Systemic distribution of different low pathogenic avian influenza (LPAI) viruses in chicken.

0.28 MB·English

by  Post, Jacob

Postetal.VirologyJournal2013,10:23
http://www.virologyj.com/content/10/1/23
SHORT REPORT Open Access
Systemic distribution of different low pathogenic
avian influenza (LPAI) viruses in chicken
Jacob Post1*, Eveline D de Geus2, Lonneke Vervelde2, Jan BWJ Cornelissen1 and Johanna MJ Rebel1
Abstract
Background: Since wewere able to isolate viable virus from brain and lung ofH7N1low pathogenic avian
influenza virus (LPAIV) infected chickens, wehere examined thedistribution of different LPAIV strains in chickens by
measuring theviral AIRNA load inmultipleorgans.Subtypes ofH5 (H5N1, H5N2), H7 (H7N1, H7N7) and H9 (H9N2),
ofchicken(H5N2, H7N1, H7N7,H9N2), or mallard (H5N1) origin were tested. The actual presence of viable virus was
evaluated withvirus isolation in organs of H7N7inoculated chickens.
Findings: Viral RNA was found by PCR inlung, brain, intestine, peripheral blood mononuclear cells, heart, liver,
kidney and spleen from chickens infectedwith chicken isolated LPAIV H5N2,H7N1, H7N7or H9N2.H7N7virus
could be isolated from lung, ileum,heart,liver, kidney and spleen, but not from brain, which was in agreement
withthedata from thePCR. Infection withmallard isolated H5N1LPAIVresulted in viral RNA detectionin lung and
peripheral blood mononuclear cellsonly.
Conclusion: We speculate that chickenisolated LPAI viruses are spreadingsystemically inchicken, independently of
thestrain.
Keywords: Lowpathogenic avian Influenza, Chickens, Systemic distribution
Findings However, some chicken-isolated LPAIV strains have been
Avian influenza (AI) A is a highly heterogeneous group isolated from a limited number of other tissues including
of viruses with varying pathogenicity in different species. the pancreas, kidneys and oviduct of intranasally (i.n.) or
Influenza virus subtypes have gained sufficient adaptive intratracheally (i.t.) inoculated chickens [5]. In addition, we
molecular changes to become established in domestic wereabletoisolateviablevirusfrombrainofH7N1LPAIV
poultry and cause mild to severe disease [1-3]. AI is inoculated chickens [6]. With the introduction of PCR
classified by the world organization for animal health several reports indicate systemic distribution of mRNA of
into two pathotypes, low pathogenicity avian influenza LPAIV strains like H7N1 and H9N2 [6-8]. In order to
viruses (LPAIV) and high pathogenicity avian influenza investigate whether systemic distribution of LPAIV was
viruses (HPAIV), based on their virulence in chickens. restricted to some individually reported strains, we tested
Pandemic influenza outbreaks of HPAIV in poultry pose thesystemicdistributionoffourLPAIVchicken(C-LPAIV)
a significant threat to public health as highlighted by the andonemallard(M-LPAIV)isolate,usingPCRinchicken.
emergence of H5N1 HPAIV [4]. In general, the HPAIV H7N1 LPAIV (A/Chicken/Italy/1067/99) was a gift from
emerged from a H5 or H7 LPAIV subtype that was Dr. Ilaria Capua (Istituto Zooprofilattico Sperimentaledelle
circulating in chickens or turkeys [1]. LPAIVhaemagglu- Venezie, Italy). H5N1 LPAIV (A/Mallard/Italy/3401/05),
tinin (HA) lacks the polybasic cleavage site that charac- H5N2 LPAIV (A/Chicken/Pennsylvania/21525/83) and
terizesmostHPAIV.ThemonobasiccleavagesiteofLPAIV H7N7 LPAIV (A/Chicken/Netherlands/06022003/06) were
favors trypsin-like proteases, which are thought to be kindly provided by Dr. Guus Koch (Department of
secretedonlybycellsoftherespiratoryandintestinaltract. Virology, Central Veterinary Institute of Wageningen UR,
The Netherlands). H9N2 (A/Chicken/Saudi Arabia/
*Correspondence:[email protected] SP02525/3AAV/2000) was obtained from the Animal
1CentralVeterinaryInstituteofWageningenUR,P.O.Box65,Lelystad,8219 Health Service (Deventer, The Netherlands). The viruses
PH,TheNetherlands
were propagated and titrated in the allantoic cavities of
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Postetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
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Lung Brain
30 30
20 20
Ct Ct
5 - 5 -
4 4
10 10
0 0
H5N1 H7N1 H7N7 H5N2 H9N2 H5N1 H7N1 H7N7 H5N2 H9N2
Exp. I Exp. II Exp. I Exp. II
Ileum PBMC
30 30
20 20
Ct Ct
5 - 5 -
4 4
10 10
0 0
H5N1 H7N1 H7N7 H5N2 H9N2 H5N1 H7N1 H7N7 H5N2 H9N2
Exp. I Exp. II Exp. I Exp. II
Heart Liver
30 30
20 20
Ct Ct
5 - 5 -
4 4
10 10
0 0
H5N1 H7N1 H7N7 H5N2 H9N2 H5N1 H7N1 H7N7 H5N2 H9N2
Exp. I Exp. II Exp. I Exp. II
Kidney Spleen
30 30
20 20
Ct Ct
5 - 5 -
4 4
10 10
0 0
H5N1 H7N1 H7N7 H5N2 H9N2 H5N1 H7N1 H7N7 H5N2 H9N2
Exp. I Exp. II Exp. I Exp. II
Figure1(Seelegendonnextpage.)
