animals
B ie Repo
A Po en ial A ypical Case o Rabbi Haemo hagic Disease in a
Dwa Rabbi
Fábio A. Abade dos San os 1,2,3,* , Ca olina Mag o 4, Ca ina L. Ca alho 2, Ped o Rui o 5, Ma ga ida D. Dua e 1,2
and Ma ia C. Pele ei o 1


Ci a ion: Abade dos San os, F.A.;
Mag o, C.; Ca alho, C.L.; Rui o, P.;
Dua e, M.D.; Pele ei o, M.C. A
Po en ial A ypical Case o Rabbi
Haemo hagic Disease in a Dwa
Rabbi . Animals 2021,11, 40.
h ps://dx.doi.o g/10.3390/
ani11010040
Recei ed: 26 Oc obe 2020
Accep ed: 21 Decembe 2020
Published: 28 Decembe 2020
Publishe ’s No e: MDPI s ays neu-
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censee MDPI, Basel, Swi ze land. This
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license (h ps://c ea i ecommons.o g/
licenses/by/4.0/).
1Cen e o In e disciplina y Resea ch in Animal Heal h (CIISA), Faculdade de Medicina Ve e iná ia,
Uni e sidade de Lisboa, A enida da Uni e sidade Técnica, 1300-477 Lisboa, Po ugal;
ma ga ida.dua e@inia .p (M.D.D.); mcpele @ m .ulisboa.p (M.C.P.)
2Ins i u o Nacional de In es igação Ag á ia e Ve e iná ia (INIAV, I.P.), A . da República, Quin a do Ma quês,
2780-157 Oei as, Po ugal; ca ina.ca alho@inia .p
3Ins i u o Uni e si a io de Bio ecnología de As u ias (IUBA), Depa amen o de Bioquímica y Biología
Molecula , Uni e sidad de O iedo, 33006 O iedo, Spain
4Ve Oei as, Hospi al Médico-Ve e iná io, Es ada de Oei as n18-20, 2780-114 Oei as, Po ugal;
ca olinamag [email p o ec ed]
5
Ins i u o de Medicina Molecula João Lobo An unes (IMM), Faculdade de Medicina, Uni e sidade de Lisboa,
1070-312 Lisbon, Po ugal; ui o_ped [email p o ec ed]
*Co espondence: abio.abade@inia .p
Simple Summa y:
We epo an unusual clinical case in a pe abbi accina ed agains abbi
haemo hagic disease i us (RHDV, GI.1), ha de eloped a p olonged hepa ic disease, and was
diagnosed RHDV2 (GI.2) posi i e pos -mo em. This inding is a wa ning o all e e ina ians ha
abbi haemo hagic disease should also be conside ed o di e en ial diagnosis despi e he his o y
o RHDV accina ion and he need o upda e accina ion p og ams agains he cu en RHDV2
ci cula ing s ains.
Abs ac :
Rabbi haemo hagic disease (RHD) is a highly con agious in ec ious disease o Eu opean
wild and domes ic abbi s. Rabbi haemo hagic disease i us (RHDV, GI.1) eme ged in 1986 in
Eu ope, apidly sp eading all o e he wo ld. Se e al geno ypes o RHDV ha e been ecognised
o e ime, bu in 2010, a new i us (RHDV2/RHDVb, GI.2) eme ged and p og essi ely eplaced he
p e ious RHDV s ains, due o he lack o c oss-immuni y con e ed be ween RHDV and RHDV2.
RHDV2 has a high mu a ion a e, simila ly o he o he cali i us and ecombines wi h s ains o
RHDV and non-pa hogenic calici i us (GI.4), ensu ing he con inuous eme gence o new ield s ains.
Al hough his poses a h ea o he al eady endange ed Eu opean abbi species, he a ailable accines
agains RHDV2 and he compliance o biosa e y measu es seem o be con olling he in ec ion in he
abbi indus y Pe abbi s, especially when kep indoo , a e conside ed a lowe isk o in ec ions,
al hough RHDV2 and myxoma i us (MYXV) cons i u e a pe manen h ea due o ansmission ia
insec s. Vaccina ion agains hese i uses is he e o e ecommended e e y 6 mon hs (myxoma osis)
o annually ( abbi haemo hagic disease). The combined immuniza ion o myxoma osis and RHDV
h ough a comme cially a ailable bi alen accine wi h RHDV an igen has been ex ensi ely used
(Nobi ac
®
Myxo-RHD, MSD, Kenilwo h, NJ, USA). This accine howe e does no con e p ope
p o ec ion agains he RHDV2, hus he need o a abbi clinical accina ion p o ocol upda e. He e we
epo a clinical case o hepa i is and al e a ion o coagula ion in a pe abbi ha had been accina ed
wi h he comme cially a ailable bi alen accine agains RHDV and es ed posi i e o RHDV2 a e
dea h. The animal de eloped a p olonged and a ypical disease, compa ible wi h RHD. The i us was
iden i ied o be an RHDV2 ecombinan s ain, wi h he s uc u al backbone o RHDV2 (GI.2) and he
non-s uc u al genes o non-pa hogenic-A1 s ains (RCV-A1, GI.4). Al hough con i ma ion o he
e iological agen was only made a e dea h, he clinical signs and analy ic da a we e e y sugges i e
o RHD.
Keywo ds:
Eu opean abbi ; O yc olagus cuniculus; pe abbi ; abbi haemo hagic disease;
a ypical clinical cou se; subacu e
Animals 2021,11, 40. h ps://dx.doi.o g/10.3390/ani11010040 h ps://www.mdpi.com/jou nal/animals
Animals 2021,11, 40 2 o 12
1. In oduc ion
O igina ing in he Ibe ian Peninsula [
1
], he Eu opean abbi was widely in oduced
i s in Eu ope, and subsequen ly in all o he con inen s excep An a c ica. The Eu opean
domes ic abbi has been used as an impo an mea and u esou ce. A i icial selec ion o
small b eeds o his species (O yc olagus cuniculus), weighing less han 2 kg, also gene a ed
an inc easingly popula pe - abbi ade. Pe abbi s can be a ec ed by he same diseases
ound in he abbi indus y o wild abbi popula ions. Howe e , he indoo li es yle
dec eases he isk o con ac wi h common abbi diseases such as myxoma osis and abbi
haemo hagic disease (RHD).
RHD is a highly con agious in ec ious disease caused by a i us om he Calici i idae
amily, genus Lago i us, which a ge s hepa ocy es and cells o he mononuclea phagocy ic
sys em (e.g., Kupp e cells and al eola mac ophages) [
2
,
3
]. The disease was i s iden i ied
in 1984 in China and quickly sp ead o o he con inen s, e ol ing in o di e en geno ypes
(GI.1a (RHDVa o G6), GI.1b (G1), GI.c (G2) and GI.1d (G3–G5)) [
4
,
5
]. Along wi h myxo-
ma osis, his disease led o an ab up dec ease in he Eu opean abbi wild popula ions [
6
].
