Co-circulation of GI.1 and GI.2 genotypes of rabbit hemorrhagic disease virus in Egypt

Abstract

Recently, Egypt has experienced an increased incidence of rabbit hemorrhagic disease virus (RHDV) infection even among vaccinated rabbits. The present study estimates the emergence of RHDV in vaccinated (n = 10) and unvaccinated (n = 8) domestic rabbitries in Beheira and Kafr El-Sheikh provinces, Egypt, during the period 2018–2020. A total of 8 out of 18 (44.4%) liver extracts were able to agglutinate human type O RBCs with HA titers ranged from 8 to 12 log2, and then subsequently confirmed for the presence of RHDV RNA using a reverse transcriptase-polymerase chain reaction (RT-PCR). The VP60 gene sequences of three selected isolates, designated Beh-1, Beh-9 and kaf-14, were submitted to the GenBank database and the accession numbers MZ782083 to MZ782085 were assigned, respectively. Phylogenetic analysis revealed that the Kaf-14 isolate was placed into the GI.1 genotype, while the Beh-1 and Beh-9 isolates were grouped into the GI.2 genotype. Overall, the three isolates shared 78.6–98.7%.nucleotide identity with previously published Egyptian sequences. In comparison with the GI.1a Giza2006 vaccine strain, the three isolates exhibited divergence ranging from 4.5 to 17.4% at the amino acid level. Approximately 55.5–87.5% of the amino acid substitutions were located in the P2 subdomain of the VP60 capsid protein which contains the main determinants of antigenicity and cellular recognition. In conclusion, our results provide crucial evidence for the co-circulation of RHDV GI.1 and GI.2 genotypes in Egypt and highlight the antigenic diversity among vaccine and field strains. Therefore, new effective vaccines are urgently required to counter the spread of GI.1 and GI.2 genotypes in Egypt.

Introduction

Rabbit hemorrhagic disease (RHD) is a fatal and highly contagious viral disease in both domestic and wild rabbits (Oryctologus cuniculus). The etiological agent, rabbit hemorrhagic disease virus (RHDV), is a small-sized (30–40 nm in diameter), non-enveloped, icosahedral-shaped, single-stranded, positive-sense RNA virus of Lagovirus genus, family Caliciviridae [29]. The virus genome comprises approximately 7437 nucleotides and possesses two slightly overlapping open reading frames (ORFs). A polyprotein that is cleaved into the major structural capsid protein VP60 and several non-structural proteins is encoded by ORF1 (nucleotides 10-7044), whereas ORF2 (nucleotides 7025–7378) encodes the small capsid protein VP10 [11, 29]. The capsid protein VP60 is responsible for determining virus pathogenicity and antigenicity and eliciting protective humoral immune response against RHDV. Therefore, the RHDV VP60 gene plays a key role in molecular diagnosis of the virus and vaccine design [15].

RHD was first reported in china in 1984 [25], and subsequently dispersed across the world within a few years [3, 6]. RHDV GI.1 infection is associated with 100% morbidity and mortality rates close to 90% in adult rabbits, whereas rabbits younger than 2 months are naturally resistant to lethal infection. RHDV replicates primarily in the liver and the incubation period ranges between 1 and 3 days. Rabbits often die suddenly within 12 to 36 h after infection without presenting any clinical symptoms, whereas some rabbits may display pyrexia, anorexia, lethargy, seizures, dyspnea, pneumonia and bleeding from the mouth [28]. Upon necropsy, the primary lesions include extensive hepatic necrosis, splenomegaly, pulmonary congestion and scattered hemorrhages in lungs, trachea and kidneys [20, 28].

In 2010, a novel pathogenic lagovirus, designated as RHDV2 or RHDVb, was detected in France [22], and subsequently spread throughout Europe, United States, Canada, Asia, Australia and Africa. This new variant differs genetically and antigenically from the classic GI.1 RHDV and is therefore included in the new genotype GI.2 [24]. Currently, the GI.2/RHDV2/b variant seems to have replaced the classic GI.1 RHDV in the majority of European countries and Australia [4, 27]. Unlike GI.1 RHDV, GI.2 RHDV is able to infect and kill kittens as young as 11 days old as well as rabbits that had been previously vaccinated against GI.1 RHDV [10, 23].

Due to the continual emergence of new genetic variants, a unified nomenclature and classification system have been proposed for lagoviruses in 2017. Based on phylogenetic analysis of VP60 gene sequences, lagoviruses have been classified into two genogroups (GI and GII). RHDV strains belong to genogroup GI, which further splits into four genotypes (Gl.1, GI.2, GI.3 and GI.4). The genotypes GI.1 and GI.2 include the classical RHDV and RHDV2/b variant strains, respectively. Furthermore, genotype GI.1 splits into various variants (GI.1a to GI.1d). Meanwhile, the genotypes GI.3 and GI.4 comprise the non-pathogenic rabbit caliciviruses (RCVs) E-1 and A-1/E-2, respectively [24].