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(Seefigureonpreviouspage.)
Figure1Scatterplotof45-CtvaluesofviralRNAfromorgansofindividualbirdsat2dayspostinoculation(d.p.i.).Chickenswere
inoculatedwithdifferentLPAIVstrainsandthepresenceofviralRNAinorganswasexaminedat2d.p.i.byqPCR.Thehorizontallinerepresents
themeanof5or6individualbirdswithSD.Trianglesrepresentthe45-Ctvalueofindividualbirds.ThedatawereexpressedasCt-45values,
whichmeansthatincreasedvaluesinthefiguresindicateincreasedamountofviralRNAandwhenCt-45isequaltozeronoviralRNAwas
detected.NoviralRNAwasdetectedincontrols(Ct=45).M-LPAIVH5N1,C-LPAIVH7N1andH7N7:n=6.C-LPAIVH5N2andH9N2:n=5.
10-day-old SPF chicken eggs to prepare stock virus. For described. Comparable Ct values for AI load were
animal experiments, the viruses were diluted in sterile obtained. Therefore both PCR methods can be used next
PBS to 106 EID per ml immediately prior to use. Non- to each other. As a control, tissue samples of the unin-
50
vaccinated AI-free chickens were randomly divided over fected chickens were included in each run of the qPCR.
four (experiment 1) or 2 (experiment 2) groups and Standardization of the PCR was done by equalizing the
housed in floor cages for 3 weeks prior to inoculation. amountofinputRNAwith200ng.Thequalityandinteg-
Food and water were provided ad libitum. In compliance rity of the RNA samples was analyzed using the Agilent
with Dutch law, all experiments were approved by the Bioanalyzer (lab on chip, Agilent). The data were
Animal Experimental committee of the institutions, in expressed as Ct-45 values, which means that increased
accordance with the Dutch regulations on experimental values in the figures indicate increased amount of viral
animals. For the first experiment one-day-old Lohmann RNA and when Ct-45 is equal to zero no viral RNA was
Brown layer chickens were obtained from a commercial detected.BecausenoCtvalueswerefoundforanyorgans
breeder (Pronk’s Broederij, Meppel, The Netherlands). of control chickens tested during a 45 cycles run in these
Chickens were inoculated with 0.2 ml (2×105 EID ) of particular PCRs, we considered Ct values ≤ 45 as positive
50
the LPAIVstrains H7N1, H7N7 or H5N1 equally divided [6]. H7N7 inoculated chickens from the first experiment
between the i.n. and i.t. route. Control chickens were were used as an example to confirm the presence of pro-
inoculatedwith0.2mlPBS.Sixchickensfromeachgroup geny virus in tissues where viral RNA was detected by
were sacrificed at 2 and 4 days post infection (d.p.i.). The PCR [6]. Briefly, the supernatant of homogenized and
body weight of the chickens was established daily and clarified organs of six H7N7 inoculated chickens (4 d.p.i.)
