15.6 yea s). The e we e pa ien s bo h om a paedia ic
ou pa ien clinic, 137 (14.3%), and a hospi al wa d, 818 (85.7%). S ool and nasal swab samples we e a ailable
om e e y child and se um samples we e om 228 child en o whom blood sampling was medically indica ed. A
w i en in o med consen was ob ained om he pa en s o all he child en en olled. The s udy was app o ed by
he E hics Commi ee o Pi kanmaa Hospi al Dis ic , and, and he s udy was conduc ed in acco dance wi h he
ele an guidelines and egula ions.
DNA ex ac ion and bu a i us quan i a i e PCR. DNA was ex ac ed om s ool and nasal swab
samples as desc ibed in Paloniemi e al., and he DNA aliquo s we e analysed wi h BuV-qPCR as desc ibed in
Väisänen e al.7,18. In qPCR-posi i e cases he amplicon was sequenced o con i m he esul ; howe e , he nea ly
iden ical sequence in he NS1 amplicon a ea p e en ed BuV geno yping.
Gene a ion o ecombinan BuV1-3- and TuV-VP2 baculo i uses. Recombinan baculo i uses we e
c ea ed wi h he Bac- o-Bac Baculo i us Exp ession Sys em (In i ogen) acco ding o he manu ac u e ’s ins uc-
ions. B ie ly, DNA was ex ac ed om he o iginal BuV and TuV DNA-posi i e s ool samples (BuV1 and 21,
BuV35 and TuV2) wi h he QIAamp DNA mini ki (Qiagen, Ge many). The pu a i e VP2 gene was ampli ied wi h
Phusion High-Fideli y DNA Polyme ase (The mo Fische Scien i ic) by using p ime s wi h designed BamHI and
SalI es ic ion si es (Table4). The amplicon was in oduced in o he pFas BacDual ec o (In i ogen) by he
es ic ion si es, and he gene a ion o ecombinan BuV1-3- and TuV-VP2 bacmids and he subsequen ans-
ec ion in o Spodop e a ugipe da S 9 insec cells we e pe o med acco ding o he manu ac u e ’s ins uc ions.
The esul ing i us om each ans ec ion was ampli ied in h ee consecu i e passages in T ichoplusia ni High5
insec cells.
BuV1-3- and TuV-VP2-VLP exp ession and pu i ica ion. Fo p o ein exp ession and VLP p oduc-
ion, High5 insec cells we e in ec ed wi h he ecombinan baculo i us and g own o 5 days a + 27 °C. The
cells we e collec ed by cen i uga ion (4300 g, 5 min) and e-suspended in 20 mM T is-HCl, pH 8.0, con aining
Comple e-EDTA- ee p o ease inhibi o cock ail (Roche), and we e ozen-s o ed a − 20 °C.
The cell suspension was ea ed wi h 0.5% sodium deoxychola e (Sigma-Ald ich) o 30 min a + 37 °C
wi h 600 pm shaking, chilled on ice, and sonica ed 8 × 10 sec wi h 50% ampli ude. The lysa e was cen i uged
a 16100 g o 30 min a + 4 °C. The supe na an was collec ed and he emaining pelle was e-suspended,
e-sonica ed and e-cen i uged. The p eclea ed lysa es om he wo sonica ion ounds we e combined and
loaded on o a CsCl double cushion wi h densi ies o 1.52 g/cm3 and 1.22 g/cm3 in 10 mM T is-HCl (pH 7.8)
con aining 1 mM EDTA. Ul acen i uga ion was pe o med a 65 000 g o 4 h a 10 °C. F ac ions we e collec ed
and subjec ed o SDS-PAGE and he VLP ac ion(s) we e dialyzed 3 imes agains PBS. The inal pu i ied and
dialyzed VLPs we e iewed unde elec on mic oscopy (Fig.1).
BuV and TuV VP2-VLP IgG EIA assays. Va ious plas ics and bu e s as well as VLP, an ibody and sub-
s a e concen a ions we e sys ema ically compa ed, and he ollowing p o ocol was chosen. The pu i ied VP2
VLPs we e bio inyla ed using he EZ-Link Sul o-NHS-LC-Bio in ki (The mo Fische Scien i ic) acco ding o he
manu ac u e ’s ins uc ions, and he bio inyla ed VLPs we e used as an igens in EIA. Sonica ed High5 insec cells
we e bio inyla ed as well, and used as con ol an igen. The bio inyla ed VLPs o insec cell lysa e (80 ng pe well in
PBS con aining 0.05% Tween 20 [PBST]) we e immobilized on s ep a idin-coa ed pla es (Uni e SA96-Lockwell,
Kai ogen) o 1 hou a oom empe a u e wi h 400 pm shaking. To minimize non-speci ic backg ound, he
pla es we e pos -coa ed 3 × 10 min wi h he Diluen (Labsys ems Diagnos ics). Plasma o se um samples we e
dilu ed 1:200 in RED bu e (Kai ogen) and incuba ed o 1 hou a oom empe a u e wi h 400 pm shaking.
