PERSPECTIVE
published: 24 Janua y 2017
doi: 10.3389/ micb.2017.00045
F on ie s in Mic obiology | www. on ie sin.o g 1Janua y 2017 | Volume 8 | A icle 45
Edi ed by:
Da id Gilme ,
Uni e si y o S asbou g, F ance
Re iewed by:
D’Ann Rochon,
Ag icul u e and Ag icul u e-Food
Canada, Canada
Ma ie-Line Isk a-Ca uana,
CIRAD, F ance
*Co espondence:
Sebas ien Massa
[email p o ec ed]
Special y sec ion:
This a icle was submi ed o
Vi ology,
a sec ion o he jou nal
F on ie s in Mic obiology
Recei ed: 10 No embe 2016
Accep ed: 06 Janua y 2017
Published: 24 Janua y 2017
Ci a ion:
Massa S, Cand esse T, Gil J,
Lacomme C, P edajna L, Ra nika M,
Reyna d J-S, Rumbou A, Salda elli P,
Ško i ´
c D, Vainio EJ, Valkonen JPT,
Vande schu en H, Va e i C and
We zel T (2017) A F amewo k o he
E alua ion o Biosecu i y, Comme cial,
Regula o y, and Scien i ic Impac s o
Plan Vi uses and Vi oids Iden i ied by
NGS Technologies.
F on . Mic obiol. 8:45.
doi: 10.3389/ micb.2017.00045
A F amewo k o he E alua ion o
Biosecu i y, Comme cial, Regula o y,
and Scien i ic Impac s o Plan
Vi uses and Vi oids Iden i ied by NGS
Technologies
Sebas ien Massa 1*, Thie y Cand esse2, José Gil3, Ch is ophe Lacomme4,
Lukas P edajna5, Maja Ra nika 6, Jean-Sébas ien Reyna d7, A emis Rumbou8,
Pasquale Salda elli9, Dijana Ško i´
c10, Ee a J. Vainio11, Ja i P. T. Valkonen12,
He é Vande schu en13, Ch is ina Va e i14 and Thie y We zel15
1Plan Pa hology Labo a o y, Gembloux Ag o-Bio Tech, Uni e si y o Liège, Gembloux, Belgium, 2Ins i u Na ional de la
Reche che Ag onomique (INRA), Uni e si y o Bo deaux, CS20032 UMR 1332 BFP, Villena e d’O non, F ance, 3Plan
Biology, Linnean Cen e o Plan Biology, Uppsala BioCen e, Swedish Uni e si y o Ag icul u al Sciences, Uppsala, Sweden,
4Vi ology and Zoology, Science and Ad ice o Sco ish Ag icul u e, Edinbou gh, UK, 5Depa men o Plan Vi ology, Ins i u e
o Vi ology, Biomedical Resea ch Cen e , Slo ak Academy o Science (SAS), B a isla a, Slo akia, 6Depa men o
Bio echnology and Sys ems Biology, Na ional Ins i u e o Biology, Ljubljana, Slo enia, 7Vi ology, Ag oscope, Nyon,
Swi ze land, 8Di ision Phy omedicine Len zeallee, Facul y o Li e Sciences, Alb ech Daniel Thae -Ins i u e o Ag icul u al and
Ho icul u al Sciences, Humbold -Uni e si y o Be lin, Be lin, Ge many, 9Na ional Resea ch Council Ins i u e o Sus ainable
Plan P o ec ion, Ba i, I aly, 10 Depa men o Biology, Facul y o Science, Uni e si y o Zag eb, Zag eb, C oa ia,
11 Managemen and P oduc ion o Renewable Resou ces, Na u al Resou ces Ins i u e Finland (Luke), Helsinki, Finland,
12 Depa men o Ag icul u al Sciences, Uni e si y o Helsinki, Helsinki, Finland, 13 Plan Gene ics, Gembloux Ag o-Bio Tech,
Uni e si y o Liège, Gembloux, Belgium, 14 Depa men o Phy opa hology, Benaki Phy opa hological Ins i u e, A hens,
G eece, 15 DLR Rheinp alz, Ins i u e o Plan P o ec ion, Neus ad an de Weins asse, Ge many
Recen ad ances in high- h oughpu sequencing echnologies and bioin o ma ics ha e
gene a ed huge new oppo uni ies o disco e ing and diagnosing plan i uses and
i oids. Plan i ology has undoub edly bene i ed om hese new me hodologies,
bu a he same ime, aces now subs an ial bo lenecks, namely he biological
cha ac e iza ion o he newly disco e ed i uses and he analysis o hei impac a he
biosecu i y, comme cial, egula o y, and scien i ic le els. This pape p oposes a scaled
and p og essi e scien i ic amewo k o e icien biological cha ac e iza ion and isk
assessmen when a p e iously known o a new plan i us is de ec ed by nex gene a ion
sequencing (NGS) echnologies. Fou case s udies a e also p esen ed o illus a e he
need o such a amewo k, and o discuss he scena ios.