from all sacrificed chickens gross pathology of the organs was divided over five embryonated eggs. The eggs were
was studied.Lung, brain,ileum, blood, liver, kidney, heart screened daily and incubated for 7 days. After 7 days or
and spleen were collected for RNA extraction, snap- earlier after embryo mortality the allantoic fluid was har-
frozen in liquid nitrogen and stored at −80°C until use. A vested. The presence of H7N7 virus in the allantoic fluid
piece of about 0.5×0.5 cm tissue was used which cor- was tested with the HA-test and sequencing. Differences
respond to approximately 200 mg. peripheral blood between the Ct values of the strains were analyzed for
mononuclear cells (PBMC) were isolated from blood statistical significance by the Mann–Whitney U test. For
by ficol-paque (Amersham Biosciences, Uppsala, Sweden). this purpose the Ct value of samples with no detectable
For the second experiment the chickens were obtained viruswassetat45.Differencesinweightwasanalyzedfor
from a commercial breeder and inoculated as in exp. 1 statisticalsignificancebytheStudent’sT-test.P≤0.05was
with LPAIV H5N2 or H9N2. Five chickens from each consideredsignificantlydifferent.
group were sacrificed at 2 and 4 d.p.i. The body weight of To determine whether the systemic detection of
theH5N2LPAIVinoculatedchickenswasestablishedat0, LPAIV RNA was restricted to a limited set of strains, we
1,2and4d.p.i. measured systemic distribution of viral RNA from five
For RNA isolation, organs were stored in trizol LPAIV strains using PCR. Subtypes of H5 (H5N1,
(Invitrogen, Breda, The Netherlands) at −80°C until H5N2), H7 (H7N1, H7N7) and H9 (H9N2), of chicken
use. Both methods of preservation were tested and (H5N2, H7N1, H7N7, H9N2), or mallard (H5N1) origin
give equal RNA values. RNA isolation of the samples were tested. Systemic distribution of virus was con-
of both experiments was performed using the phe- firmed by virus isolation of H7N7 C-LPAIV virus parti-
nol/chloroform method as previously described [6]. cles from organs that were positive for viral RNA by
H7N1, H7N7, H5N1 and H5N2 were analyzed using the PCR. Low pathogenicity was confirmed by sequencing
quantitativePCR(qPCR)[6].ForH9N2cDNAwasgener- over the HA cleavage site [6]. Body weight of the chick-
atedfrom500ngRNAwithreversetranscriptionusingan ens was measured as an indicator of severity of the
iScript cDNA Synthesis kit (Biorad, Veenendaal, The disease after inoculation. Systemic distribution of LPAIV
Netherlands). H9N2 viral cDNA was amplified with RNA was found for the C-LPAIV strains H7N1, H7N7,
specific primers and conditions as described [6]. RNA H5N2 and H9N2. For these strains viral RNA was found
samples of day 2 p.i. of H9N2 infected chickens were in multiple organs (Figures 1 and 2). In contrast to the
tested using an one and two step PCR with the primers C-LPAIVstrains, viral RNA of the mallard isolated H5N1
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Lung Brain
30 30
20 20
Ct Ct
5 - 5 -
4 4
10 10
0 0
H5N1 H7N1 H7N7 H5N2 H9N2 H5N1 H7N1 H7N7 H5N2 H9N2
Exp. I Exp. II Exp. I Exp. II
Ileum PBMC
30 30
20 20
Ct Ct
5 - 5 -
4 4
10 10
0 0
H5N1 H7N1 H7N7 H5N2 H9N2 H5N1 H7N1 H7N7 H5N2 H9N2
Exp. I Exp. II Exp. I Exp. II
Heart Liver
30 30
20 20
Ct Ct
5 - 5 -
4 4
10 10
0 0
H5N1 H7N1 H7N7 H5N2 H9N2 H5N1 H7N1 H7N7 H5N2 H9N2
Exp. I Exp. II Exp. I Exp. II
Kidney Spleen
30 30
20 20
Ct Ct
5 - 5 -
4 4
10 10
0 0
H5N1 H7N1 H7N7 H5N2 H9N2 H5N1 H7N1 H7N7 H5N2 H9N2
Exp. I Exp. II Exp. I Exp. II
Figure2Scatterplotof45-CtvaluesofviralRNAfromorgansofindividualbirdsat4dayspostinoculation(d.p.i.).Chickenswere
inoculatedwithdifferentLPAIVstrainsandthepresenceofviralRNAinorganswasexaminedat4d.p.i.byqPCR.Thehorizontallinerepresents
themeanof5or6individualbirdswithSD.Trianglesrepresentthe45-Ctvalueofindividualbirds.ThedatawereexpressedasCt-45values,
whichmeansthatincreasedvaluesinthefiguresindicateincreasedamountofviralRNAandwhenCt-45isequaltozeronoviralRNAwas
detected.NoviralRNAwasdetectedincontrols(Ct=45).M-LPAIVH5N1,C-LPAIVH7N1andH7N7:n=6.C-LPAIVH5N2andH9N2:n=5.