Since attempts to isolate RHDV in a convenient cell culture system or chick embryo have been universally unsuccessful, using an in vivo rabbit model remains the only method that permits isolation, propagation and titration of RHDV [2]. Currently, the definitive diagnosis of RHDV relies largely on detection of the VP60 gene using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay [7]. In addition, the ability of RHDV to agglutinate human type O red blood cells (RBCs) is still widely used as a preliminary diagnostic test [10].

In Egypt, GI.1 RHDV was firstly reported in Sharkia governorate in 1991 [16]. In the following years, the virus dispersed rapidly throughout the country and caused serious production losses. In 2006, the GI.1a variant started to circulate and subsequently became more prevalent than the GI.1d variant. Therefore, the GI.1d Giza97 strain has been totally replaced by the GI.1a Giza2006 strain in vaccine manufacture since 2008. However, both the GI.1a and GI.1d variants continue to circulate in the following years [1, 26]. In addition, the emergence and spread of the GI.2 genotype was first reported in 2018 [14, 18]. The present study aimed to monitor the emergence of RHDV in vaccinated rabbitries in different Egyptian provinces and to determine the genetic diversity between circulating RHDV strains and the currently used vaccine strain in order to provide a better evaluation of the current vaccination strategy.

Materials and methods

Flock history and clinical samples

During 2018–2020, a total of 18 domestic rabbitries (Oryctologus cuniculus) were considered to be infected with RHD in Beheira (14 rabbitries) and Kafr El-Sheikh (4 rabbitries) provinces. A total of 10 out of 18 rabbitries had previously received a single subcutaneous dose (0.5 ml/rabbit) of a locally produced inactivated vaccine (SERVAC RHDV®), containing the GI.1a Giza2006 strain. Overall, the rabbits suffered up to 70% mortality. Before death, they showed struggling motion, low spirit, urgent breath and occasionally bloody discharges from nostrils. Necropsy revealed serosal hemorrhages in the lumen of trachea, pulmonary congestion, friable and enlarged livers with scattered necrosis and congested kidneys. In each rabbitry, five liver samples were collected aseptically from freshly dead rabbits of both sexes at various ages, pooled and homogenized in sterile phosphate-buffered saline (PBS) to obtain a 10% (w/v) suspension as previously described [21]. The cloudy suspension was centrifuged at 900 xg for 10 min and the clarified supernatant was kept at − 20˚C until use.

Hemagglutination (HA) test

The HA activity of the liver extracts was assessed as previously described [21]. Briefly, a 50 µl aliquot of the supernatant of each liver homogenate was diluted with PBS in a series of twofold dilutions using a 96-well U-bottom microtiter plate. Each dilution was then incubated with an equal volume of 0.75% human type O RBCs at 4 °C for 1 h. Samples exhibited HA at an end point dilution greater than 1:160 were considered positive. The HA titer was recorded as the reciprocal log₂ value of the highest dilution that exhibited complete HA.

RNA extraction and RHDV detection

Total RNA was extracted from the clarified liver homogenates using a QIAamp Viral RNA Mini Kit (Qiagen, Germany, Catalog No. 52,904) in accordance with the manufacturer’s protocol. The extracted RNA was further subjected to RT-PCR assay using the oligonucleotide primers P33-F (5-CCACCACCAACACTTCAGGT-3) and P34-R (5-CAGGTTGAACACGAGTGTGC-3) to amplify 538 bp (nucleotides 6473 to 7011, Genbank accession number Z29514) of the VP60 gene of RHDV [32]. One-step RT-PCR assay was conducted in a 25 µl reaction mixture containing 12.5 µl of 2x MyTaq One-Step Mix (Bioline, USA), 6 µl of RNA, 1 µl (20 pmol) of each primer, 0.5 µl of RiboSafe RNase Inhibitor, 0.25 µl of reverse transcriptase and 3.75 µl of diethyl pyrocarbonate (DEPC)-treated water. The amplification profile comprised reverse transcription at 45 °C for 30 min, initial denaturation at 95 °C for 15 min, followed by 40 cycles (denaturation at 95 °C for 1 min, annealing at 56 °C for 1 min and extension at 72 °C for 2 min) and a final extension at 72 °C for 10 min. The products of amplification were electrophoresed through a 1% agarose gel containing ethidium bromide (0.5 µg/ml) and visualized by UV transillumination.