Keywo ds: NGS, pes isk analysis, i us diseases, biological cha ac e iza ion, plan heal h, egula o y agencies
INTRODUCTION
Un il ecen ly, plan i us disco e y appea ed as a long and as idious ask, mainly d i en by he
need o iden i y he e iology o diseases o unknown o igin. In he las ew yea s, howe e , he ad en
o nex gene a ion sequencing (NGS) has e olu ionized he s udy o plan i uses by p o iding a
powe ul al e na i e o hei de ec ion and iden i ica ion, wi hou any a p io i knowledge. NGS
echnologies a e p og essi ely eaching he diagnos ic ield (Massa e al., 2014), impac ing also
qua an ine egula ions (Ma in e al., 2016). Thei use allows he con inuous disco e y o new plan
Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses
i uses, he obse a ion o an inc easing di e si y o a ian s
o known i uses and he equen exis ence o a complex o
di e en i uses. Downs eam o hese indings, he main
challenge o be add essed is he biological signi icance
(pa hogenici y, hos s, ansmission, epidemiology...) o he
disco e y o a no el i us species o s ain in single plan s,
pa icula ly in asymp oma ic plan s and o i uses ha a e he
ounding membe s o new i us gene a o amilies and sha e
li le o no sequence simila i y wi h known i uses (Wu e al.,
2015). Plan i ologis s a e now challenged o do he wo k
backwa ds, namely o cha ac e ize new i uses, wi h genome
sequence in o ma ion as he unique s a ing poin . Fu he mo e,
he abili y o NGS o de ec c yp ic (symp omless) i uses
in cul i a ed and wild plan s, and he epo ed mu ualis ic
in e ac ions challenging he adi ional dogma ha all i uses a e
pa hogens (Roossinck, 2011), a e adding new di icul ies o he
p edic ion o he impac o new i uses.
Undoub edly, hese new scien i ic ou comes will ha e o be
handled wi h ex eme ca e in e ms o dissemina ion om basic
esea ch o applied ag onomy, and o plan heal h, ag icul u e
and o es y policy make s and egula o s. Indeed, he amoun o
in o ma ion needed o assess he isk posed by a new i us species
o a ce ain commodi y o egion is huge. Scien is s may indeed
ha e o wo k o yea s o p o ide he answe s needed o conduc
a ho ough Pes Risk Analysis (PRA) acco ding o in e na ional
phy osani a y s anda ds (ISPM 2 and ISPM 11) (FAO, 2004,
2007). The e o e, he main challenge o scien is s a ising om
he disco e y o a new i al sequence is o e icien ly cha ac e ize
he biological p ope ies h ough e icien sho , mid-, and
long- e m s a egies while c ea ing app op ia e communica ion
channels wi h he egula o y au ho i ies. In his pape , we aim
o discuss he new challenges aised by NGS as illus a ed by
case s udies, and o sugges guidelines o esea che s, policy
make s, plan heal h au ho i ies and plan inspec ion se ices by
desc ibing he necessa y s eps, he app op ia e in e ac ions and
he inhe en p io i iza ion o be ollowed a e disco e ing a new
i us sequence [o a new isola e o a ian , o (a) new hos (s)].
O e all, he ad en o new NGS echnologies wisely
complemen ed by adi ional o classical i us s udy me hods is
expec ed o globally p omo e plan heal h and plan p o ec ion,
bu may also cause mayhem in ade and ag icul u e i challenges
and ques ions a e no add essed p ope ly.
