Disease X Testing: The results of an international external quality assessment exercise

The United Nations Secretary-General Mechanism (UNSGM) for investigation of the alleged use of chemical and biological weapons is the only established international mechanism of this type under the UN. The UNGSM may launch an international investigation, relying on a roster of expert consultants, qualified experts, and analytical laboratories nominated by the member states. Under the framework of the UNSGM, we organized an external quality assurance exercise for nominated laboratories, named the Disease X Test, to improve the ability to discover and identify new pathogens that may cause possible epidemics and to determine their animal origin. The "what-if" scenario was to identify the etiological agent responsible for an outbreak that has tested negative for many known pathogens, including viruses and bacteria. Three microbes were added to the samples, Dabie bandavirus, Mammarenavirus, and Gemella spp., of which the last two have not been taxonomically named or published. The animal samples were from Rattus norvegicus, Marmota himalayana, New Zealand white rabbit, and the tick Haemaphysalis longicornis. Of the 11 international laboratories that participated in this activity, six accurately identified pathogen X as a new Mammarenavirus, and five correctly identified the animal origin as R. norvegicus. These results showed that many laboratories under the UNSGM have the capacity and ability to identify a new virus during a possible international investigation of a suspected biological event. The technical details are discussed in this report.Copyright © 2022

A rabbit model of tracheal collapse for optimal self-expanding metal stents.

Investigating the characteristics of tracheas can help the understanding of diseases related to the trachea, particularly tracheal collapse (TC) in dogs. This study aimed to compare the mechanical properties of tracheas from New Zealand White (NZW) rabbits and dogs and to introduce a method for inducing a model of TC in the normal trachea. Tracheal samples were obtained from NZW rabbit cadavers (n=5) weighing 3.62-3.92 kg and from dog cadavers (n=5) weighing 2.97-3.28 kg. Three live NZW rabbits weighing 3.5-4.0 kg were used to establish the model. The radial forces of both sample sets were measured using a digital force gauge and statistically compared. Subsequently, TC was surgically induced in three female NZW rabbits by physically weakening their tracheal cartilage under general anesthesia. Their clinical signs were monitored for 3 months, and radiographic examinations were performed monthly for 3 months. The mean radial forces of the two sample sets were comparable (P>0.05). The clinical signs, radiographic examinations, and macroscopic examinations were all comparable to those of dogs with TC. The cadaveric study between the rabbits and dogs demonstrated that the surgically induced rabbit model of TC is an excellent candidate for the experimental study of dogs with TC. This study also provides a reference of tracheal radial force values to enable selection of appropriate mesh types and wire diameters of self-expanding metal stents.

Gastrointestinal helminths increase Bordetella bronchiseptica shedding and host variation in supershedding.

Co-infected hosts, individuals that carry more than one infectious agent at any one time, have been suggested to facilitate pathogen transmission, including the emergence of supershedding events. However, how the host immune response mediates the interactions between co-infecting pathogens and how these affect the dynamics of shedding remains largely unclear. We used laboratory experiments and a modeling approach to examine temporal changes in the shedding of the respiratory bacterium Bordetella bronchiseptica in rabbits with one or two gastrointestinal helminth species. Experimental data showed that rabbits co-infected with one or both helminths shed significantly more B. bronchiseptica, by direct contact with an agar petri dish, than rabbits with bacteria alone. Co-infected hosts generated supershedding events of higher intensity and more frequently than hosts with no helminths. To explain this variation in shedding an infection-immune model was developed and fitted to rabbits of each group. Simulations suggested that differences in the magnitude and duration of shedding could be explained by the effect of the two helminths on the relative contribution of neutrophils and specific IgA and IgG to B. bronchiseptica neutralization in the respiratory tract. However, the interactions between infection and immune response at the scale of analysis that we used could not capture the rapid variation in the intensity of shedding of every rabbit. We suggest that fast and local changes at the level of respiratory tissue probably played a more important role. This study indicates that co-infected hosts are important source of variation in shedding, and provides a quantitative explanation into the role of helminths to the dynamics of respiratory bacterial infections.