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LPAIVstrain was only found in the lung at 2 and 4 d.p.i. (H7N1 and H9N2) and kidney (H9N2). The cause of the
and in PBMC at 4 d.p.i. Furthermore, the viral RNA load differences in distribution between the C-LPAIV strains
in in the lung and PBMC of H5N1 M-LPAIV inoculated has not been studied here, but might for instance be
chickens was generally low. In the lung, significant differ- related to receptor affinity [9,10] or replication efficiency
ences were found at 2 d.p.i. (compared to H7N1, H7N7 assuggestedfor differences betweenHPAIV[11].
and H5N2 (p ≤ 0.05)) and at 4 d.p.i. (compared to H7N1 In agreement with the viral RNA distribution, a delay
and H5N2 (p ≤ 0.01)). For PBMC significant differences in bodyweight gain was found for C-LPAIV H5N2,
were found at 4 d.p.i. (compared to H5N2 (p ≤ 0.01) and H7N1 and H7N7 but not for H5N1 M-LPAIV infected
H9N2(p≤0.05)).AclearcontrastbetweenM-LPAIVand chickens (Figure 3). The decrease in weight gain was
C-LPAIV was also seen in the viral RNA load in the only significant for H5N2 C-LPAIV, which corresponded
spleen. While in the majority of the C-LPAIV inoculated with the highest number of organs in which viral RNA
chickensviral RNA was detected inthe spleen, viral RNA was detected. Although bodyweight was not measured
could not be detected in the spleen of H5N1 M-LPAIV after H9N2 inoculation in this experiment, weight loss
inoculatedchickens(Figures1and2). waspreviouslyfoundafterinfectionwiththisvirusstrain
Although viral RNA was detected in most lungs and (pers. comm. C.A. Jansen) and also in relation to other
spleens after inoculation with C-LPAIV strains, differ- H9N2strains [12-14].
ences were found between the C-LPAIV strains. After a In contrast to the review of Spickler et al. (2008) we
H5N2 C-LPAIV infection all chickens become positive here show a wide viral RNA distribution over multiple
for viral RNA and almost all evaluated organs of these organs of different LPAIVstrains isolated form chickens.
chickens were infected (Figures 1 and 2). At 2 d.p.i. a The differences in amount of viral RNA between strains
significant higher viral load was found in PBMC (com- in tissues might indicate that not all C-LPAIV could be
pared to H7N1, H7N7 (p ≤ 0.01) and H9N2 (p ≤ 0.05)), detected systemically. However, since our study shows
heart (compared to H7N1 and H9N2 (p ≤ 0.01)) and that different LPAI strains were able to spread systemic-
kidney (compared to H9N2 (p ≤ 0.01)). At 4 d.p.i. a sig- ally beyond the respiratory and gastrointestinal tract,
nificant higher viral load was found in brain (compared systemic distribution of LPAI viruses could very well be
to H7N1, H7N7 (p ≤ 0.01) and H9N2 (p ≤ 0.05)) and a general phenomenon. In a parallel experiment we were
PBMC (compared to H7N1 (p ≤ 0.01) and H7N7 (p ≤ able to isolate H7N1 LPAI virus from the chicken brain,
0.05)). A typically high viral RNA load was found in the where the necessary proteases to replicate are absent [6].
kidney of H7N1 inoculated chickens at 4 d.p.i. (signifi- Here we isolated viable H7N7 LPAIV from lung, intes-
cant differences compared to H7N7, H9N2 (p ≤ 0.01) tine, heart, liver,kidney and spleen (Table 1). Viruscould
and H5N2 (p ≤ 0.05)), in the ileum of H7N7 inoculated only be isolated from organs with a high viral RNA load
chickens at 2 d.p.i. (significant differences compared to (Ct≤35)asmeasuredwiththeqPCR.Differencesinvirus
H7N1 (p ≤ 0.05)) and in the PBMC of H9N2 inoculated detection between virus isolation and qPCR is considered
chickens (significant differences compared to H7N1 and to be due to sensitivity differences in assays used [6,15].
H7N7 (p ≤ 0.05)). Figures 1 and 2 show that not all NoviruscouldbeisolatedfromthebrainofH7N7LPAIV
chickens were positive in brain (H7N1 and H7N7), heart inoculated chickens, which was in agreement with the
Figure3Weightgainduringtheexperimentalperiod.Thebodyweightofthechickenswasestablishedpre-andpost-infection.Symbols
representthemeanofindividualchickens.PBScontrol,M-LPAIVH5N1,C-LPAIVH7N1andH7N7:n=6.C-LPAIVH5N2andH9N2:n=5.**:P≤0.01.