CURRENT SITUATION WITH KNOWN
VIRUSES
Qua an ine and ce i ica ion lis s a e egula ly upda ed ollowing
new PRAs. They a e based on 6 ca ego ies o in o ma ion:
(i) knowledge o he iden i y o he pes (and he e o e being
able o di e en ia e i om o he i al agen s); (ii) da a on i s
dis ibu ion and (iii) hos ange; (i ) in o ma ion on he modes
and e iciency o sp ead and on he iden i y o any ec o (s); ( )
sui abili y o he local ag o-en i onmen al condi ions o he pes
[and ec o (s)]; and ( i) he abili y o cause a disease and impac
he de elopmen , ep oduc ion, o p oduc i i y o cul i a ed o
wild hos plan s. Fu he , e inemen o he isk assessmen can
be based on addi ional in o ma ion such as he a ailabili y o
e icien and easy- o-implemen con ol me hods.
The PRA p o ides he isk assesso wi h a isk assessmen ool
whose de ails will depend bo h on da a a ailabili y conce ning
he six poin s lis ed abo e and on he needs o he isk manage ,
whose ole is o conside he a ailable scien i ic in o ma ion
( isk assessmen ) as well as o he ac o s (economical, accep able
poli ical isk, easibili y, and impac o measu es) in eaching
a decision on whe he o egula e o add ess he pes in any
speci ic way.
EMERGING CHALLENGES FOR
SCIENTISTS AND AUTHORITIES
When a known i us is de ec ed by NGS echnologies, he
main ques ions a e ela ed o i s legal s a us (see Figu e 1). The
decision scheme o be applied is based on exis ing qua an ine
o ce i ica ion egula ions and is simila o he p ocess
ou inely ca ied ou wi h PCR o ELISA-based me hodologies.
Ne e heless, NGS may also e eal addi ional complexi y and
e en eo ien disease in es iga ion o known species (see he
S udy Case N◦1 in Supplemen a y Ma e ial).
I should be s essed ha isk assessmen is some imes needed
o eques ed by Na ional Au ho i ies in si ua ions whe e ha dly
any in o ma ion is a ailable, a seen in ecen examples in 2016
o Peppe ein yellow i us (PeVYV) o G ape ine pino g is
i us (Ge many). In such a case, isk assesso s canno wai
un il enough da a is a ailable and i s conclusions ha e o be
eached based on wha e e in o ma ion is a hand. This mus
be done cau iously, while simul aneously aking in o accoun he
unce ain ies associa ed o o caused by he lack o da a and
making hem clea o he isk manage .
In his con ex , he inc easing iden i ica ion o new i al
sequences by NGS echnologies may complica e he decision-
making p ocess o ce i ica ion p og ams, qua an ine p ocesses,
and mo e gene ally he ading o plan ma e ials (see he S udy
Case N◦2 in Supplemen a y Ma e ial). The only in o ma ion
a ailable may be he ull o pa ial genome sequence wi h
he addi ional complexi y ha i may ha e been de ec ed in
asymp oma ic samples o in combina ion wi h o he i uses. I
is he e o e inc easingly impo an o unde s and he biology
o any new i al sequence o p o ide a basis o assessing he
isk hey pose and ake scien i ically-based decisions. Scien is s
a e he e o e now challenged o p o ide biological da a on hese
newly desc ibed i al sequences, in a sho ime ame and wi h
limi ed unding. The ollowing chap e s p opose a amewo k
o scaled ac ions o e icien ly add ess he need o PRA o
egula o y au ho i ies.
EARLY STEPS OF BIOLOGICAL
CHARACTERIZATION
The de ec ion o a new i al sequence should di ec ly be
ollowed by se e al immedia e ac ions o i s ea ly biological
cha ac e iza ion o each a i s decision le el o no i ica ion o
au ho i ies (see Figu e 2).
F on ie s in Mic obiology | www. on ie sin.o g 2Janua y 2017 | Volume 8 | A icle 45
Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses
FIGURE 1 | F amewo k o known i uses. Y means posi i e esponse and N nega i e esponse (do ed lines co espond o op ional analyses o non qua an ine
pa hogens depending on he eques o he cus ome ).
Con i ma ion o De ec ion
To dispel a labo a o y o bioin o ma ics e o o con amina ion,
NGS-based iden i ica ion o pu a i e new i al sequence o new
s ains o exis ing i al species equi es u he con i ma ion by
complemen a y me hods, usually by (RT)-PCR using speci ic
p ime s. I he concen a ion is e y low, he sample can be
en iched in i al pa icles h ough homogeniza ion, il a ion,
and/o ul acen i uga ion p io o nucleic acid ex ac ion.