P ime Sequence
BuV1 VP2 wd BamHI TAgga ccATGACTGACACACAAGATGTATCTGA
BuV1 VP2 e SalI ATTg cgacTCCATTTTAGATTGTGTAGTTAGGCATAC
BuV2 VP2 wd BamHI TAgga ccATGTCTGAAAGCAATGAAATTGGAG
BuV2 VP2 e SalI ATTg cgacTTACATTGTGTAGTTAGGCATGGCTCT
BuV3 VP2 wd BamHI TAgga ccATGTCCGAAAGCAATGAAATTGACG
BuV3 VP2 e SalI ATTg cgacTTAGTATGTGTAGTTTGGCATTGCTC
TuV VP2 wd BamHI TAgga ccATGGCAGCCTCTAGCTCAGACAGTG
TuV VP2 e SalI ATTg cgacTTAGTAAACAGTAGAGGGTACAATTCTTC
Table 4. P ime s used o ampli ying he VP2 gene o BuV1, 2 and 3 and TuV. The es ic ion si es a e
w i en in lowe case.
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Scien i ic RepoR s | 6:39267 | DOI: 10.1038/s ep39267
Wells we e washed 4 imes wi h PBST and ho se adish-pe oxidase conjuga ed an i-human IgG (DAKO), dilu ed
1:4000 in he Diluen , was applied o 1 hou . A e 4x PBST washes, 3,3′ ,5,5′ - e ame hylb enzidine (TMB,
DAKO) was applied o 10 min, he eac ion was s opped wi h 0.5 M H2SO4, and he ODs we e measu ed a
450 nm (Mul iskan EX, The mo Fische Scien i ic). Blank ODs we e sub ac ed om he es ODs.
IgG es ing o human se a. IgG EIA analyses o BuV1, BuV2, BuV3 and TuV as well as o High5 insec
cells we e ca ied ou in panels, in which each sample was analysed o all i e an igens simul aneously in adja-
cen wells (one well pe an igen). This p ocedu e would e eal any unspeci ic backg ound due o a ious ac o s,
allowing hei assessmen in mo e de ail. To con i m he IgG esul s and de e mine he an igenic c oss- eac i i y
o BuV1-3 and TuV, all samples showing measu able OD alues (≥ 0.1) we e subjec ed o homologous and he -
e ologous compe i ion, as desc ibed o human boca i uses19. In b ie , he plasma o se um sample was mixed
wi h an unbio inyla ed an igen (VP2-VLP a 20 μ g/ml o High5 insec cell lysa e a 0.5 mg/ml) p io o he EIA
o in e es . Each eac i e sample was sepa a ely blocked bo h wi h he homologous an igen, which should ully
block a speci ic eac ion, and wi h all ou he e ologous an igens, which should no block he speci ic eac ion.
The o e all BuV-IgG esul a e compe i ion was conside ed posi i e when no he e ologous blocking occu ed
and he esidual OD ell upon homologous blocking (i) below OD 0.2, when he non-blocked OD was > 0.5; (ii)
below OD 0.15, when he non-blocked OD was 0.3–0.5; and (iii) below 50% o he non-blocked OD, when he
non-blocked ODs we e low, 0.1–0.3. Examples o blocking esul s a e shown in Table3. Fo e ec i e blocking
in TuV EIA, a 5- old highe concen a ion o homologous blocking an igen, compa ed o ha in BuV EIA, was
equi ed (Case 18, Table3).
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Acknowledgemen s
This s udy was suppo ed by g an s om he Academy o Finland (g an #1257964), he Jane and Aa os E kko
Founda ion, he Sig id Jusélius Founda ion, he Medical Socie y o Finland (FLS), he Resea ch Funds o he
Uni e si y o Helsinki, he Helsinki Uni e si y Hospi al Resea ch and Educa ion Fund, and he Li e and Heal h
Medical G an Associa ion, he Paulo Founda ion, he Founda ion o Resea ch on Vi al Diseases, he Clinical
Chemis y Founda ion, he Osca Ö lund Founda ion, he Finnish-No wegian Medical Founda ion, he Jenny
and An i Wihu i Founda ion, he O ion Resea ch Founda ion, and he Finnish Socie y o S udy o In ec ious
Diseases.
Au ho Con ibu ions
E.V. de eloped me hods o and pe o med he EIA expe imen s, E.V., I.K. and V.L. pe o med he qPCR
expe imen s, M.P. analysed he clinical da a, E.V., M.P., A.K., F.R., K.A., E.D. and T.V. collec ed he samples and
conduc ed he sample p ocessing, E.V. and M.S.V. designed he esea ch and analysed he da a, E.V. p epa ed he
igu es, E.V., K.H. and M.S.V. w o e he manusc ip , T.V., K.H. and M.S.V. supe ised he esea ch. All au ho s
e iewed he manusc ip du ing i s p epa a ion.
Addi ional In o ma ion
Accession Codes: The VP2 gene sequences o he BuV1-3 and TuV cons uc s can be ound in GenBank,
accession nos. KX856937-KX856940.
Compe ing inancial in e es s: The au ho s decla e no compe ing inancial in e es s.
How o ci e his a icle: Väisänen, E. e al. Epidemiology o wo human p o opa o i uses, bu a i us and
usa i us. Sci. Rep. 6, 39267; doi: 10.1038/s ep39267 (2016).
Publishe 's no e: Sp inge Na u e emains neu al wi h ega d o ju isdic ional claims in published maps and
ins i u ional a ilia ions.
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