In 2010, a dis inc i us (bo h gene ic and an igenically) ela ed o RHDV eme ged in
F ance [
7
]. This new i us, e e ed o as RHDV2 o RHDVb, induced a disease e y simila
o ha caused by RHDV s ains and i became also known as Lago i us eu opaeus GI.2 [5].
Mo e ecen ly, na u al ecombinan RHDV2 s ains we e iden i ied con aining he
s uc u al p o eins o RHDV2 (VP60 and mino p o ein-encoding genes), bu he non-
s uc u al p o eins om ei he non-pa hogenic calici i us (NP-CV GI.4 and GI.3)). Fu -
he mo e, he non-s uc u al p o eins om he RHDV s ains (GI.1b) we e also ound in
RHDV2 ecombinan s [8]. Figu e 1 ep esen s he genomic o ganiza ion o RHDV2 RNA.
Animals 2020, 10, x 2 o 12
Keywo ds: Eu opean abbi ; O yc olagus cuniculus; pe abbi ; abbi haemo hagic disease; a ypical
clinical cou se; subacu e
1. In oduc ion
O igina ing in he Ibe ian Peninsula [1], he Eu opean abbi was widely in oduced
i s in Eu ope, and subsequen ly in all o he con inen s excep An a c ica. The Eu opean
domes ic abbi has been used as an impo an mea and u esou ce. A i icial selec ion
o small b eeds o his species (O yc olagus cuniculus), weighing less han 2 kg, also gene -
a ed an inc easingly popula pe - abbi ade. Pe abbi s can be a ec ed by he same dis-
eases ound in he abbi indus y o wild abbi popula ions. Howe e , he indoo li e-
s yle dec eases he isk o con ac wi h common abbi diseases such as myxoma osis and
abbi haemo hagic disease (RHD).
RHD is a highly con agious in ec ious disease caused by a i us om he Calici i idae
amily, genus Lago i us, which a ge s hepa ocy es and cells o he mononuclea phagocy ic
sys em (e.g., Kupp e cells and al eola mac ophages) [2,3]. The disease was i s iden i ied
in 1984 in China and quickly sp ead o o he con inen s, e ol ing in o di e en geno ypes
(GI.1a (RHDVa o G6), GI.1b (G1), GI.c (G2) and GI.1d (G3–G5)) [4,5]. Along wi h myxoma-
osis, his disease led o an ab up dec ease in he Eu opean abbi wild popula ions [6].
In 2010, a dis inc i us (bo h gene ic and an igenically) ela ed o RHDV eme ged in
F ance [7]. This new i us, e e ed o as RHDV2 o RHDVb, induced a disease e y simila
o ha caused by RHDV s ains and i became also known as Lago i us eu opaeus GI.2 [5].
Mo e ecen ly, na u al ecombinan RHDV2 s ains we e iden i ied con aining he
s uc u al p o eins o RHDV2 (VP60 and mino p o ein-encoding genes), bu he non-
s uc u al p o eins om ei he non-pa hogenic calici i us (NP-CV GI.4 and GI.3)). Fu -
he mo e, he non-s uc u al p o eins om he RHDV s ains (GI.1b) we e also ound in
RHDV2 ecombinan s [8]. Figu e 1 ep esen s he genomic o ganiza ion o RHDV2 RNA.
Figu e 1. Simpli ied schema ic ep esen a ion o he genomic RNA o RHDV2. Nucleo ide posi-
ions we e calcula ed om sequence KF442964.2. The genome includes wo ORFs. ORF1 encodes a
polyp o ein ha is clea ed o o m he non-s uc u al p o eins p16, p23, he helicase, p29, VPg, a
p o ease and he i al polyme ase, and he majo s uc u al p o ein VP60. ORF 2 encodes a mino
s uc u al p o ein VP10.
Di e en clinical p esen a ions and disease p og ession can occu wi h RHDV2 [9–
12] and hose a ia ions can be associa ed wi h s ain. The incuba ion pe iod o RHD
anges be ween 1 and 3 days. In pe acu e o ms, sudden dea h may occu wi hin 12 hou s
a e in ec ion. In acu e and subacu e o ms o RHD, di e en clinical signs can be ob-
se ed, such as ano exia, mucosal conges ion, neu ologic signs, cyanosis, dyspnoea,
oamy haemo hagic epis axis, ocula haemo hage and o he s [13]. In ch onically in-
ec ed abbi s, he onse o ano exia, le ha gy and jaundice usually p ecedes dea h in abou
1 o 3 weeks [13]. Ch onic o subclinical cou ses a e mo e equen in abbi s in ec ed by
RHDV2 han by RHDV.
Haemo hagic dia hesis is ound du ing he disease, wi h dissemina ed in a ascula
coagula ion (DIC), associa ed wi h al e a ions in blood coagula ion and e mina ing in
mul i-o gan ailu e and dea h [14]. The pa hogenesis o DIC in RHD is s ill unknown. The
DIC is assumed o occu by a dis o ion o bo h ex e nal and in e nal blood coagula ion
ac i a ion pa hs, leading o ex ended One-S age P o h ombin Time (OSPT) and Ac i a ed
Pa ial Th omboplas in Time (APTT) [15–17]. Du ing he disease, a h ombocy openia and
Figu e 1.
Simpli ied schema ic ep esen a ion o he genomic RNA o RHDV2. Nucleo ide posi ions
we e calcula ed om sequence KF442964.2. The genome includes wo ORFs. ORF1 encodes a
polyp o ein ha is clea ed o o m he non-s uc u al p o eins p16, p23, he helicase, p29, VPg,
a p o ease and he i al polyme ase, and he majo s uc u al p o ein VP60. ORF 2 encodes a mino
s uc u al p o ein VP10.
Di e en clinical p esen a ions and disease p og ession can occu wi h RHDV2
[9–12]
and hose a ia ions can be associa ed wi h s ain. The incuba ion pe iod o RHD anges
be ween 1 and 3 days. In pe acu e o ms, sudden dea h may occu wi hin 12 hou s
a e in ec ion. In acu e and subacu e o ms o RHD, di e en clinical signs can be ob-
se ed, such as ano exia, mucosal conges ion, neu ologic signs, cyanosis, dyspnoea, oamy
haemo hagic epis axis, ocula haemo hage and o he s [
13
]. In ch onically in ec ed ab-
bi s, he onse o ano exia, le ha gy and jaundice usually p ecedes dea h in abou 1 o 3
weeks [
13
]. Ch onic o subclinical cou ses a e mo e equen in abbi s in ec ed by RHDV2
han by RHDV.
Haemo hagic dia hesis is ound du ing he disease, wi h dissemina ed in a ascula
coagula ion (DIC), associa ed wi h al e a ions in blood coagula ion and e mina ing in
mul i-o gan ailu e and dea h [
14
]. The pa hogenesis o DIC in RHD is s ill unknown.