En iched i al pa icles can also be obse ed by elec on
mic oscopy, e en i elec on mic oscopy lacks sensi i i y and
i al pa icles can be missed. Fo DNA i uses, especially
Caulimo i idae, addi ional esea ch is also needed o e alua e
whe he he de ec ed i al sequences exis in an episomal o m
and no only as he ace o a “ ossilized” i us in eg a ed in
he plan genome (Liu e al., 2010; Aiewsakun and Ka zou akis,
2015).
P o isional Taxonomy Assignmen
The assembled con ig will gene ally allow o p o isional
axonomic assignmen o he i al sequence. Ne e heless, his
can u n ou o be a e y di icul ask, made e en mo e
complex by (i) he equen incomple eness o he genome
sequence da a ob ained, (ii) he a iable and i egula co e age
o i al genomes, (iii) he incomple eness o i al axonomy,
and (i ) he a iabili y o species, genus, o amily molecula
disc imina ion c i e ia. Speci ic guidelines can be ound in he
la es ICTV epo (h p://www.ic online.o g). The de ec ed
i al sequences may also co espond o a bona ide i us in ec ing
o he o ganisms associa ed wi h he sample, including bac e ia,
ungi o a h opods (Al Rwahnih e al., 2011; Ma zano and
Domie , 2015) o o i al sequences in eg a ed in o he plan
genome. The u u e solu ion o his p oblem is o es ablish he
baseline o i us p esence h ough an imp o ed knowledge o he
i omes o ag onomically impo an plan s as well as i omes o
na u al ecosys ems, including he in eg a ed i al sequences.
Bibliog aphy
O e all, p o isional axonomic assignmen should always be
conside ed wi h cau ion bu can gi e i s clues on he physical
and/o chemical p ope ies o a i us as well as on i s
biology, including (i) he pu a i e hos ange o he en a i e
iden i ica ion o al e na i e hos s, (ii) he p edic ion o he
pa hogenic po en ial in hose hos s, (iii) he modes o ho izon al
and/o e ical ansmission, including he iden i ica ion o
candida e ec o s. These hypo heses ha e p ac ical consequences
such as he selec ion o app op ia e hos s and expe imen al
modes o inocula ion, a i s assessmen o he possible epidemic
po en ial o a new i al agen , o a en a i e iden i ica ion o
possible al e na i e hos s in ecosys ems. Al oge he , his ea ly
s ep, which equi es expe ise in molecula biology, in plan
i ology, and basic bioin o ma ics skills, migh lead o he
elabo a ion o a d a epidemiological scena io (Loconsole e al.,
2012; Adams e al., 2014). Ne e heless, his app oach can be
biased as o example in he genus Clos e o i idae, which includes
economically impo an i uses wi h simila i ion mo phology,
genome size, and o ganiza ion bu wi h con as ed biological
p ope ies (Ma elli e al., 2012).
Sample Documen a ion
In addi ion, all he me ada a abou he sample should be collec ed
p ecisely o eed he isk assessmen p ocess. I is impo an o
documen symp oms (i any) and ime o appea ance, o iden i y
he plan species and cul i a , o desc ibe he sampled plan
F on ie s in Mic obiology | www. on ie sin.o g 3Janua y 2017 | Volume 8 | A icle 45
Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses
FIGURE 2 | F amewo k p oposal ollowing disco e y o new i uses. Y means posi i e esponse and N nega i e esponse. The numbe s be ween b acke s
co espond o he chap e s in he ex . *The absence o i al pa icle should be eco ded and u he expe imen s ca ied ou i new cases a e disco e ed.
pa (lea es, ui , seeds, e c.), he geog aphical o igin o he
sample, i s in ended use e c... Any missing in o ma ion should
be comple ed a his s age so as o be included in he i s
no i ica ion.
Full Genome Sequencing and Anno a ion
The assembly o i al sequence(s) based on NGS eads can
esul in h ee scena ios o genome co e age: (i) comple e,
(ii) incomple e bu con inuous, o (iii) a se o sca olds and
con igs. The econs uc ion o he ull genome sequence o a
candida e new i us is mainly based on a ge ed (RT)-PCR
ampli ica ion and sequencing o ill he sequence gaps be ween de
no o gene a ed con igs, e i y hei exclusi e i al o igin and he
absence o hos -de i ed sequences. Taxonomic assignmen can
also be ine- uned hanks o he whole genome sequence; bu in a
wo s case si ua ion, e en he a ailabili y o a comple e genomic
sequence may be insu icien o se le such a ques ion (Ma ais
e al., 2016).
