Increased pathogenicity in rabbit haemorrhagic disease virus type 2 (RHDV2)

Abstract

RABBIT haemorrhagic disease (RHD) is an acute and lethal form of viral hepatitis in rabbits ( Oryctolagus cuniculus ) with a mortality rate in adults ranging from 70 per cent to 100 per cent. RHD was first reported in China in 1984, in Europe in 1986, where it caused severe losses to rabbit, and in Australia in 1996 (Abrantes and others 2012). RHD is caused by the lagovirus RHD virus (RHDV) in the family Caliciviridae. In the course of its evolution, RHDV split into six genotypes (Kerr and others 2009), all highly pathogenic and virulent. Genotype 6 is the antigenic subtype (RHDVa) that became prevalent in certain countries, including the USA (McIntosh and others 2007). In addition, other enteric non-pathogenic rabbit caliciviruses (RCVs) related to RHDV have been identified in Europe and Australia (Capucci and others 1996, Strive and others 2009, Le Gall-Recule and others 2011a).

In 2010, a new lagovirus was identified in France. This virus showed a capsid protein sequence identity of about 80 per cent with RHDV and was able to cause RHD in vaccinated and young rabbits (15–25 days old) (Le Gall-Recule and others 2011a, b). In addition, it showed a distinct antigenic profile and induced an average mortality rate of 20–30 per cent in both experimental infections and natural cases; such a low mortality rate was never observed in the many experimental rabbit infections carried out with other strains of RHDV. The remaining 70–80 per cent of the rabbits survived the infection without showing typical signs of RHD (Le Gall-Recule and others 2011b). Unexpectedly, in autumn 2011, this new virus also caused fatal cases in cape hares ( Lepus capensis var meditteraneus) (Puggioni and others 2013). All these features strongly suggested that the virus was not derived from RHDV but …

Full text

Short Communication
Increased pathogenicity
in rabbit haemorrhagic
disease virus type 2
(RHDV2)
L. Capucci, P. Cavadini, M. Schiavitto,
G. Lombardi, A. Lavazza
RABBIT haemorrhagic disease (RHD) is an acute and lethal
form of viral hepatitis in rabbits (Oryctolagus cuniculus) with a
mortality rate in adults ranging from 70 per cent to 100 per cent.
RHD was first reported in China in 1984, in Europe in 1986,
where it caused severe losses to rabbit, and in Australia in 1996
(Abrantes and others 2012). RHD is caused by the lagovirus
RHD virus (RHDV) in the family Caliciviridae. In the course of
its evolution, RHDV split into six genotypes (Kerr and others
2009), all highly pathogenic and virulent. Genotype 6 is the anti-
genic subtype (RHDVa) that became prevalent in certain coun-
tries, including the USA (McIntosh and others 2007). In
addition, other enteric non-pathogenic rabbit caliciviruses
(RCVs) related to RHDV have been identified in Europe and
Australia (Capucci and others 1996,Strive and others 2009,
Le Gall-Reculé and others 2011a).
In 2010, a new lagovirus was identified in France. This virus
showed a capsid protein sequence identity of about 80 per cent
with RHDV and was able to cause RHD in vaccinated and
young rabbits (15–25 days old) (Le Gall-Reculé and others 2011a,b).
In addition, it showed a distinct antigenic profile and induced an
average mortality rate of 20–30 per cent in both experimental
infections and natural cases; such a low mortality rate was never
observed in the many experimental rabbit infections carried out
with other strains of RHDV. The remaining 70–80 per cent of
the rabbits survived the infection without showing typical signs
of RHD (Le Gall-Reculé and others 2011b). Unexpectedly, in
autumn 2011, this new virus also caused fatal cases in cape hares
(Lepus capensis var meditteraneus) (Puggioni and others 2013). All
these features strongly suggested that the virus was not derived
from RHDV but rather that it had recently emerged from an
unknown source; for this reason, we named it RHDV type 2
(RHDV2). Within a couple of years, RHDV2 spread throughout
Europe (Le Gall-Reculé and others 2013,Dalton and others
2014), where it caused outbreaks on rabbit farms and in wild
populations. Over time, the mortality rate of RHDV2 cases in
Italy appeared to increase, as did the proportion of cases result-
ing in acute RHD.