The DIC is assumed o occu by a dis o ion o bo h ex e nal and in e nal blood coagula ion
ac i a ion pa hs, leading o ex ended One-S age P o h ombin Time (OSPT) and Ac i a ed
Pa ial Th omboplas in Time (APTT) [
15
–
17
]. Du ing he disease, a h ombocy openia and
educed pla ele agg ega ion is obse ed, al e a ion o he ac i i y o coagula ion ac o s
Animals 2021,11, 40 3 o 12
V, VII and X, as well as inc eased olume o soluble ib in and i s deg ada ion p oduc s
(D-dime s) [15–17].
Du ing an RHDV2 ou b eak, mo e han 10% o he in ec ed abbi s may show a
ch onic o subclinical e olu ion o he disease, wi h se e e and gene alised jaundice, loss o
weigh , and le ha gy. These animals o en die some weeks la e , due o li e disease [
18
].
Al hough he s udy o he disease has been expanded by animal expe imen a ion, he cha -
ac e isa ion o na u al in ec ion is a less known [10,11,19,20].
The e ms pe acu e, acu e and ch onic a e o clinical use bu no always e lec ed
in dis inc nec opsy indings and speci ic his opa hological pa e ns, meaning ha i is
possible o ind sudden dea h associa ed wi h pe acu e widesp ead hepa ocellula nec osis
o wi h mo e ch onic changes such as in lamma ion o ib oplasia [10].
Ve e ina ians should be awa e ha despi e nec opsy and his opa hology indings may
be sugges i e o RHD, since mic oscopic changes a e usually p esen and, in many cases,
mac oscopic changes can also be seen [
10
], labo a o y con i ma ion is always equi ed o a
conclusi e diagnosis.
Labo a o y es ing can be done by molecula me hods (e.g., RT-PCR), ELISA o elec on
mic oscopy. Howe e , he apid and debili a ing e olu ion o he disease, oge he wi h
he sho age o di ec access o hese echniques by clinicians, poo epo ing o RHD in
pe abbi s and possible lack o awa eness among clinicians, con ibu es o he almos
non-exis en knowledge o he p e alence o RHD in pe s in Po ugal.
A e he eme gence o RHDV2 in 2010, his i us apidly eplaced he p e ious s ains
ha soon we e no longe epo ed in Eu ope. The humo al immuni y acqui ed by na u al-
o accine-induced RHDV s ains do no con e p ope c oss-p o ec ion agains RHDV2
s ains [
21
,
22
]. Un il 2016, when ERAVAC, Hip a, Gi ona, Spain was comme cialised,
all accines a ailable agains RHDV2 we e di ec ed o he indus ial ma ke (sold in mul i-
dose bo les), and he e o e no adequa e o accina ion o pe abbi s. In No embe 2019,
a new single-dose bo le accine agains myxoma i us, RHDV and RHDV2 was also made
a ailable in Eu ope (Nobi ac®Myxo-RHD Plus, MSD, Kenilwo h, NJ, USA).
2. Clinical P esen a ion
We p esen he case o a 2-yea -old spayed emale Ne he land dwa abbi . This ab-
bi was dewo med yea ly wi h enbendazole, (Panacu
®
, Me ck Animal Heal h, Gi-
alda Fa ms, Madison, WI, USA, 20 mg/kg) and wice a yea wi h a 15 mg selamec in
spo -on (S onghold
®
, Zoe is, Belgium accina ed yea ly (Nobi ac
®
Myxo-RHD, MSD,
Kenilwo h, NJ, USA), and was kep indoo s wi h an owne esiding in he Oei as Dis ic ,
Po ugal. A he age o 18 mon hs, he pa ien was p esen ed o annual accina ion.
Physical examina ion a ha poin was un ema kable and he abbi was clinically no mal.
In Feb ua y 2020, 2 mon hs a e he p e ious isi , he abbi p esen ed o ano exia,
cop os asis and le ha gy. The physical examina ion only e ealed a pain ul condi ion as
he abbi exhibi ed b uxism ( ee h g inding) a e c anial abdominal palpa ion. The es
o he physical examina ion was un ema kable and he abbi weighed 1580 g a he
ime. A comple e blood coun (CBC, de ailed in Table 1) e ealed leukopenia (2.53 K/
µ
L)
wi h he e openia (0.71 K/
µ
L) and h ombocy openia (40 K/
µ
L). Alanine amino ans e ase
(ALT) le els we e ele a ed (240 U/L), indica ing li e damage and adiog aphs showed a
mo e adio-opaque c anial abdomen. The pa ien was admi ed and s a ed on s anda d
in a enous luid he apy wi h NaCl 0.9%, was adminis e ed bup eno phine (0.03 mg/kg
IV TID) o analgesia and p o ided nu i ional suppo (Oxbow’s he bi o e c i ical ca e),
eco e ing appe i e abou 6 hou s la e .
Animals 2021,11, 40 4 o 12
Table 1.
Summa y o he abbi haema ological and biochemical pa ame e s and espec i e e e ence alues e ie ed
om Ca pen e ’s 4 h ed o he Exo ic animal o mula y [
23
] and Idexx’s e e ence alues o he P oCy e Dx* Haema ology
Analyze (as o IDEXX Ve Lab* S a ion so wa e e sion 4.48) and Ca alys one Biochemis y analyse , he equipmen used
in his s udy.
Haema ology Feb ua y Ma ch June
Re e ence Values
Ca pen e , 2018 Idexx Lab., 2017
Haema oc i (%) 30.9 36.3 37.6 30–50 29.4–40.9
Haemoglobin (g/dL) 10.7 11.8 13.3 8–17.5 9.8–13.2
E y h ocy es (×106/µL) 5.21 5.67 6.45 4–8 4.45–6.71
MCV ( L) 59.3 64.0 58.3 58–75 58.1–69.6
MCH (pg) 20.5 20.8 20.6 17.5–23.5 18.9–22.1
MCHC (g/dL) 34.6 32.5 35.4 29–37 31.6–33.6
Re iculocy es (%) 2.4 5.4 1.4 2–4 -
Re iculocy es (K/µL) 122.4 306.7↑88.4 - 69.5–242.7
Pla ele s (103/µL) 40↓370 97↓290–650 219–521
WBC (103/µL) 2.53↓5.15 1.86↓5–12 4.54–10.22
He e ophils (%) 28.1↓28.8↓47.9 35–55 -
Lymphocy es (%) 64.4↑60.0 41.9 25–60 -
Monocy es (%) 4.7 5.4 4.8 2–10 -
Eosinophils (%) 0.8 0.8 0.0 0–5 -
Basophils (%) 2.0 5.0 5.4 2–8 -
He e ophils (K/µL) 0.71↓1.48 0.89↓- 0.96–3.34
Lymphocy es (K/µL) 1.63 3.09 0.78↓- 1.49–5.21
Monocy es (K/µL) 0.12↓0.28↓0.09↓- 0.31–0.99
Eosinophils (K/µL) 0.02↓0.04↓0.00↓- 0.05–2.12
Basophils (K/µL) 0.05↓0.26↓0.10↓- 0.56–2.12
Biochemis y
Glucose 194↑-164↑75–150 75–145
C ea inine (CREA) 1.0 - 1.2 0.5–2.6 0.8–1.8
BUN 12 - 22 15–50 10–24
BUN/CREA 12 - 18 - -
PHOS - - 3.7 2.3–6.9 1.2–4.9
CA - - 11.7 8–14.8 5.6–12.0
TP 6.6 - 7.7↑5.4–7.5 5.5–7.2
ALB 4.4 - 4.4 2.5–5 2.7–4.6
GLOB 2.2 - 3.3↑1.5–3.5 1.5–2.8
ALB/GLOB 2.0 - 1.3 - -
ALT 240↑-586↑14–80 31–53
ALKP 99 - 104 4–70 70–145
GGT - - 2 - -
TBIL - - 0.9 0–0.75 0.3–0.8
CHOL - - 30 12–16 35–53
The alues highligh ed in bold ep esen he main de ia ions obse ed and he a ows indica e i he alues a e abo e (
↑
) o below (
↓
)
he e e ences.