F on ie s in Mic obiology | www. on ie sin.o g 4Janua y 2017 | Volume 8 | A icle 45
Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses
De elopmen o a Diagnos ic P o ocol
Iden i ying a new i us species, e en om a pa ial genome
sequence, igge s he immedia e need o de elop a diagnos ic
p o ocol. This is a undamen al s ep owa d he managemen
o i al diseases in cul i a ed c ops o he un eiling o a new
i us eco-epidemiological ole in wild plan s. The diagnos ic o
newly iden i ied and con i med i uses is usually done using
PCR-based me hods ( he mos popula ones o new i uses) o
wi h he LAMP echnology, as a ecen al e na i e (Boonham
e al., 2014). Nucleic acid-based me hods a e cu en ly p e e ed
o diagnose new i uses because hey a e quicke , easie and
cheape o de elop han an ibody p oduc ion o ELISA.
Field/Ba ch Obse a ion
Small-scale epidemiological su eys a he disco e y loca ion
can be unde aken. Such su eys will also ake in o accoun he
hypo heses ha may ha e a isen a e bibliog aphical esea ch
and should be ocused on he plan species whe e he i us
was de ec ed; bu hey could also ake in o accoun , whene e
possible, he po en ial hos (s) and ec o (s), hei geog aphical
sp ead and seasonali y. Likewise, sampling me hods enabling o
he s a is ical linking o indings and ield obse a ions should be
selec ed. Se e al scena ios a e possible depending on he o igin
o he sample: ield o ade/qua an ine/ce i ica ion.
The p esence o symp oms in he ini ial ield hos has o en
been pe cei ed as a s a ing poin o u he in es iga ions abou
he p esence o a i us, bu asymp oma ic plan s mus also
be included in he sampling because symp oma ology e lec s
he complex in e play o in ec ious agen (s), he hos plan
me abolism, i s de ense sys ems and a ie al speci ici ies, as well
as abio ic ac o s. The ad en o wide me agenomic s udies o
en i onmen al i omes (K is ensen e al., 2010; Mohiuddin and
Schellho n, 2015) allows o he iden i ica ion o new i uses
om any sample, including asymp oma ic ield samples. Thus,
e en i ocused symp om obse a ions in he ield canno be
neglec ed, a su ey canno be led by symp oma ology alone.
Hence, i us sp ead o o he ag o-ecological niches whe e hey
may encoun e new hos s, co-in ec ing i uses o ec o s, may
cause a shi in hei pa hogenic po en ial and esul in a se ious
disease.
IN-DEPTH BIOLOGICAL
CHARACTERIZATION
The in-dep h biological cha ac e iza ion o a new i us
is en isioned as a mid- o long- e m goal in o de o
g adually educe he unce ain ies associa ed wi h he ea ly isk
assessmen and decision-making p ocesses. Mo e speci ically, in-
dep h biological cha ac e iza ion aims o build comp ehensi e
knowledge on he symp oma ology/e iology, epidemiology and
ecology o a new i us by ga he ing in o ma ion on i s hos ange,
i s symp oma ology on a ious cul i a s and hos species, i s
ec o s and modes o ansmission, i s geog aphical dis ibu ion,
and i s in e ac ions wi h o he i uses. Ful illing Koch’s pos ula es
can be conside ed as a co ne s one and is pi o al in his in-
dep h biological cha ac e iza ion. An addi ional di icul y a ises
in he case o a i us iden i ied om asymp oma ic issue
o i uses wi h po en ially neu al o bene icial in e ac ions
wi h he plan s (Xu e al., 2008; an Molken e al., 2012).
In hese cases, Koch’s pos ula es may p o e useless, because
hey p esume he de elopmen o disease symp oms ollowing
in oduc ion o he pa hogen in o a heal hy hos . Bu labo a o y
and g eenhouse expe imen s as well as ield su eys will b ing
mo e in o ma ion and migh b ing he p oo o mu ualis ic o
syne gis ic in e ac ions be ween he i us and he plan . These
da a will be necessa y o isk assessmen o make u he
p og ess by de e mining whe he he i us is p esen (locally o
widesp ead) o no in a gi en geog aphical a ea, i s po en ial
o sp ead ( ype o ansmission, hos ange), and ul ima ely o
e alua e i s economic impac (symp oms alone o in associa ion).