With the aim of establishing the pathogenicity (ie, the pro-
portion of infected rabbits that develop RHD) of recent field
strains, we infected three groups, each consisting of five healthy,
seronegative, adult New Zealand rabbits. Each rabbit received,
orally, 1 ml of 0.5 per cent w/v liver homogenate from rabbits
that died of acute RHD caused by the following strains:
RHDVBs89 (Capucci and others 1996), which is the reference
RHDV strain, RHDV2Ta14 from an outbreak in the Puglia
region in 2014 and RHDV2Ch15 from an outbreak in the
Abruzzo region in 2015. Trials were performed in a Biosecurity
Level 3-designated area. All animal work was approved by the
Ministry of Health and conducted according to the requirements
of national (DM 4/3/2014 n. 26) and European (2010/63/EU)
laws regarding the care and use of animals.
The three different isolates caused similar RHD symptoms
and degrees of pathogenicity (ie, 80 per cent of rabbits developed
acute RHD) (Fig 1).
The average time to death postinfection ( p.i.) was similar for
RHDVBs89 and RHDV2Ta14, approximately 70 hours, and
slightly higher for RHDV2Ch15, 85 hours. RHDV and
RHDV2-specific sandwich ELISAs (OIE –Rabbit Haemorrhagic
Disease 2012) performed on 10 per cent w/v liver homogenates,
revealed viral titres (ie, ELISA endpoints) ranging from 10
−2
to
10
−3
, typical of acute RHD. During the experiments, we also
recorded the death of three rabbits, one from each group,
without signs of RHD. During necropsy, we found relatively
severe enteritis with fluid faecal contents, likely due to a bacter-
ial infection; this outcome may have been a consequence of the
sudden environmental change from the farm to the experimental
area. The livers of these rabbits were negative for RHDV based
on an ELISA but positive based on RT-PCR, indicating that the
infection was under way. Since 1990, we have performed several
experimental infections of rabbits with RHDV; the present
experiments were the first in which some rabbits died from
causes other than RHD. Although the unusual deaths were
neither desired nor planned, they did permit us to observe a new
and interesting phenomenon. In the three rabbits suffering from
severe enteric bacterial infection, the replication of highly patho-
genic RHDV was strongly inhibited until at least 96 hours p.i., a
time point at which all others infected rabbits had already died
from RHD. Such acquired ‘resistance’to RHD in these three
rabbits could be due to the prompt activation and action of the
innate immune system stimulated by the bacterial infection,
which indirectly limited RHDV replication. Studies on innate
immunity in young rabbits have previously suggested the direct
involvement of the innate immune system in resistance and sus-
ceptibility to RHD (Marques and others 2014).
This virulence trial, the first described so far to use RHDV2
strains identified after 2011, showed that the two Italian RHDV2
strains isolated in 2014 and 2015 induced at least 80 per cent
mortality, which approaches the usual mortality rate of RHDV
and is four times higher than that found in the early RHDV2 iso-
lates (Le Gall-Reculé and others 2013). Considering that we
recently observed several natural RHDV2 outbreaks in farmed
rabbits characterised by higher mortality rates, we conclude that
highly pathogenic RHDV2 strains have emerged during the
virus’s evolution and have become prevalent in the field.
The demonstrated significant increase in RHDV2 pathogen-
icity within a few years supports two hypotheses. The first has
already been put forth to explain RHDV evolution in wild
rabbits in Australia (Elsworth and others 2014): high pathogen-
icity and virulence in RHDV are traits that undergo positive
selection. Presumably, they allow for RHDV’s rapid spread and
sustained presence in rabbit host populations. The second
Veterinary Record (2017) doi: 10.1136/vr.104132
L. Capucci, BSc,
P. Cavadini, BSc, PhD,
G. Lombardi, DVM,
A. Lavazza, DVM,
Istituto Zooprofilattico Sperimentale
della Lombardia e dell’Emilia
Romagna and OIE Reference
Laboratory for Rabbit Hemorrhagic
Disease, Brescia, Italy
M. Schiavitto, DVM,
Centro Genetico Associazione
Nazionale Coniglicoltori Italiani
(ANCI), Volturara Appula,
Foggia, Italy
E-mail for correspondence:
lorenzo.capucci@izsler.it
Provenance: not commissioned;
externally peer reviewed
Accepted February 16, 2017
10.1136/vr.104132 | Veterinary Record |1of2
Short Communication
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hypothesis relates to the origin of RHDV2 and its status as a
newly emerging virus and not as a direct variant of former
RHDVs (Le Gall-Reculé and others 2013). Indeed, our results
indicate that the first RHDV2 isolates from 2010 to 2011 are,
among the known RHDVs, the only mildly pathogenic ones.