Ul asound e ealed a sligh ly he e ogeneous li e wi h a hypoechoic cauda e lobe
and a kidney s one conside ed meaningless o his case. The e we e no ob ious signs o
ascula comp omise. Howe e , sha ing he abdomen o he ul asound exam e ealed a
la ge haema oma ha ex ended om he neck o o elimbs, ho ax and uppe -hal o he
abdomen. This was assumed o ha e o igina ed om he jugula blood collec ion ca ied
ou in he p e ious day, despi e he seemingly a auma ic punc u e.
Th oughou he 5 h day o hospi alisa ion, he pa ien main ained a pa e n o cu a-
neous haemo hagic dysc asia, wi h ascula agili y, easy b uising and equi ing he need
o p olonged comp ession o punc u e si es. Coagula ion es s we e ca ied ou and bo h
p o h ombin ime (PT) and ac i a ed pa ial h omboplas in ime (aPTT) we e p olonged,
a 25 and 195 s, espec i ely.
Animals 2021,11, 40 5 o 12
A CT scan showed no signs o hepa ic abno mali ies o ascula comp omise. The pa-
ien was s a ed on injec able i amin K1 (10 mg/kg SC SID) and kep on bup eno phine
(0.03 mg/kg SC TID) and IV NaCl 0.9% a a a e o 5mL/h. B uising was ully eabso bed
by he 8 h day and he pa ien was sen home. Be o e elease, he physical exam was
un ema kable. The abbi was b igh and ale and had ully eco e ed his appe i e, weigh -
ing a he ime 1500 g. No mo e blood collec ions we e possible due o lack o enous
accesses, seconda y o mul iple hema omas and b uising in he p e ious blood collec ion
si es. Two weeks la e , in Ma ch 2020, ollow up blood es s we e pe o med, including a
CBC. The e alua ion hemog am was un ema kable, as e iculocy osis was conside ed a
no mal inding gi en he e en s in he p e ious hospi alisa ion. No o he clinical ollow
up could be pe o med due o inancial limi a ions and lockdown policies associa ed wi h
he COVID-19 wo ld pandemic.
In June 2020, he abbi was eadmi ed due o ano exia, le ha gy, cop os asis and
abdominal discom o . The abbi had los 80 g, weigh ing hen 1420 g, and o he i s ime,
mildly hype he mia (39.4
◦
C) was egis e ed. The blood panel e ealed se e e leukope-
nia (1.86 K/
µ
L) associa ed wi h he e openia (0.89 K/
µ
L) and lymphopenia (0.78 K/
µ
L).
Th ombocy openia (97 K/
µ
L) was also obse ed. ALT was hen 586 U/L and he e was
also he ele a ion o o al bili ubin (0.9 mg/dL) and o al p o eins (7.7 g/dL) due o hy-
pe globulinemia (3.3 g/dL) (Table 1). The blood d aw esul ed once again in cu aneous
haemo hagic dysc asia and he e o e he abbi was s a ed on i amin K1 (10 mg/kg
SC BID), en o loxacin (5 mg/kg SC SID), bup eno phine (0.03 mg/kg SC TID), me oclo-
p amide (0.5 mg/kg SC BID), lac ulose (0.5 mL/kg PO BID) and agg essi e luid he apy.
Ul asound was compa ible wi h se e e hepa i is and pe i-lobula pe i oni is (Figu e 2),
wi h mesen e ic eac i i y and ee luid in he abdomen. The abbi was in he hospi al
o 3 days showing no signs o imp o emen . The empe a u e kep inc easing eaching
a peak o 40.2
◦
C on he second day o hospi alisa ion, a e which i s a ed d opping
and he abbi became p og essi ely hypo he mic. On he 3 d day o hospi aliza ion,
he empe a u e d opped o 36.9
◦
C, despi e he ac i e hea ing e o s. The abbi became
le ha gic, jaundiced and de eloped e ical nys agmus. The dea h occu ed abou 6 h a e
he onse o hese symp oms.
Animals 2020, 10, x 4 o 12
abdomen. This was assumed o ha e o igina ed om he jugula blood collec ion ca ied
ou in he p e ious day, despi e he seemingly a auma ic punc u e.
Th oughou he 5 h day o hospi alisa ion, he pa ien main ained a pa e n o cu a-
neous haemo hagic dysc asia, wi h ascula agili y, easy b uising and equi ing he
need o p olonged comp ession o punc u e si es. Coagula ion es s we e ca ied ou and
bo h p o h ombin ime (PT) and ac i a ed pa ial h omboplas in ime (aPTT) we e p o-
longed, a 25 and 195 s, espec i ely.
A CT scan showed no signs o hepa ic abno mali ies o ascula comp omise. The
pa ien was s a ed on injec able i amin K1 (10 mg/kg SC SID) and kep on bup eno -
phine (0.03 mg/kg SC TID) and IV NaCl 0.9% a a a e o 5mL/h. B uising was ully eab-
so bed by he 8 h day and he pa ien was sen home. Be o e elease, he physical exam
was un ema kable. The abbi was b igh and ale and had ully eco e ed his appe i e,
weigh ing a he ime 1500 g. No mo e blood collec ions we e possible due o lack o e-
nous accesses, seconda y o mul iple hema omas and b uising in he p e ious blood col-
lec ion si es. Two weeks la e , in Ma ch 2020, ollow up blood es s we e pe o med, in-
cluding a CBC. The e alua ion hemog am was un ema kable, as e iculocy osis was con-
side ed a no mal inding gi en he e en s in he p e ious hospi alisa ion. No o he clinical
ollow up could be pe o med due o inancial limi a ions and lockdown policies associ-
a ed wi h he COVID-19 wo ld pandemic.