Chapter 5 - Coronaviruses of wild and semidomesticated animals with the potential for zoonotic transmission

Coronaviruses are present in most animal species. Some animals may then serve as a reservoir or intermediate hosts of viruses causing mild or severe to fatal diseases in humans and other animals. Infected humans may also transmit coronaviruses, such as severe acute respiratory syndrome virus (SARS-CoV)-2, to animals, including captive endangered animal species. This chapter focuses on coronaviruses of wild and semidomesticated animals, including viruses from bats, rodents, nonhuman primates, ferrets, minks, and rabbits. The ability of coronaviruses to rapidly mutate and to exchange their genetic material with other coronaviruses leads to the production of variants able to infect and adapt to new host species. Special attention is given to coronaviruses of bats and rodents since they appear to have hosted ancestral coronaviruses that indirectly lead to zoonotic transmission of highly pathogenic human viruses, including SARS-CoV, the closely related SARS-CoV-2, and Middle East respiratory syndrome virus. The RNA genomes of several bat coronaviruses, such as WIV1 and WIV16, are very similar to SARS-CoV. Coronaviruses in animals primarily cause severe disease in the respiratory, central nervous, and digestive systems but may damage other organ systems as well. Further studies on wildlife coronaviruses are advisable to avoid human epidemics or pandemics as well as to protect endangered animal species.

Spreading of Pasteurella multocida Infection in a Pet Rabbit Breeding and Possible Implications on Healed Bunnies.

The number of pets such as dogs, cats, rabbits, and parrots has increased in European families. Social benefits to owners such as decreasing feelings of loneliness and anxiety are provided by pets which are also used in Animal-assisted Therapy (AAT). Nevertheless, human-animal interactions are also associated with health problems including allergies, asthma, and zoonosis. Rabbits may carry potential pathogens for humans. One of the most common bacteria that colonizes the oro-pharynx and the upper respiratory tract of rabbits is Pasteurella (P.) multocida. Transmission of the infection to humans results from scratches, licks, and bites but it also can occur from the inhalation of air particles containing the microorganism. Immunocompromised people or persons with pulmonary disorders are particularly susceptible to the infection. Infected rabbits may carry P. multocida with or without clinical signs. In this paper, the sensitivity to antibiotics and the invasiveness ability of P. multocida identified in a farm of pet rabbits affected by severe pasteurellosis were investigated. The strain was P. multocida belonging to capsular type A which is the type most often detected in humans. The identified strain was susceptible to the tested antibiotics, but it appeared equipped with several virulence genes which are responsible for fimbriae production, adhesion processes to host cells, enzyme production, and are involved in iron acquisition processes. These findings are of particular interest because rabbits recovered from pasteurellosis very often become carriers of the bacteria. Therefore, we suggest considering P. multocida screening in the routine medical checks of rabbits, especially if they are meant to be companion animals for children and elder people, given that the transmission of the pathogen cannot be excluded.

Potent Protease Inhibitors of Highly Pathogenic Lagoviruses: Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus.

Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family. These infectious diseases are associated with high mortality and a serious threat to domesticated and wild rabbits and hares, including endangered species such as riparian brush rabbits (Sylvilagus bachmani riparius). In the United States (U.S.), only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by GI.2/rabbit hemorrhagic disease virus (RHDV)2/b was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several U.S. states, infecting wild rabbits and hares and making it highly likely that RHD will become endemic in the U.S. Vaccines are available for RHD; however, there is no specific treatment for this disease. Lagoviruses encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small-molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses encoding 3CLpro, including caliciviruses and coronaviruses. Here, we report the development of the enzyme and cell-based assays for the 3CLpro of GI.1c/RHDV, recombinant GI.3P-GI.2 (RHDV2/b), and GII.1/European brown hare syndrome virus (EBHSV) as well as the identification of potent lagovirus 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis. In addition, structure-activity relationship study and homology modeling of the 3CLpro and inhibitors revealed that lagovirus 3CLpro share similar structural requirements for inhibition with other calicivirus 3CLpro. IMPORTANCE Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are viral diseases that affect lagomorphs with significant economic and ecological impacts. RHD vaccines are available, but specific antiviral treatment for these viral infections would be a valuable addition to the current control measures. Lagoviruses encode 3C-like protease (3CLpro), which is essential for virus replication and an attractive target for antiviral drug discovery. We have screened and identified potent small-molecule inhibitors that block lagovirus 3CLpro in the enzyme- and cell-based assays. Our results suggest that these compounds have the potential for further development as antiviral drugs for lagoviruses.

