Introduction
Rabbit haemorrhagic disease (RHD) is a fatal infectious disease that
primarily affects adult rabbits and causes great economic losses in the
rabbit industry (Mitro and Krauss, 1993).
The causative pathogen for RHD is RHD virus (RHDV), which belongs to the
genus Lagovirus and family Calicivirus
(Moussa et al., 1992). In 1984, it was
first reported in China within a group of
commercially-bred
Angora rabbits imported from Germany. However, some studies suggested
that it had spread across Europe for some time; therefore, it is
difficult to clarify whether those rabbits were infected with RHDV
before importation from Germany (Kerr et
al., 2009). All RHDV strains are classified into one serotype; however,
several different subtypes have been identified, including classic RHDV
(GI.1/G1-6) and RHDV2 (GI.2/RHDVb). RHDV2 is a new variant of classic
RHDV, which was first reported in north-western France in 2010
(Le Gall-Reculé et al., 2011). It has now
spread across many countries in Europe, Australia, and parts of Africa
(Abrantes et al., 2013; Kerr et al., 2009;
Rouco et al., 2019). More recently, RHDV2
outbreaks have been reported in the United States and Canada. Compared
with RHDV, RHDV2 is hosted in a wider range of rabbits and can not only
infect domestic rabbits and European hares, but also a wider range of
hare species, including the Sardinian cape hare (L. capensis)
(Puggioni et al., 2013), Corsican hare
(L. corsicanus)(Camarda et al., 2014), and
European hare (L. europaeus) (Neimanis et
al., 2018b). Notably, rabbits immunized with the classic RHDV vaccine
can become infected with RHDV2 and die, which indicates a significant
antigenic difference between RHDV2 and classic RHDV. The mechanism of
its mutation remains unclear. Another unique characteristic of RHDV2 is
that it can infect young rabbits and cause death with a high mortality
rate (70%-100%). Although China is the largest rabbit breeder in the
world and was the first to report RHDV, it has had no previous RHDV2
outbreak. However, on May 2020, there was a sudden RHDV2 outbreak in
Sichuan Province, China, which killed thousands of rabbits. Here, we
report and describe the first RHDV2 case in China.
A large rabbit population died in two rabbit farms in Sichuan province,
China (Fig 1A). Most of the dead rabbits were young rabbits (age: 30
days); however, others were adult female rabbits. Moreover, all the dead
rabbits showed typical clinical signs of RHD; including
anorexia,
cachexia, opisthotonos, and bloody nasal discharge (Fig. 1B). The dead
rabbits were autopsied to detect pathological tissue changes. The major
autopsy findings were haemorrhages and congestions in the lungs, heart,
and kidneys, as well as acute hepatitis and splenomegaly (Fig. 1C). For
subsequent analysis, tissues, including the heart, liver, spleen, lung,
kidney, and serum, were collected and stored at -80 °C.
To identify causative pathogens, the livers of the dead rabbits were
homogenized with phosphate-buffered saline (PBH; pH 7.4), subjected to
three freeze-thaw cycles, and centrifuged for supernatant collection. A
haemagglutination assay (HA) test was performed using these supernatants
and 1% human erythrocytes of blood group O. The supernatants were found
to significantly agglutinate human type O erythrocytes and the HA titre
was 1:800. Subsequently, the supernatants were clarified with 30%
sucrose solution and washed with PBS to remove the sucrose. The
collected supernatants were analysed using transmission electron
microscopy (TEM), which revealed icosahedral symmetry viral particles
with a 30-40 nm diameter that were consistent with those of RHDV (Fig.
2). The obtained RHDV isolation strain was named as SC20-01.
To explore the pathogen source, liver total RNA from the dead rabbits
was extracted
using
Trizol Reagent (Invitrogen, USA) and reverse transcribed using the M-MLV
reverse transcriptase (Promega, USA) The complete sequence of
SC20-01
was amplified and submitted to GenBank
(accession
no. MT737965). From GenBank, 21 and 19 genome sequences of RHDV2 and
classic RHDV, respectively, were selected. Three genome sequences of
Rabbit Calicivirus (RCV) were chosen as the out-groups. A
phylogenetic
tree was constructed using the maximum likelihood statistical methods
based on VP60 protein using MEGA5.0 software (www.megasoftware.net). The
SC20-01 strain was found to belong to the RHDV2 subtype; moreover, its
VP60 protein shared 97.2%~99.5% and
86.8%~87.8% amino acid identity with other RHDV2
subtypes and classic RHDV strains, respectively (Fig. 3A). Based on the
phylogenetic tree,
the
SC20-01 strain was most closely related with RHDV2-NL2016 strain
(accession no. MN061492) and Bremerhaven-17 strain (accession no.
MN901451), which were all isolated from rabbits in Europe.