In June 2020, he abbi was eadmi ed due o ano exia, le ha gy, cop os asis and
abdominal discom o . The abbi had los 80 g, weigh ing hen 1420 g, and o he i s
ime, mildly hype he mia (39.4 °C) was egis e ed. The blood panel e ealed se e e leu-
kopenia (1.86 K/µL) associa ed wi h he e openia (0.89 K/µL) and lymphopenia (0.78
K/µL). Th ombocy openia (97 K/µL) was also obse ed. ALT was hen 586 U/L and he e
was also he ele a ion o o al bili ubin (0.9 mg/dL) and o al p o eins (7.7 g/dL) due o
hype globulinemia (3.3 g/dL) (Table 1). The blood d aw esul ed once again in cu aneous
haemo hagic dysc asia and he e o e he abbi was s a ed on i amin K1 (10 mg/kg SC
BID), en o loxacin (5 mg/kg SC SID), bup eno phine (0.03 mg/kg SC TID), me oclo-
p amide (0.5 mg/kg SC BID), lac ulose (0.5 mL/kg PO BID) and agg essi e luid he apy.
Ul asound was compa ible wi h se e e hepa i is and pe i-lobula pe i oni is (Figu e 2),
wi h mesen e ic eac i i y and ee luid in he abdomen. The abbi was in he hospi al
o 3 days showing no signs o imp o emen . The empe a u e kep inc easing eaching a
peak o 40.2 °C on he second day o hospi alisa ion, a e which i s a ed d opping and
he abbi became p og essi ely hypo he mic. On he 3 d day o hospi aliza ion, he em-
pe a u e d opped o 36.9 °C, despi e he ac i e hea ing e o s. The abbi became le ha -
gic, jaundiced and de eloped e ical nys agmus. The dea h occu ed abou 6 h a e he
onse o hese symp oms.
Figu e 2. Ul asound is compa ible wi h hepa i is. The image on he le shows pe i-lobula mesen-
e ic eac i i y (a ow). On he igh , ee luid in he abdomen is isible (a ow).
Th oughou he clinical e olu ion o his case, se e al di e en ial diagnoses we e
conside ed. These included hepa ic lipidosis, hepa ic coccidiosis, li e lobe o sion, bac e-
ial, ungal, and pa asi ic hepa i is, Encephali ozoon cuniculi in ec ion, Tyzze ’s disease
Figu e 2.
Ul asound is compa ible wi h hepa i is. The image on he le shows pe i-lobula mesen-
e ic eac i i y (a ow). On he igh , ee luid in he abdomen is isible (a ow).
Th oughou he clinical e olu ion o his case, se e al di e en ial diagnoses we e
conside ed. These included hepa ic lipidosis, hepa ic coccidiosis, li e lobe o sion, bac e-
ial, ungal, and pa asi ic hepa i is, Encephali ozoon cuniculi in ec ion, Tyzze ’s disease
and neoplasia. All hese possibili ies we e excluded by he abbi ’s clinical his o y, semes-
al cop ology esul s and dewo ming his o y, along wi h he blood wo ks, adiog aphs,
ul asounds, CT scan and pos mo em da a.
The nec opsy indings ein o ced ha he in lamma o y and/o in ec ious p ocess
was he mos likely di e en ial diagnosis o his pa ien .

Animals 2021,11, 40 6 o 12
3. Ma e ial and Me hods
3.1. Nec opsy and His opa hology
The nec opsy was pe o med acco ding o ou ine p ocedu es, and samples we e
collec ed o bac e iology (li e , spleen and lung), his opa hology (li e , spleen, s omach,
small in es ine, panc eas and kidney) and i ology (li e ). All analyses ollowed ou ine
p ocedu es.
His opa hology agmen s we e ixed in 10% neu al bu e ed o malin, ou inely pa a in-
embedded, sec ioned a 4 µm, and s ained wi h Haema oxylin and Eosin (H&E).
3.2. Molecula Analysis
Fo nucleic acid ex ac ion, a esh sample o li e was homogenized a 10% (w/ )
wi h phospha e-bu e ed saline (PBS) and cla i ied a 3000 g o 5 min. To al nucleic acids
we e ex ac ed om 200
µ
L o he cla i ied supe na an s, using he MagA ac 96 cado
Pa hogen Ki (Qiagen, Hilden, Ge many) in a BioSp in 96 nucleic acid ex ac o (Qiagen,
Hilden, Ge many), acco ding o he manu ac u e ’s p o ocol.
The abbi was es ed o abbi haemo hagic disease i us (RHDV) by con en ional
PCR [
24
] and abbi haemo hagic disease i us 2 (RHDV2) and myxoma i us (MYXV)
by eal- ime PCR [
25
,
26
]. Ampli ica ions we e ca ied ou in a Bio-Rad CFX96
™
The mal
Cycle (Bio-Rad Labo a o ies S l, Redmond, WA, USA), using he One-S ep RT-PCR ki
(Qiagen, Hilden, Ge many) o RHDV2, and he NZYTaq II 2x Colou less Mas e Mix
(Nzy ech, Lisbon, Po ugal) o MYXV.
cDNA was syn hesised wi h he Supe Sc ip
™
IV Fi s -S and Syn hesis Sys em (InVi -
ogen, Ca lsbad, CA, USA) acco ding o he manu ac u e ’s ecommenda ions, using ei he
oligo(dT)12-18 and andom hexame s. Ampli ica ion o ull VP60 gene and pa ial RdRp
gene was achie ed using p ime s, ki s and p o ocols a ailable in appendix Table S1.
The PCR p oduc s we e isualised in 2% ho izon al elec opho esis aga ose gel, pu i-
ied using he NZYGelpu e ki (NZYTECH), and di ec ly sequenced using he ABI P ism
BigDye Te mina o 3.1 Cycle sequencing ki on a 3130 Gene ic Analyse (Applied Biosys-
ems, Fos e Ci y, CA, USA). Nucleo ide sequences we e analysed and assembled in o
consensus sequences using he Seqscape So wa e 2.7 (Applied Biosys ems, Fos e Ci y,
CA, USA)., The inal 2176 nucleo ide sequence including he VP60 comple e gene (1740 n -
long) and a 436 n -long egion o he RdRp gene, was deposi ed in GenBank da abase and
gi en he accession numbe MT829254.
3.3. Phylogene ic Analysis
To u he in es iga e he ela ion o his s ain o o he ecombinan s cha ac e ised
p e iously in Po ugal [
8
], a phylogene ic analysis was ca ied ou by Maximum Likelihood
(ML) esou cing o he R so wa e (R De elopmen Co e Team, 2009) [
27
]. Th ee ypes o
mul iple sequence alignmen s (msa) we e used o phylogene ic in e ence, encompassing a
436 n egion wi hin he RdRp gene, he comple e 1740 n p60 gene sequences, and he
2176 n long sequence comp ising he wo egions men ioned.
Mul iple sequence alignmen s (msa) we e gene a ed by MUSCLE h ough he R so -
wa e (R De elopmen Co e Team, 2009). Fo each alignmen , he app op ia e subs i u ion
model o ML analysis was de e mined using he unc ion model es . Fo RdRp ee,
he Hasengawa–Kishino–Yano (HKY) model [
28
] an allowance o he inco po a ion o
in a ian si es (I) (HKY+I), showed he lowe AIC alue and was used o in e phylogene ic
ela ionships. Fo he ML phylogene ic ee based on he ull p60 gene (1740 n long)
he Gene al Time Re e sible (GTR) model [
29
] wi h a disc e e gamma dis ibu ion (+G),
(GTR+G) showed he lowe AIC alue was used o in e phylogene ic ela ionships. Fo he
ML phylogene ic ee based on a 2176 n long sequence comp ising he e minal 436 n
long egion wi hin he RdRp gene and he comple e p60 gene he Gene al Time Re e sible
(GTR) model [
29
] wi h a disc e e gamma dis ibu ion (+G) and/o an allowance o he
inco po a ion o in a ian si es (+I) (GTR+G+I) showed he lowe AIC alue and was used
o in e phylogene ic ela ionships.