Interleukin - 2 Receptor Antagonists Induction Therapy in Simultaneous Heart - Kidney Transplantation

SRTR data currently suggests that induction therapy in simultaneous heart-kidney transplantation (SHKT) with rabbit antithymoglobulin (ATG) provides survival advantage compared to interleukin-2 receptor antagonist (IL2-RA). We are reporting the outcomes of recipients with SHKT treated with IL2-RA as induction therapy. This is a single center, retrospective study of 26 patients who received SHKT at our institution from Dec 2018 to Oct 2021. A multidisciplinary team composed of heart and kidney transplant medical and surgical members determined appropriate recipient-donor SHKT candidate pairs. The majority of patients received IL2-RA induction therapy, and all patients received triple immunosuppression therapy with prednisone, mycophenolate mofetil and tacrolimus. Adjustments in long term therapy were made in collaboration between the heart and kidney transplant teams. From Dec 2018 to Oct 2021, 26 patients underwent SHKT. 23 patients (88%) were male, the median age was 57 years, and 5.4% were ≥ 65 years. 18 patients (69%) had non ischemic cardiomyopathy and 24 patients (92%) had CKD (mean GFR ≤ 35%). 18 patients were listed Status 2 and 2 patient Status 5. One patient received a DCD donor and 12 patients (46%) received hep C donors. 25 patients (96%) received induction therapy with IL2-RA. During the first 3 months post-transplant, the only patient who received ATG had 7 severe infections;11 patients (44%) and 13 patients (52%) who received IL2 -RA had no infections and ≤ 4 mild infections, respectively. One patient died due to COVID 19 pneumonia complicated by multisystem organ failure. For a median follow up period of 410 (187-707) days, 8% patients in the IL2-RA induction cohort experienced a 2R/3A heart rejection, 8% patients remained on HD due to primary kidney graft non-function, and the survival rate was 96%. Compared with present literature, our data support the use of IL2- RA as an induction strategy in SHKT with excellent patient survival. [ FROM AUTHOR] Copyright of Journal of Heart & Lung Transplantation is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

Efficacy and safety testing of a COVID-19 era emergency ventilator in a healthy rabbit lung model.

BACKGROUND: The COVID-19 pandemic revealed a substantial and unmet need for low-cost, easily accessible mechanical ventilation strategies for use in medical resource-challenged areas. Internationally, several groups developed non-conventional COVID-19 era emergency ventilator strategies as a stopgap measure when conventional ventilators were unavailable. Here, we compared our FALCON emergency ventilator in a rabbit model and compared its safety and functionality to conventional mechanical ventilation. METHODS: New Zealand white rabbits (n = 5) received mechanical ventilation from both the FALCON and a conventional mechanical ventilator (Engström Carestation™) for 1 h each. Airflow and pressure, blood O2 saturation, end tidal CO2, and arterial blood gas measurements were measured. Additionally, gross and histological lung samples were compared to spontaneously breathing rabbits (n = 3) to assess signs of ventilator induced lung injury. RESULTS: All rabbits were successfully ventilated with the FALCON. At identical ventilator settings, tidal volumes, pressures, and respiratory rates were similar between both ventilators, but the inspiratory to expiratory ratio was lower using the FALCON. End tidal CO2 was significantly higher on the FALCON, and arterial blood gas measurements demonstrated lower arterial partial pressure of O2 at 30 min and higher arterial partial pressure of CO2 at 30 and 60 min using the FALCON. However, when ventilated at higher respiratory rates, we observed a stepwise decrease in end tidal CO2. Poincaré plot analysis demonstrated small but significant increases in short-term and long-term variation of peak inspiratory pressure generation from the FALCON. Wet to dry lung weight and lung injury scoring between the mechanically ventilated and spontaneously breathing rabbits were similar. CONCLUSIONS: Although conventional ventilators are always preferable outside of emergency use, the FALCON ventilator safely and effectively ventilated healthy rabbits without lung injury. Emergency ventilation using accessible and inexpensive strategies like the FALCON may be useful for communities with low access to medical resources and as a backup form of emergency ventilation.

Reproductive and developmental safety of nirmatrelvir (PF-07321332), an oral SARS-CoV-2 Mpro inhibitor in animal models.