VP60 gene sequencing and phylogenetic analysis

The products of the PCR were purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA) and then sequenced in both sense and antisense directions using the amplification primers and a BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City). The three nucleotide sequences were edited and aligned with 32 publicly available VP60 sequences of different genotypes (11 GI.1, 17 GI.2, 2 GI.3 and 2 GI.4 strains) using BioEdit software v7.2.5.0 [17]. In addition, RCV was included as an out-group. The phylogenetic tree was constructed on the NGphylogeny.fr platform (https://ngphylogeny.fr/) through a fully automatic one click workflow (default tools and default parameters) using MAFFT for sequence alignment, BMGE for alignment curation, PhyML for maximum likelihood-based inference of large phylogenetic trees and Newick display for tree rendering.

Experimental RHDV infection of rabbits

To evaluate the pathogenicity of the three isolates, twenty crossbred 12-week-old rabbits were randomly allotted into four groups of 5 rabbits each. Each rabbit in the groups I, II and III was inoculated intramuscularly with 1ml of the clarified liver homogenate of Kaf-14, Beh-1 and Beh-9, respectively. Meanwhile, rabbits in group IV were inoculated with sterile PBS and kept as negative controls. Each group was housed in a separate cage and monitored twice daily for clinical signs and mortality. Dead rabbits were necropsied and inspected for gross lesions. In each group the two surviving rabbits were euthanized 10 days post-inoculation and necropsied. Liver homogenates were prepared from dead and euthanized rabbits and subjected to RT-PCR using the same primers and procedure as described above.

Results

RHDV identification and HA activity

A total of 18 liver homogenate samples were firstly screened for the presence of HA activity, and then subjected to RT-PCR for detection of RHDV RNA. Results revealed the presence of HA activity in 5 out of 14 (36%) and 3 out of 4 (75%) tested samples in Beheira and Kafr El-Sheikh provinces, respectively. The HA titers were ranged from 8 to 12 log₂. In agreement with the HA results, RT-PCR was only able to detect RHDV RNA in the HA-positive samples. As expected, the amplicons produced had the size of 538 bp following agarose gel electrophoresis (data not shown).

Sequencing and phylogenetic analysis

The sequences of two isolates from Beheira province, designated Beh-1and Beh-9, and one isolate from Kafr El-Sheikh province, designated Kaf-14, were submitted to the GenBank database and assigned the accession numbers MZ782083 to MZ782085 (Table 1). Alignment analysis of the three nucleotide sequences with 33 publicly available VP60 gene sequences revealed clustering of the three isolates into two distinct genotypes (GI.1 and GI.2). The Kaf-14 isolate shared 95.7%, 94.6% and 91.2% nucleotide identities with RHDV GI.1 strains reported in Egypt (MN397253), Saudi Arabia (KJ949620) and Bahrain (DQ189077), respectively. Meanwhile, the Beh-1 and Beh-9 isolates shared 94.6% nucleotide identity with each other and 95.9–98.7%, 94.6–97.4% and 94.2–97% nucleotide identities with RHDV GI.2 strains reported in Egypt (MN276176), Germany (LR899145) and Nigeria (MW123059), respectively. The alignment results and clustering of the three isolates were clearly confirmed by phylogenetic analysis (Fig. 1). The deduced amino acid sequences alignment analysis of the VP60 gene between residues 390 and 597 revealed the presence of only 4 substitutions in the sequence of the Kaf-14 isolate. These substitutions were clustered within the hypervariable regions V5 (residues 411–417), V6 (residues 431–435) and V7 (residues 476–480). Meanwhile, a total of 26 and 21 substitutions were recorded throughout the sequences of the Beh-1 and Beh-9 isolates, respectively. Only 28.5–30.7% of these substitutions were clustered in the hypervariable regions V5-V7 of both isolates (Fig. 2). Furthermore, alignment analysis of the current amino acid sequences with previously published Egyptian sequences revealed an identity of 95.5–98.3% between the Kaf-14 isolate and the other GI.1 isolates. In addition, the Beh-1 and Beh-9 isolates were 91-99.4% identical with the GI.2 isolates. However, the commonly used GI.1a Giza2006 vaccine strain (JQ995154) exhibited 4.5%, 17.4% and 15.1% of divergence at the amino acid level from the Kaf-14, Beh-1 and Beh-9 isolates, respectively.

Table 1.

Details of RHDV isolates obtained in the current study

Isolate	Genotype	Province	Vaccination status	Year	HA titer with human type O RBCs (log2 )	GenBank accession number
Beh-1	GI.2	Beheira	Vaccinated	2019	11	MZ782083
Beh-9	GI.2	Beheira	Unvaccinated	2020	12	MZ782084
Kaf-14	GI.1	Kafr El-Sheikh	Vaccinated	2019	12	MZ782085

Fig. 1.

Maximum-likelihood phylogenetic tree based on 36 partial VP60 gene sequences of RHDV. RCV strain was included as an out-group to root the tree. The black square indicates the three isolates reported in the present study, meanwhile the black triangle indicates the previously reported Egyptian isolates

Fig. 2.