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Table1DetectionofvirusinorgansofH7N7LPAIV M-LPAIVinfected chickensdid haveahigh RNAload in
inoculatedchickensbyvirusisolationandHA thelungisunclear.
Organ Viruspresent(pos.chickens/totaltested) In summary, RNA of a panel of LPAI viruses isolated
Lung ++(4/6) fromchickenscanbedetectedinmultipleorgansofchick-
ens. Viral RNA might indicate viable virus in organs
Brain -(0/6)
beyond the respiratory tract as was shown for H7N1 and
Ileum ++(3/6)
H7N7 LPAIV. Differences in systemic distribution be-
PBMC N.D.
tween the LPAIVstrains isolated from chickens exist but
Heart +(2/6) larger differences are found between the chicken isolated
Liver +(4/6) strains and the mallard isolated strain. These differences
Kidney +(1/6) are likely to be dependent on host preference and might
eventually be related to replication efficiency of the virus
Spleen +(5/6)
indifferenthosts.
++virusdetected(HAtiterofpositivesamples128–512).
+virusdetected(HAtiterofpositivesamples16–64).
-Novirusdetected;N.D.notdone. Abbreviations
LPAIV:Lowpathogenicavianinfluenzavirus;C-LPAIV:Chickenisolatedlow
pathogenicavianinfluenzavirusstrain;M-LPAIV:Mallardisolatedlow
negativeresult inthePCR atday4.Altogether,viralRNA
pathogenicavianinfluenzavirusstrain;d.p.i:Dayspostinfection;
indifferentorgansmightimplicatethepresenceofpropa- i.n:Intranasalinoculation;i.t:Intratrachealinoculation.
gatedvirus.
Whilethereissufficientliterature onC-LPAIV,reports Competinginterests
Theauthorsdeclarethattheyhavenocompetinginterests.
studying M- LPAIV infections are scarce. Ladman et al.
[16] examined the upper respiratory and the intestinal
Authors’contributions
tract of turkeys and chickens after inoculation of differ-
EDdGcarriedoutanimalexperimentIIandparticipatedintheinterpretation
ent LPAI viruses, including the mallard isolates H5N1 ofthedata.LVwasresponsibleforthedesignofanimalexperimentII.JBWJC
andH7N3.H5N1 LPAIVcould only beisolatedfrom the assistedinanimalexperimentI.JMJRwasresponsibleforthestudydesign
andinterpretationofthedata.JPwasresponsibleforanimalexperimentI,
upper respiratory tract of turkeys, while H7N3 LPAIV
carriedouttheqPCRs,participatedintheinterpretationofthedataand
was isolated from the upper respiratory and the intes- draftedthemanuscript.Allauthorsreadandapprovedthefinalmanuscript.
tinal tract of both turkeys and chickens. The fact that
H5N1LPAIVwasdetected inturkeysandnotinchicken Acknowledgements
FinanciallysupportedbyEU6thframeworkFlupath(grant044220)and
might be explained by a difference in susceptibility for
program“ImpulseVeterinaryavianinfluenzaresearchintheNetherlands”
AI between turkeys and chickens [17]. Although the tis- DutchMinistryofAgriculture,NatureandFoodQuality.
sue tropism of the mallard isolates LPAIV H5N1 and
Authordetails
H7N3 might differ in chicken, we speculate that differ-
1CentralVeterinaryInstituteofWageningenUR,P.O.Box65,Lelystad,8219
ences between H5N1 M- LPAIVand the C- LPAIVsub- PH,TheNetherlands.2FacultyVeterinaryMedicine,Dept.InfectiousDiseases
types that were tested in our study were related to andImmunology,UtrechtUniversity,Yalelaan1,Utrecht3584CL,The
Netherlands.
differences in adaptation to the host. Starting from the
primary infected organs, systemic distribution is among Received:26June2012Accepted:15January2013
others facilitated by lymphocytes [18,19]. In chickens Published:17January2013
withviralRNAofH5N1M-LPAIVinthelung(Figure 2)
we also found viral RNA in PBMC at 4 d.p.i., confirming References
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doi:10.1186/1743-422X-10-23
Citethisarticleas:Postetal.:Systemicdistributionofdifferentlow
pathogenicavianinfluenza(LPAI)virusesinchicken.VirologyJournal
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