Nirmatrelvir (PF-07321332; NMV) the antiviral component of PAXLOVID™ is a potent and selective inhibitor of the SARS-CoV-2 main protease (Mpro), which plays a critical role in viral replication. PAXLOVID, comprised of nirmatrelvir and ritonavir (used as a pharmacokinetic enhancer), is an oral therapy currently in development as a therapeutic option for those infected with SARS-CoV-2 to prevent progression to severe disease, hospitalization, and death. PAXLOVID has been shown to be efficacious against hospitalization and death in two Phase 2/3 clinical studies that evaluated non hospitalized patients both with and without high risk factors for progression to severe illness. Given that males and females of reproductive age are included in the intended patient population, we assessed the potential effects of NMV up to the limit dose of 1000 mg/kg/day in ICH guideline embryo-fetal development studies in rats and rabbits, and a fertility and early embryonic development study in rats. There were no effects on male and female fertility or early embryonic development in rats, and no severe manifestations of developmental toxicity in rats or rabbits. The lack of adverse findings reported here in nonclinical species is consistent with the intended therapeutic target of NMV (a virus specific protein not present in mammalian cells), the favorable off-target selectivity profile, and lack of genetic toxicity. The results of these nonclinical studies with NMV along with existing ritonavir safety information indicate that there are no clinically relevant risks associated with PAXLOVID administration during pregnancy and in males and females of reproductive age.

The dynamic nature of the coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in differentiating airway epithelia.

Once inhaled, SARS-CoV-2 particles enter respiratory ciliated cells by interacting with angiotensin converting enzyme 2 (ACE2). Understanding the nature of ACE2 within airway tissue has become a recent focus particularly in light of the COVID-19 pandemic. Airway mucociliary tissue was generated in-vitro using primary human nasal epithelial cells and the air-liquid interface (ALI) model of differentiation. Using ALI tissue, three distinct transcript variants of ACE2 were identified. One transcript encodes the documented full-length ACE2 protein. The other two transcripts are unique truncated isoforms, that until recently had only been predicted to exist via sequence analysis software. Quantitative PCR revealed that all three transcript variants are expressed throughout differentiation of airway mucociliary epithelia. Immunofluorescence analysis of individual ACE2 protein isoforms exogenously expressed in cell-lines revealed similar abilities to localize in the plasma membrane and interact with the SARS CoV 2 spike receptor binding domain. Immunohistochemistry on differentiated ALI tissue using antibodies to either the N-term or C-term of ACE2 revealed both overlapping and distinct signals in cells, most notably only the ACE2 C-term antibody displayed plasma-membrane localization. We also demonstrate that ACE2 protein shedding is different in ALI Tissue compared to ACE2-transfected cell lines, and that ACE2 is released from both the apical and basal surfaces of ALI tissue. Together, our data highlights various facets of ACE2 transcripts and protein in airway mucociliary tissue that may represent variables which impact an individual's susceptibility to SARS-CoV-2 infection, or the severity of Covid-19.

First Evidence of Natural SARS-CoV-2 Infection in Domestic Rabbits.

We tested 144 pet rabbits sampled in France between November 2020 and June 2021 for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by microsphere immunoassay. We reported the first evidence of a natural SARS-CoV-2 infection in rabbits with a low observed seroprevalence between 0.7% and 1.4%.

Susceptibility of livestock to SARS-CoV-2 infection.

We report pilot studies to evaluate the susceptibility of common domestic livestock (cattle, sheep, goat, alpaca, rabbit, and horse) to intranasal infection with SARS-CoV-2. None of the infected animals shed infectious virus via nasal, oral, or faecal routes, although viral RNA was detected in several animals. Further, neutralizing antibody titres were low or non-existent one month following infection. These results suggest that domestic livestock are unlikely to contribute to SARS-CoV-2 epidemiology.

Rabbit Monoclonal Antibody Specifically Recognizing a Linear Epitope in the RBD of SARS-CoV-2 Spike Protein.

To date, SARS-CoV-2 pandemic has caused more than 188 million infections and 4.06 million deaths worldwide. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein has been regarded as an important target for vaccine and therapeutics development because it plays a key role in binding the human cell receptor ACE2 that is required for viral entry. However, it is not easy to detect RBD in Western blot using polyclonal antibody, suggesting that RBD may form a complicated conformation under native condition and bear rare linear epitope. So far, no linear epitope on RBD is reported. Thus, a monoclonal antibody (mAb) that recognizes linear epitope on RBD will become valuable. In the present study, an RBD-specific rabbit antibody named 9E1 was isolated from peripheral blood mononuclear cells (PBMC) of immunized rabbit by RBD-specific single B cell sorting and mapped to a highly conserved linear epitope within twelve amino acids 480CNGVEGFNCYFP491 on RBD. 9E1 works well in Western blot on S protein and immunohistochemistry on the SARS-CoV-2 infected tissue sections. The results demonstrated that 9E1 can be used as a useful tool for pathological and functional studies of SARS-CoV-2.

Recombinant myxoma virus infection associated with high mortality in rabbit farming (Oryctolagus cuniculus).