Thus, in this respect, they resemble the Michigan rabbit calici-
virus (MRCV), identified in 2001 at a USA rabbit farm, which
was associated with episodes of RHD-like disease and had a
total case-fatality rate of approximately 30 per cent (Bergin and
others 2009). Because the MRCV genome is unique among the
lagoviruses and no later outbreaks were reported nor was it iden-
tified again, MRCV could be considered an ‘attempt’at emer-
gence by a new RHDV-like virus. The reason for the
transmission and evolutionary ‘success’of RHDV2, compared
with the ‘failure’of MRCV, could be the strong presence in
Europe of susceptible hosts (Oryctolagus cuniculus) in the wild
and on farms; this may have allowed RHDV2 to complete its
transition to a highly pathogenic virus. In contrast, in the USA,
European rabbits are present only on a few farms and there are
no wild populations, a situation not sufficient to support a lago-
virus diffusion and persistence. This is also demonstrated by the
fact that the pathogenic RHDVa is not endemic in USA in spite
of the occurrence of some outbreaks.
In light of these results and the fact that the immunogenic
differences between RHDV and RHDV2 occur at the serotype
level, there is an urgent need for homologous RHDV2 vaccines
that can fully protect rabbits from RHD and limit the environ-
mental contamination and diffusion of RHDV2 in affected coun-
tries. RHDV2 has been present since 2011 in several European
countries but, as suggested by this study, it is evolving rapidly,
and the distribution of antigenic variants (subtypes) is likely
shifting. Therefore, the specific choice of RHDV2 isolate will be
important in the production of specific vaccines.
Finally, although RHDV2 has been the dominant virus
causing RHD in most European countries in recent years, the
fact that RHDVa still coexists in some areas (ie, Italy) makes it
advisable to vaccinate animals against RHDV2 and the ‘original’
RHDV/RHDVa strains.
Acknowledgements
The authors thank Giuliana Botti, Alessandra Previdi and Dante
Pedretti for careful technical support. The authors thank Jennie
S. Lavine (ScienceDocs) for editing of this manuscript.
Funding Funding for this study was provided by the Italian Ministry of Health, project
PRC IZSLER 14/2013.
Open Access This is an Open Access article distributed in accordance with the
Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits
others to distribute, remix, adapt, build upon this work non-commercially, and license
their derivative works on different terms, provided the original work is properly cited
and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
References
ABRANTES, J., VAN DER LOO, W., LE PENDU, J. & ESTEVES, P. J. (2012) Rabbit
haemorrhagic disease (RHD) and rabbit haemorrhagic disease virus (RHDV): a
review. Veterinary Research 43,12–31
BERGIN, I. L., WISE, A. G., BOLIN, S. R., MULLANEY, T. P., KIUPEL, M. & MAES,
R. K. (2009) Novel calicivirus identified in rabbits, Michigan, USA. Emerging
Infectious Diseases 15(12), 1955–1962
CAPUCCI, L., FUSI, P., LAVAZZA, A., PACCIARINI, M. L. & ROSSI C. (1996)
Detection and preliminary characterization of a new rabbit calicivirus related to
rabbit hemorrhagic disease virus but nonpathogenic. Journal of Virology 70,
8614–8623
DALTON, K. P., NICIEZA, I., ABRANTES, J., ESTEVES, P. J. & PARRA, F. (2014)
Spread of new variant RHDV in domestic rabbits on the Iberian Peninsula.
Veterinary Microbiology 169,67–73
ELSWORTH, P., COOKE, B. D., KOVALISKI, J., SINCLAIR, R., HOLMES, E. C. &
STRIVE, T. (2014) Increased virulence of rabbit haemorrhagic disease virus asso-
ciated with genetic resistance in wild Australian rabbits (Oryctolagus cuniculus).