Animals 2021,11, 40 7 o 12
4. Resul s
4.1. Nec opsy and His opa hology
The nec opsy e ealed li e conges ion and ma ked lobula pa e n, p esence o a
small amoun o ee pe i oneal luid and conges ed lungs wi h haemo hagic oci. Bo h kid-
neys showed a eas o su ace e ac ion accoun ing o abou 20% o he o al su ace.
The ollowing mic oscopic lesions we e obse ed:
Li e
: se e e gene alized pe ilob-
ula haemo hagic nec osis (Figu es 3and 4). Disc e e in il a ion by mononuclea ed in-
lamma o y cells, mainly mac ophages and lymphocy es, a ound po al iads. Fine b own
pigmen in he cells o he po al bile duc s.
Spleen
: di use nec osis o he en i e ed pulp
e ealed by deposi ion o ib inoid acidophilic ma e ial d awing se piginous pa e ns in
he pa enchyma (Figu e 5). The egula p esence o lymphoid ollicles a ound cen al
a e ioles.
Panc eas
: in e lobula oedema and nec osis o adipocy es, bo h in alobula
and in e lobula . No changes we e p esen in he sec e o y cells.
S omach
: no signi ican
changes we e obse ed.
Small in es ine
: Nec o ic en e i is, pa icula ly in he duodenum,
wi h loss o illi and deposi ion o ib in in he p oximal mucosa (Figu e 6).
Kidneys
:
he a eas o su ace e ac ion in bo h kidneys co esponded o segmen al ib osis a ec ing
co ex and medulla. In hese a eas, he e was a loss o ubules and glome uli, which we e
mode a ely conges ed. No mic obial agen s we e iden i ied in any o gan and he esul s o
he mic obiological analysis we e also nega i e.
Animals 2020, 10, x 7 o 12
o he inco po a ion o in a ian si es (+I) (GTR+G+I) showed he lowe AIC alue and
was used o in e phylogene ic ela ionships.
4. Resul s
4.1. Nec opsy and His opa hology
The nec opsy e ealed li e conges ion and ma ked lobula pa e n, p esence o a
small amoun o ee pe i oneal luid and conges ed lungs wi h haemo hagic oci. Bo h
kidneys showed a eas o su ace e ac ion accoun ing o abou 20% o he o al su ace.
The ollowing mic oscopic lesions we e obse ed: Li e : se e e gene alized pe i-
lobula haemo hagic nec osis (Figu es 3 and 4). Disc e e in il a ion by mononuclea ed
in lamma o y cells, mainly mac ophages and lymphocy es, a ound po al iads. Fine
b own pigmen in he cells o he po al bile duc s. Spleen: di use nec osis o he en i e
ed pulp e ealed by deposi ion o ib inoid acidophilic ma e ial d awing se piginous
pa e ns in he pa enchyma (Figu e 5). The egula p esence o lymphoid ollicles a ound
cen al a e ioles. Panc eas: in e lobula oedema and nec osis o adipocy es, bo h in a-
lobula and in e lobula . No changes we e p esen in he sec e o y cells. S omach: no sig-
ni ican changes we e obse ed. Small in es ine: Nec o ic en e i is, pa icula ly in he du-
odenum, wi h loss o illi and deposi ion o ib in in he p oximal mucosa (Figu e 6). Kid-
neys: he a eas o su ace e ac ion in bo h kidneys co esponded o segmen al ib osis
a ec ing co ex and medulla. In hese a eas, he e was a loss o ubules and glome uli,
which we e mode a ely conges ed. No mic obial agen s we e iden i ied in any o gan and
he esul s o he mic obiological analysis we e also nega i e.
Figu e 3. Li e . Se e e haemo hagic nec osis consis en ly a ec s pe ilobula a eas o acina zone
1, conspicuous due o he b igh ed colou close o he po al a eas (H&E, 40×).
Figu e 4. Li e . Apa om pe ilobula haemo hagic nec osis, single-cell nec osis is p esen in
dispe sed hepa ocy es in acina zones 2 and 3, which su ound he pe iacina ein in he uppe
Figu e 3.
Li e . Se e e haemo hagic nec osis consis en ly a ec s pe ilobula a eas o acina zone 1,
conspicuous due o he b igh ed colou close o he po al a eas (H&E, 40×).
Animals 2020, 10, x 7 o 12
o he inco po a ion o in a ian si es (+I) (GTR+G+I) showed he lowe AIC alue and
was used o in e phylogene ic ela ionships.
4. Resul s
4.1. Nec opsy and His opa hology
The nec opsy e ealed li e conges ion and ma ked lobula pa e n, p esence o a
small amoun o ee pe i oneal luid and conges ed lungs wi h haemo hagic oci. Bo h
kidneys showed a eas o su ace e ac ion accoun ing o abou 20% o he o al su ace.
The ollowing mic oscopic lesions we e obse ed: Li e : se e e gene alized pe i-
lobula haemo hagic nec osis (Figu es 3 and 4). Disc e e in il a ion by mononuclea ed
in lamma o y cells, mainly mac ophages and lymphocy es, a ound po al iads. Fine
b own pigmen in he cells o he po al bile duc s. Spleen: di use nec osis o he en i e
ed pulp e ealed by deposi ion o ib inoid acidophilic ma e ial d awing se piginous
pa e ns in he pa enchyma (Figu e 5). The egula p esence o lymphoid ollicles a ound
cen al a e ioles. Panc eas: in e lobula oedema and nec osis o adipocy es, bo h in a-
lobula and in e lobula . No changes we e p esen in he sec e o y cells. S omach: no sig-
ni ican changes we e obse ed. Small in es ine: Nec o ic en e i is, pa icula ly in he du-
odenum, wi h loss o illi and deposi ion o ib in in he p oximal mucosa (Figu e 6). Kid-
neys: he a eas o su ace e ac ion in bo h kidneys co esponded o segmen al ib osis
a ec ing co ex and medulla. In hese a eas, he e was a loss o ubules and glome uli,
which we e mode a ely conges ed. No mic obial agen s we e iden i ied in any o gan and
he esul s o he mic obiological analysis we e also nega i e.
Figu e 3. Li e . Se e e haemo hagic nec osis consis en ly a ec s pe ilobula a eas o acina zone
1, conspicuous due o he b igh ed colou close o he po al a eas (H&E, 40×).
Figu e 4. Li e . Apa om pe ilobula haemo hagic nec osis, single-cell nec osis is p esen in
dispe sed hepa ocy es in acina zones 2 and 3, which su ound he pe iacina ein in he uppe
Figu e 4.