In u n, local au ho i ies may de ine app op ia e egula o y
measu es in ela ion o Plan Heal h p o ec ion and con ol i
equi ed. The S udy Case N◦3 on G ape ine Pino G is i us (see
Supplemen a y Ma e ial) exempli ies his complex app oach.
T ansmission Expe imen s
The inocula ion o a new i us o es plan s in
labo a o y/g eenhouse condi ions is he basis o any biological
cha ac e iza ion and may be pa icula ly complex. Depending
on he objec i e, he i us can be ansmi ed o indica o plan s,
hos plan candida es o o he cul i a s o a same species. The
modes o e ical and ho izon al ansmission o i uses a e
di e se, so ha he in eg a ion o axonomic (Figu e 2 and
Sec ion P o isional Taxonomy Assignmen ), bibliog aphical
(Figu e 2 and Sec ion Bibliog aphy), and ield su ey da a
(Figu e 2 and Sec ion Field Su eys) is o u mos impo ance o
he p ope design o expe imen al ansmission p ocedu es in
he labo a o y o he g eenhouse.
Plan s can be inocula ed by se e al echniques, he mos
common ones being mechanical inocula ion, g a ing, ec o
inocula ion o he use o dodde , bu all hese echniques
ha e po en ial limi a ions as hey may no sepa a e i uses in
case o a mix u e o i uses. A e y in e es ing and uni e sal
echnique o an in-dep h cha ac e iza ion o a i us is he
p epa a ion o in ec ious clones. The in ec ious clones, equi ing
he comple e i al genome sequence, o e aluable in o ma ion
abou (i) symp oma ology o indi idual i uses and o mix u e
o i uses, (ii) hos ange s udies and ansmissibili y, (iii) he
assessmen o na u al o induced mu a ion/ ecombina ion a es
in hos plan s, and (i ) he e ec s o a ge ed modi ica ions
on i us-hos in e ac ions and symp oma ology. Mas e ing i us
ansmission will also be key o he de elopmen o esea ch
e o s o unde s and he po en ially posi i e e ec s o i uses
on plan s. Inocula ed plan s om a ious species o cul i a s can
be submi ed o bio ic o abio ic s esses, and hei eac ion can
be s udied in labo a o y and/o in g eenhouse expe imen s o
p o ide e idence o bene icial in e ac ions.
Field Su eys
La ge-scale su eys, o ganized on a na ional o in e na ional
scale and based on he diagnos ic echnique(s) de eloped o
he new agen , should be unde aken o e alua e i s p e alence
and dis ibu ion wi hin a gi en ecosys em (K enz e al., 2014).
These s udies will aim o moni o he candida e/demons a ed
hos plan s/ ec o s. They should be ca ied ou in ag icul u al
F on ie s in Mic obiology | www. on ie sin.o g 5Janua y 2017 | Volume 8 | A icle 45

Massa e al. P oposal: F amewo k o Cha ac e isa ion o New Vi uses
ecosys ems ( ields, comme cial o cha ds o ineya ds, nu se ies,
and ge mplasm collec ions), and in na u al ecosys ems, o
enable a holis ic cha ac e iza ion o he pa hosys ems wi h
pa icula a en ion o symp oma ology. They will complemen
he p elimina y in o ma ion gained a a smalle scale (See
Sec ion Field/Ba ch Obse a ion) o in he labo a o ies and
g eenhouses (See Sec ion T ansmission Expe imen s). The
in e play be ween ield su eys and labo a o y expe imen s is
pa icula ly impo an a his s age because i will allow a ge ing
he mos app op ia e hos plan (s) and ec o (s), and hus
inc eases knowledge on i us biology.
La ge-scale su eys will also p o ide insigh s in o he gene ic
a iabili y o a new i us wi hin he su eyed e i o ies.
This knowledge will be key o e o s o op imize diagnos ic
echniques by selec ing p ime s co e ing he gene ic di e si y o
he species (Massa e al., 2014). In addi ion, he iden i ica ion
o new a ian s may b ing new undamen al hypo heses on he
pa hogenici y o he i uses, and hese new a ian s could be
cha ac e ized unde labo a o y and g eenhouse condi ions (See
Sec ion T ansmission Expe imen s).
The Speci ic Challenge o Vi us Complexes
The biological cha ac e iza ion o new i uses may be pa icula ly
challenging wi h complex o di e en i uses, whe e he
combina ion o wo o mo e i uses can signi ican ly modi y
hei pa hogenic po en ial h ough syne gis ic o an agonis ic
in e ac ions (Ma in and Elena, 2009; Sylle and G upa, 2016).