Virology 464-465, 415–423
KERR, P. J., KITCHEN, A. & HOLMES, E. C. (2009) Origin and phylodynamics of
rabbit hemorrhagic disease virus. Journal of Virology 83, 12129–12138
LE GALL-RECULÉ, G., LAVAZZA, A., MARCHANDEAU, S., BERTAGNOLI, S.,
ZWINGELSTEIN, F., CAVADINI, P., MARTINELLI, N., LOMBARDI, G.,
GUÉRIN, J. L., LEMAITRE, E., DECORS, A., BOUCHER, S., LE NORMAND, B.
& CAPUCCI, L. (2013) Emergence of a new lagovirus related to Rabbit
Haemorrhagic Disease Virus. Veterinary Research 44,81–94
LE GALL-RECULÉ, G., ZWINGELSTEIN, F., FAGES, M. P., BERTAGNOLI, S.,
GELFI, J., AUBINEAU, J., ROOBROUCK, A., BOTTI, G., LAVAZZA, A. &
MARCHANDEAU S. (2011a) Characterisation of a non-pathogenic and non-
protective infectious rabbit lagovirus related to RHDV. Virology 410, 395–402
LE GALL-RECULÉ, G., ZWINGELSTEIN, F., BOUCHER, S., LE NORMAND, B.,
PLASSIART, G., PORTEJOIE, Y., DECORS, A., BERTAGNOLI, S., GUÉRIN, J. L.
& MARCHANDEAU, S. (2011b) Detection of a new variant of rabbit haemor-
rhagic disease virus in France. Veterinary Record 168, 137–138
MARQUES, R. M., TEIXEIRA, L., AGUAS, A. P., RIBEIRO, J. C., COSTA-E-SILVA,
A. & FERRERIRA, P. G. (2014). Immunosuppression abrogates resistance of
young rabbits to Rabbit Haemorrhagic Disease (RHD). Veterinary Research 45,
14–19
MCINTOSH, M. T., BEHAN, S. C., MOHAMED F. M., LU, Z., MORAN, K. E.,
BURRAGE, T. G., NEILAN, J. G., WARD, G. B., BOTTI, G., CAPUCCI, L. &
METWALLY, S. A. (2007) A pandemic strain of calicivirus threatens rabbit indus-
tries in the Americas. Virology Journal 4,96–110
OIE –RABBIT HAEMORRHAGIC DISEASE Chapter 2.6.2 (2012). OIE Manual of
Diagnostic Tests and Vaccines for Terrestrial Animals. Paris, France: OIE, pp 941–955
(nb: version adopted in May 2016)
PUGGIONI, G., CAVADINI, P., MAESTRALE, C., SCIVOLI, R., BOTTI, G.,
LIGIOS, C., LE GALL-RECULÉ, G., LAVAZZA, A. & CAPUCCI, L. (2013) The
new French 2010 Rabbit Hemorrhagic Disease Virus causes an RHD-like disease
in the Sardinian Cape hare (Lepus capensis mediterraneus). Veterinary Research 44,
96–103
STRIVE, T., WRIGHT, J. D. & ROBINSON, A. J. (2009) Identification and partial
characterization of a new Lagovirus in Australian wild rabbits. Virology 384,
97–105
0-36 48 72 96 AT
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
RHDV 2 Ch15
86
Hours post infection
RHDV Bs89
68
RHDV2 Ta14
75
FIG 1: Time of death, expressed in hours postinfection, in the three
experimental groups (five rabbits each) due to rabbit haemorrhagic
disease virus type 2 (RHDV2) ( full light grey box) or from other
causes (dashed boxes). AT, average time of survival.
2of2|Veterinary Record | 10.1136/vr.104132
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haemorrhagic disease virus type 2 (RHDV2)
Increased pathogenicity in rabbit
L. Capucci, P. Cavadini, M. Schiavitto, G. Lombardi and A. Lavazza
published online March 24, 2017Veterinary Record
http://veterinaryrecord.bmj.com/content/early/2017/03/24/vr.104132
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