Li e . Apa om pe ilobula haemo hagic nec osis, single-cell nec osis is p esen in
dispe sed hepa ocy es in acina zones 2 and 3, which su ound he pe iacina ein in he uppe igh .
Inse -magni ica ion o he acina zones 2 and 3, showing a ious cells wi h he agmen a ion o he
nucleus-ka yo hexis (a ows) (H&E, 100×, inse 400×).
Animals 2021,11, 40 8 o 12
Animals 2020, 10, x 8 o 12
igh . Inse -magni ica ion o he acina zones 2 and 3, showing a ious cells wi h he agmen a-
ion o he nucleus-ka yo hexis (a ows) (H&E, 100×, inse 400×).
Figu e 5. Spleen. Se e e di use ib inoid nec osis o he ed pulp. The eosinophilic ma e ial i-
b inoid ma e ial is e y abundan be ween he lymphoid issue ha su ounds blood essels
(H&E, 40×).
Figu e 6. Duodenum. Nec o ic en e i is a ec ing he uppe mucosa. No e he absence o illi and
he eosinophilic deposi s o ib in in he uppe mucosa. The in es inal glands a e diso ganized and
he in lamma o y in il a e o he mucosal lamina p op ia by mononuclea cells (lymphocy es
mos ly) is scan (H&E, 100×).
4.2. Vi ology
In es iga ions o MYXV and RHDV (geno ypes GI.1b ( o me G1) o GI.1a ( o me
RHDVa o G6)) [19] we e nega i e. Howe e , he RT-qPCR speci ic o RHDV2 e ealed
a high i al load in he li e (app oxima ely 9.0 × 1011 copies o RHDV2 RNA pe mg o
li e ), highe han he usual i al load ound in in ec ed abbi li e , including bo h ac-
cina ed and un accina ed abbi s [30]. The ampli ica ion o he RdRp pa ial gene and
VP60 comple e gene was success ul, gene a ing amplicons wi hin he expec ed sizes
(a ailable in access numbe MT829254).
4.3. Phylogene ic Analysis
BLAST analysis o VP60 nucleo ide sequence showed 96.88% simila i y wi h RHDV2
sequences cha ac e ised ea lie in 2013 om mainland Po ugal, namely wi h h ee s ains
(KF44962, KF44963 and KF44964) collec ed om wild abbi s o igina ing om he sou h
egion, Alen ejo and Alga e. BLAST analysis o he pa ial RdRp gene showed 96.56%
simila i y wi h a ecombinan RHDV2/NP1 s ain (MG763952) collec ed om a wild ab-
bi om he no h o mainland Po ugal in 2015.
Figu e 5.
Spleen. Se e e di use ib inoid nec osis o he ed pulp. The eosinophilic ma e ial ib inoid
ma e ial is e y abundan be ween he lymphoid issue ha su ounds blood essels (H&E, 40×).
Animals 2020, 10, x 8 o 12
igh . Inse -magni ica ion o he acina zones 2 and 3, showing a ious cells wi h he agmen a-
ion o he nucleus-ka yo hexis (a ows) (H&E, 100×, inse 400×).
Figu e 5. Spleen. Se e e di use ib inoid nec osis o he ed pulp. The eosinophilic ma e ial i-
b inoid ma e ial is e y abundan be ween he lymphoid issue ha su ounds blood essels
(H&E, 40×).
Figu e 6. Duodenum. Nec o ic en e i is a ec ing he uppe mucosa. No e he absence o illi and
he eosinophilic deposi s o ib in in he uppe mucosa. The in es inal glands a e diso ganized and
he in lamma o y in il a e o he mucosal lamina p op ia by mononuclea cells (lymphocy es
mos ly) is scan (H&E, 100×).
4.2. Vi ology
In es iga ions o MYXV and RHDV (geno ypes GI.1b ( o me G1) o GI.1a ( o me
RHDVa o G6)) [19] we e nega i e. Howe e , he RT-qPCR speci ic o RHDV2 e ealed
a high i al load in he li e (app oxima ely 9.0 × 1011 copies o RHDV2 RNA pe mg o
li e ), highe han he usual i al load ound in in ec ed abbi li e , including bo h ac-
cina ed and un accina ed abbi s [30]. The ampli ica ion o he RdRp pa ial gene and
VP60 comple e gene was success ul, gene a ing amplicons wi hin he expec ed sizes
(a ailable in access numbe MT829254).
4.3. Phylogene ic Analysis
BLAST analysis o VP60 nucleo ide sequence showed 96.88% simila i y wi h RHDV2
sequences cha ac e ised ea lie in 2013 om mainland Po ugal, namely wi h h ee s ains
(KF44962, KF44963 and KF44964) collec ed om wild abbi s o igina ing om he sou h
egion, Alen ejo and Alga e. BLAST analysis o he pa ial RdRp gene showed 96.56%
simila i y wi h a ecombinan RHDV2/NP1 s ain (MG763952) collec ed om a wild ab-
bi om he no h o mainland Po ugal in 2015.
Figu e 6.
Duodenum. Nec o ic en e i is a ec ing he uppe mucosa. No e he absence o illi and he
eosinophilic deposi s o ib in in he uppe mucosa. The in es inal glands a e diso ganized and he
in lamma o y in il a e o he mucosal lamina p op ia by mononuclea cells (lymphocy es mos ly)
is scan (H&E, 100×).
4.2. Vi ology
In es iga ions o MYXV and RHDV (geno ypes GI.1b ( o me G1) o GI.1a ( o -
me RHDVa o G6)) [
19
] we e nega i e. Howe e , he RT-qPCR speci ic o RHDV2
e ealed a high i al load in he li e (app oxima ely 9.0
×
10
11
copies o RHDV2 RNA pe
mg o li e ), highe han he usual i al load ound in in ec ed abbi li e , including bo h
accina ed and un accina ed abbi s [
30
]. The ampli ica ion o he RdRp pa ial gene
and VP60 comple e gene was success ul, gene a ing amplicons wi hin he expec ed sizes
(a ailable in access numbe MT829254).
4.3. Phylogene ic Analysis
BLAST analysis o VP60 nucleo ide sequence showed 96.88% simila i y wi h RHDV2
sequences cha ac e ised ea lie in 2013 om mainland Po ugal, namely wi h h ee s ains
(KF44962, KF44963 and KF44964) collec ed om wild abbi s o igina ing om he sou h
egion, Alen ejo and Alga e. BLAST analysis o he pa ial RdRp gene showed 96.56%
simila i y wi h a ecombinan RHDV2/NP1 s ain (MG763952) collec ed om a wild abbi
om he no h o mainland Po ugal in 2015.
BLAST analysis o he 2176 n long sequence e ealed highe simila i y wi h sequence
KF442964, ob ained om a wild abbi om Sou h o mainland Po ugal sampled in 2013.