Mix u e o i us species is indeed e y equen , especially
in woody plan s like g ape ine (Joos e e al., 2015), o en
leading o unp edic able a ia ions in symp oms, in ec i i y,
accumula ion, and/o ec o ansmissibili y. Biological and
echnological solu ions o isola ing and de e mining he ole
o indi idual i uses a e some imes possible using selec i e
expe imen al hos s ollowed by back-inocula ion o “pu i ied”
isola es o he o iginal hos (Ali and Roossinck, 2008), pa ial
sani a ion (Maliogka e al., 2015), in ec ious clones (Nagyo á
and Šub , 2007), o he use o ion-exchange ch oma og aphy
on monoli hic suppo s o sepa a e i al pa icles (Rušˇ
ci´
c e al.,
2015). The biological p ope ies o i uses alone o in mix u e
can he e o e be compa ed in dep h (see Sec ion Field Su eys).
The complexi y o disease e iology in ield condi ions migh pose
addi ional challenges, as exempli ied in he Case S udy N◦4 on
ca o (See Supplemen a y Ma e ial).
CONCLUSIONS
NGS echnologies pose new challenges o scien is s and o plan
heal h au ho i ies when i comes o analyzing he isks associa ed
wi h a new i us o pa hogen, i s po en ial o sp ead, o i s
economic impac and when ying o ake a imely decision
on whe he o le go o des oy con amina ed plan ma e ials.
As sequencing h oughpu and bioin o ma ics analyses a e less
and less limi ing, downs eam epidemiology and disease e iology
s udies will be he ob ious bo lenecks in abili y o conclude
on he biological signi icance and impac o no el i uses o
o complex o di e en i uses. While exhaus i e knowledge
on he e iology o a disease o he epidemiology o a i us
is e y di icul i no impossible o achie e, we p opose a
amewo k o expe imen s and in es iga ions o cha ac e ize
hese newly disco e ed i uses and o unde ake ele an ac ions
in a imely ashion in ela ion o he con ex o he inding.
In he ame o he isks posed by g owing ade and clima e
change, he aim is o p og essi ely educe he unce ain ies
linked wi h isk managemen and o help plan heal h p o ec ion
au ho i ies decide on he impo ance o a new i us in a
qua an ined ma e ial o a ce i ied seed s ock. Howe e , his
decision will also always be in luenced by local legisla ion and he
socio-economic and poli ical in e p e a ion o he p og essi ely
gene a ed in o ma ion. Once adop ed by au ho i ies and ade,
his amewo k should help o ake a ionalized decisions on he
mos ele an ac ions o ake (i.e., con i ma ion using speci ic
me hods, ways o assess impac o a i us in i s en i onmen ) and
po en ially p e en “con lic s” be ween impo /expo pa ne s.
AUTHOR CONTRIBUTIONS
All he au ho s signi ican ly con ibu ed o he w i ing and
edi ing o he manusc ip . SM c ea ed he igu es. SM and TW
coo dina ed he w i ing and edi ing o he manusc ip .
FUNDING
This a icle is based upon wo k om COST Ac ion FA1407
(DIVAS), suppo ed by COST (Eu opean Coope a ion in Science
and Technology).
ACKNOWLEDGMENTS
We hank F ancoise Pe e and Baldisse a Gio anni (EPPO) o
hei inpu and c i ical eading o he manusc ip .
SUPPLEMENTARY MATERIAL
The Supplemen a y Ma e ial o his a icle can be ound
online a : h p://jou nal. on ie sin.o g/a icle/10.3389/ micb.
2017.00045/ ull#supplemen a y-ma e ial
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Con lic o In e es S a emen : The au ho s decla e ha he esea ch was
conduc ed in he absence o any comme cial o inancial ela ionships ha could
be cons ued as a po en ial con lic o in e es .
Copy igh © 2017 Massa , Cand esse, Gil, Lacomme, P edajna, Ra nika , Reyna d,
Rumbou, Salda elli, Ško i´
c, Vainio, Valkonen, Vande schu en, Va e i and We zel.
This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons
A ibu ion License (CC BY). The use, dis ibu ion o ep oduc ion in o he o ums
is pe mi ed, p o ided he o iginal au ho (s) o licenso a e c edi ed and ha he
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F on ie s in Mic obiology | www. on ie sin.o g 7Janua y 2017 | Volume 8 | A icle 45