There are many genetic
recombination
events among RHDV; further, the inter-subtype genetic recombination
events are mainly located in the ORF1s (Qi
et al., 2019). We examined recombination events in the SC20-01 strain
using the RDB, GEGECONV, Bootscan, Maximum Chi-Square, Chimaera, Sister
Scanning, and 3seq methods of the Recombination Detection Program (RDP)
v4.56 software after selecting 324 genome sequences of RHDV and 3 genome
sequences of RCV, except for SC20-01, from GenBank. The recombination
area of the SC20-01 strain was found to be from nucleotide 5248 to 5387;
moreover, its related strains were P19 (accession no. KY765610, G6) and
CBAnd1 (accession no. KP090976, RHDV2) from Portugal and Spain (Table
1). Furthermore, a recombination event of the SC20-01 strain was
confirmed using SimPlot with the observation of an apparent breakpoint
that separated the SC20-01 genome into two regions, which was consistent
with the RDP results (Fig. 3B). Two phylogenetic trees were constructed
using maximum likelihood statistical methods based on two different
nucleotide recombination regions (non-structural and structural
protein-coding regions: 10~5304bp and
5304~7378bp, respectively) of the SC20-01 strain using
MEGA5.0 software
(www.megasoftware.net). The
non-structural protein-coding region of SC20-01 was more closely related
with that of classic RHDV than that of RHDV2. This demonstrated that the
SC20-01 strain was a classic RHDV/RHDV2 recombination strain (Fig.
3C-D).
To confirm infection with the SC20-01 strain, six 6-week-old SPF rabbits
were randomly divided into two groups (n = 3) and injected with 1 ml of
the supernatants of homogenized livers or PBS followed by observation
for three days. All animal experimental procedures were approved and
performed in compliance with the guidelines of the Animal Research
Ethics Board of Shanghai Veterinary Institute (Shanghai, China), CAAS
(no. SHVRI-SZ-20200709-01). After 24 post-infection hours, two rabbits
in the experimental group showed typical clinical symptoms of RHD with
one dying (Fig. 4A). The two surviving rabbits in the experimental group
were euthanized using an intravenous injection of sodium pentobarbital
to examine the tissue distribution of pathogens in various
post-infection stages. After observation for 7 days, all rabbits in the
control group survived and did not show any clinical RHD symptoms. All
the surviving rabbits were euthanized with an intravenous injection of
sodium pentobarbital. Subsequently, they were autopsied to examine
tissue pathological changes and tissues were collected and stored in 4%
paraformaldehyde or -80 °C for subsequent analysis. The tissues of
rabbits infected with SC20-01 strain showed RHDV pathological changes
(Fig. 4A).
To examine the viral distribution, tissue and body-fluid total RNA of
rabbits infected with SC20-01 strain were extracted with Trizol Reagent
(Invitrogen, USA) and reverse transcribed using the M-MLV reverse
transcriptase (Promega, USA). Real-time quantitative polymerase chain
reaction was used to detect viral loads in the tissues and body fluids
of rabbits infected with SC20-01. Viral RNA was detected in all tissues
and body fluids, including urine, the gonads, and the brain. Among those
tissues and body fluids, there was a higher viral load in the liver,
cholecysts, spleen, and lung, which was consistent with previous
findings (Liu et al., 2015; Neimanis et
al., 2018a). Furthermore, there was a high viral load in the
serums
of the infected rabbits (Table 2). The viral antigen was detected in
both groups using immunohistochemical (IHC) analysis. The primary
antibodies were mouse monoclonal antibodies targeting VP60 of RHDV2
(prepared and preserved by our laboratory) while the secondary antibody
was horseradish peroxidase-conjugated goat anti-mouse antibody (Jackson
ImmunoResearch, USA). Viral antigen was only detected in the tissues of
rabbits infected with SC20-01 and was mainly located in the cellular
cytoplasm (Fig. 4B).
In summary, we reported and identified the first RHD case caused by
RHDV2, a new RHDV variant, in China through RT-PCR, HA, and IHC.
Subsequently, we amplified the complete genome sequence of the SC20-01
strain and conducted a phylogenetic tree based on the amino acid
sequence of VP60. We found that the strains most related to the SC20-01
strain were isolated in Europe and speculated that SC20-01 might have
spread from Europe to China.
Recombination
detection indicated that the SC20-01 strain is an inter-subtype
recombination strain composed of two different RHDV subtypes. This
indicated that there were genetic recombination events between classic
RHDV and RHDV2. Six SPF rabbits were used to monitor the pathogenicity
of the SC20-01 strain, which was found to cause death with typical
clinical symptoms and RHD tissue lesions after 24 post-infection hours.
Viral RNA could be detected in all tissues and body fluids of infected
rabbits, including urine, which suggested viral pollution in the
environments by urine and faeces. Therefore, there is a need to
disinfect the environments of rabbits farms upon identification of RHD
cases. Moreover, the high viral load in the brains and gonads suggested
that RHDV2 could cross the blood-brain barrier or blood testes barrier.
This indicated that RHDV2 might could be vertically transmitted from
female rabbits to young rabbits. There is a need to monitor the RHDV2
epidemiology in China since RHDV2 could cause death among rabbits
vaccinated against classic RHDV. Moreover, it is necessary to develop
new diagnostic methods and vaccines for preventing the spread of RHDV2.