Animals 2021,11, 40 9 o 12
The ee based on he 3
0
end sequence o RpRd gene ( agmen 436 bp), con i med ha
he RHDV2 s ain om he dwa abbi sha ed high simila i y wi h he homologous
egion o he RdRp om o he RHDV2 ecombinan s classi ied as GI.4 (NP1)/RHDV2
(
Figu e 7A
). The VP60 gene base ee and phylogene ic analysis including he comple e
agmen showed ha he dwa sequence RHDV2 is in an isola ed b anch and does no
g oup wi hin he de ined clus e s (Figu e 7B) and (Figu e 7C).
Animals 2020, 10, x 9 o 12
BLAST analysis o he 2176 n long sequence e ealed highe simila i y wi h sequence
KF442964, ob ained om a wild abbi om Sou h o mainland Po ugal sampled in 2013.
The ee based on he 3′ end sequence o RpRd gene ( agmen 436 bp), con i med
ha he RHDV2 s ain om he dwa abbi sha ed high simila i y wi h he homologous
egion o he RdRp om o he RHDV2 ecombinan s classi ied as GI.4 (NP1)/RHDV2 (Fig-
u e 7A). The VP60 gene base ee and phylogene ic analysis including he comple e ag-
men showed ha he dwa sequence RHDV2 is in an isola ed b anch and does no g oup
wi hin he de ined clus e s (Figu e 7B) and (Figu e 7C).
Figu e 7. Phylogene ic analysis. (A) Maximum likelihood analysis using 14 RdRp gene pa ial (436 n ) nucleo ide se-
quences, namely GI.4 (NP1–non-pa hogenic calici i uses simila o RCV-A1 a e he mos likely dono s o non-s uc u al
p o eins) ep esen a i es (a-MG763946, MG763944, MG763954 and a’-MG763952), GI.1b (G1) ep esen a i es (c-
MG763939, MG763938, MG763947 and MG763953), GI.3 (NP2-non-pa hogenic calici i uses simila o CBAnd1 a e he
mos likely dono s o non-s uc u al p o eins) ep esen a i es (e-MG763942, MG763949, MG763943 and MG763945; GI.1d
(G3-G5) ep esen a i es (d-MH190418) and non-pa hogenic abbi calici i us Aus alia 1 (RCV-A1) ep esen a i es (b-
EU871528). (B) Maximum likelihood analysis using 14 comple e VP60 gene nucleo ide sequences including, GI.1b ep e-
sen a i es (a-MG763939 and MG763938 and a’-MG763953 and MG763947), GI.4 (NP1) ep esen a i es (b-MG763952 and
MG763954 and b’-MG763946 and MG763944), GI.3 (NP2) ep esen a i es (c-MG763949, MG763942, MG763945 and
MG763943; GI.1d ep esen a i es (d- MH190418) and RCVA-A1 ep esen a i es (e-EU871528). (C) Maximum likelihood
analysis using 14 sequences comp ising he e minus o he RdRp gene and he comple e VP60 gene, namely GI.1b ep e-
sen a i es (a-MG763938 and MG763939, a’-MG763947, and a’’-MG763953), GI.3 (NP2) ep esen a i es (c-MG763949,
MG763942, MG763945 and MG763943), GI.4 (NP1) ep esen a i es (d-MG763954, MG763946, MG763944 and d’-
MG763952) GI.1d ep esen a i es (b-MH190418) and RCVA1 ep esen a i es (e-EU871528). The nomencla u e used is in
acco dance wi h Sil é io e al., 2018 [8], Ab an es e al., 2020 [31]. Designa ions only used by Sil é io e al., 2018 a e un-
de lined in he phylogene ic ees.
Robus ness o he ee nodes was assessed by boo s apping 1000 imes. Only boo -
s ap (BS) alues g ea e o equal o 70 a e shown. The g aphical ep esen a ion and edi-
ion o he phylogene ic ees we e pe o med wi h FigT ee 1.3.1
(h p:// ee.bio.ed.ac.uk/so wa e/ ig ee/).
5. Discussion
Rabbi haemo hagic disease a ec s bo h domes ic and wild abbi s causing a sys-
emic disease usually wi h a le hal ou come. A e i s eme gence, RHDV2 s ains e ol ed
quickly, wi h some a ia ion o amino acids in he capsid p o ein bu main aining he
Figu e 7.
Phylogene ic analysis. (
A
) Maximum likelihood analysis using 14 RdRp gene pa ial (436 n ) nucleo ide se-
quences, namely GI.4 (NP1–non-pa hogenic calici i uses simila o RCV-A1 a e he mos likely dono s o non-s uc u al
p o eins) ep esen a i es (a-MG763946, MG763944, MG763954 and a’-MG763952), GI.1b (G1) ep esen a i es (c-MG763939,
MG763938, MG763947 and MG763953), GI.3 (NP2-non-pa hogenic calici i uses simila o CBAnd1 a e he mos likely
dono s o non-s uc u al p o eins) ep esen a i es (e-MG763942, MG763949, MG763943 and MG763945; GI.1d (G3-G5)
ep esen a i es (d-MH190418) and non-pa hogenic abbi calici i us Aus alia 1 (RCV-A1) ep esen a i es (b-EU871528).
(
B
) Maximum likelihood analysis using 14 comple e VP60 gene nucleo ide sequences including, GI.1b ep esen a i es
(a-MG763939 and MG763938 and a’-MG763953 and MG763947), GI.4 (NP1) ep esen a i es (b-MG763952 and MG763954 and
b’-MG763946 and MG763944), GI.3 (NP2) ep esen a i es (c-MG763949, MG763942, MG763945 and MG763943; GI.1d ep e-
sen a i es (
d-MH190418
) and RCVA-A1 ep esen a i es (e-EU871528). (
C
) Maximum likelihood analysis using 14 sequences
comp ising he e minus o he RdRp gene and he comple e VP60 gene, namely GI.1b ep esen a i es (a-MG763938
and MG763939, a’-MG763947, and a”-MG763953), GI.3 (NP2) ep esen a i es (c-MG763949, MG763942, MG763945 and
MG763943), GI.4 (NP1) ep esen a i es (d-MG763954, MG763946, MG763944 and d’-MG763952) GI.1d ep esen a i es
(b-MH190418) and RCVA1 ep esen a i es (e-EU871528). The nomencla u e used is in acco dance wi h Sil é io e al.,
2018 [8], Ab an es e al., 2020 [31]. Designa ions only used by Sil é io e al., 2018 a e unde lined in he phylogene ic ees.
Robus ness o he ee nodes was assessed by boo s apping 1000 imes. Only boo s ap
(BS) alues g ea e o equal o 70 a e shown. The g aphical ep esen a ion and edi ion
o he phylogene ic ees we e pe o med wi h FigT ee 1.3.1 (h p:// ee.bio.ed.ac.uk/
so wa e/ ig ee/).
5. Discussion
Rabbi haemo hagic disease a ec s bo h domes ic and wild abbi s causing a sys emic
disease usually wi h a le hal ou come. A e i s eme gence, RHDV2 s ains e ol ed quickly,
wi h some a ia ion o amino acids in he capsid p o ein bu main aining he o iginal