Alignment of partial VP60 protein sequences of the three isolates reported in the present study (highlighted by a black square) and previously reported Egyptian isolates

Experimental infection

Overall, mortality rates at day 10 post infection were 60% (3/5), 20% (1/5) and 40% (2/5) among rabbits infected with the RHDV Kaf-14, Beh-1 and Beh-9 isolates, respectively. All dead rabbits exhibited typical and similar clinical signs and gross lesions of RHD, moreover, RHDV RNA was detected in their liver homogenates by RT-PCR. The remaining rabbits survived the infection for 10 days and displayed no clinical signs until being euthanized. At necropsy, no gross lesions were observed and no viral RNA was detected by RT-PCR. None of the control rabbits exhibited clinical signs nor died throughout the entire experiment.

Discussion

Since 1991, RHD has become a serious economic threat to the Egyptian rabbit industry. Despite extensive vaccination, several RHD outbreaks are still reported. Moreover, the increased occurrence of these outbreaks among previously vaccinated rabbits raises serious concerns about the genetic diversity between RHDV field and vaccine strains. Therefore, the present study aimed to monitor the emergence of RHDV in vaccinated rabbitries and to explore the genetic diversity between RHDV field isolates and the currently used Giza2006 vaccine strain. RHDV infection was suspected on the basis of clinical signs and gross pathological lesions and their consistency with previous reports on RHD outbreaks [2]. A total of 8 out of 18 (44.4%) liver extracts were able to agglutinate human type O RBCs with HA titers ranged from 8 to 12 log2. Interestingly, the HA test results were completely consistent with the ability of RT-PCR to detect RHDV RNA in liver extracts. In agreement with previous reports [12, 25], these findings reinforce the importance of HA test as a simple and readily available technique for rapid diagnosis of RHDV infection in rabbits.

The VP60 gene was chosen as the target for genetic characterization and phylogenetic analysis of RHDV isolates as it plays a key role in the induction of a defensive host response against RHDV infection and vaccine design [15, 19, 34]. Maximum likelihood phylogenetic analysis based on partial VP60 gene sequences revealed that the Kaf-14 isolate was placed into the GI.1 genotype, while the Beh-1 and Beh-9 isolates were located into the GI.2 genotype (Fig. 1). These findings clearly confirm the ongoing circulation of the GI.1 genotype, which is thought to be completely replaced by the GI.2 genotype in many countries [8, 27]. The Kaf-14 isolate shared 87.8–95.7% nucleotide identity with previously reported GI.1 isolates in Egypt. The highest identity (95.7%) was recorded with a previously identified isolate (MN397253) in the same province [13]. Interestingly, a very high identity (94.6%) was recorded with the Saudi Arabian isolate RHD/2/SA/2012 (KJ949620). This observation can be probably attributed to the wide dispersion of RHDV through human commercial activities, birds, insects, and contaminated vehicles [30].

Although our GI.2 isolates were detected in the same province, they shared only 94.6% nucleotide identity with each other. Moreover, they shared the highest identity of 95.9–98.7% with the Vet-Abotaleb isolate (MN276176), despite not being geographically close. Overall, the Egyptian GI.2 isolates shared 92.3–98.7% nucleotide identity with each other and classified phylogenetically into two distinct clusters. These findings may suggest multiple events of viral incursion into Egypt or virus evolution or both. In agreement with previous reports [31], our results demonstrated that both GI.1 and GI.2 isolates cause a similar disease in experimentally infected rabbits. However, GI.2-infected rabbits had lower mortality rates than those infected with the GI.1 isolate [5].

The P2 subdomain of the VP60 capsid protein (amino acid residues 287–449 and 467– 483) is believed to contain the main determinants of antigenicity and cellular recognition [9]. Therefore, accumulation of mutations in the P2 subdomain could lead to evasion of the protective potential of the established vaccines. Compared with the GI.1a Giza2006 vaccine strain, a total of 7, 20 and 15 amino acid substitutions were recorded in the P2 subdomain of the Kaf-14, Beh-1 and Beh-9 isolates, respectively (Fig. 2). Approximately 50-71.4% of these substitutions were located in the hypervariable regions V5-V7 [33]. In conclusion, our results provide crucial evidence for the emergence of RHDV GI.1 and GI.2 strains in rabbitries where the GI.1a Giza2006 strain has historically been applied as a vaccine against RHDV and highlight the antigenic diversity among vaccine and field strains. Therefore, new effective vaccines are urgently required to counter the spread of GI.1 and GI.2 genotypes in Egypt.

Declarations

Conflict of interest

The authors declare no competing interests.

Ethics approval

The experimental procedures were conducted according to the ethical standards applied in Veterinary Serum and Vaccine Research Institute, Abbassia, Egypt.