Myxomatosis is an emergent disease in the Iberian hare, having been considered a rabbit disease for decades. Genome sequencing of the strains obtained from Iberian hares with myxomatosis showed these to be distinct from the classical ones that circulated in rabbits since the virus introduction in Europe, in 1952. The main genomic difference in this natural recombinant hare myxoma virus (ha-MYXV) is the presence of an additional 2.8 kb region disrupting the M009L gene and adding a set of genes homologous to the myxoma virus (MYXV) genes M060R, M061R, M064R, M065R and M066R originated in Poxviruses. After the emergence of this recombinant virus (ha-MYXV) in hares, in the summer of 2019, the ha-MYXV was not detected in rabbit surveys, suggesting an apparent species segregation with the MYXV classic strains persistently circulating in rabbits. Recently, a group of six unvaccinated European rabbits (Oryctolagus cuniculus cuniculus) from a backyard rabbitry in South Portugal developed signs of myxomatosis (anorexia, dyspnoea, oedema of eyelids, head, ears, external genitals and anus, and skin myxomas in the base of the ears). Five of them died within 24-48 hr of symptom onset. Molecular analysis revealed that only the recombinant MYXV was present. This is the first documented report of a recombinant hare myxoma virus in farm rabbits associated with high mortality, which increases the concern for the future of both the Iberian hare and wild rabbits and questions the safety of the rabbit industry. This highlights the urgent need to evaluate the efficacy of available vaccines against this new MYXV.

A perspective on the ORS Spine Section initiative to develop a multi-species JOR Spine histopathology series.

This perspective summarizes the genesis, development, and potential future directions of the multispecies JOR Spine histopathology series.

Peridomestic Mammal Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Wild animals have been implicated as the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but it is largely unknown how the virus affects most wildlife species and if wildlife could ultimately serve as a reservoir for maintaining the virus outside the human population. We show that several common peridomestic species, including deer mice, bushy-tailed woodrats, and striped skunks, are susceptible to infection and can shed the virus in respiratory secretions. In contrast, we demonstrate that cottontail rabbits, fox squirrels, Wyoming ground squirrels, black-tailed prairie dogs, house mice, and racoons are not susceptible to SARS-CoV-2 infection. Our results expand the knowledge base of susceptible species and provide evidence that human-wildlife interactions could result in continued transmission of SARS-CoV-2.

Potent RBD-specific neutralizing rabbit monoclonal antibodies recognize emerging SARS-CoV-2 variants elicited by DNA prime-protein boost vaccination.

Global concerns arose as the emerged and rapidly spreading SARS-CoV-2 variants might escape host immunity induced by vaccination. In this study, a heterologous prime-boost immunization strategy for COVID-19 was designed to prime with a DNA vaccine encoding wild type (WT) spike protein receptor-binding domain (RBD) followed by S1 protein-based vaccine in rabbits. Four vaccine-elicited rabbit monoclonal antibodies (RmAbs), including 1H1, 9H1, 7G5, and 5E1, were isolated for biophysical property, neutralization potency and sequence analysis. All RmAbs recognized RBD or S1 protein with KD in the low nM or sub nM range. 1H1 and 9H1, but neither 7G5 nor 5E1, can bind to all RBD protein variants derived from B.1.351. All four RmAbs were able to neutralize wild type (WT) SARS-CoV-2 strain in pseudovirus assay, and 1H1 and 9H1 could neutralize the SARS-CoV-2 WT authentic virus with IC50 values of 0.136 and 0.026 µg/mL, respectively. Notably, 1H1 was able to neutralize all 6 emerging SARS-CoV-2 variants tested including D614G, B.1.1.7, B.1.429, P.1, B.1.526, and B.1.351 variants, and 5E1 could neutralize against the above 5 variants except P.1. Epitope binning analysis revealed that 9H1, 5E1 and 1H1 recognized distinct epitopes, while 9H1 and 7G5 may have overlapping but not identical epitope. In conclusion, DNA priming protein boost vaccination was an effective strategy to induce RmAbs with potent neutralization capability against not only SARS-CoV-2 WT strain but also emergent variants, which may provide a new avenue for effective therapeutics and point-of-care diagnostic measures.

Neutralizing Monoclonal Anti-SARS-CoV-2 Antibodies Isolated from Immunized Rabbits Define Novel Vulnerable Spike-Protein Epitope.

Monoclonal antibodies represent an important avenue for COVID-19 therapy and are routinely used for rapid and accessible diagnosis of SARS-CoV-2 infection. The recent emergence of SARS-CoV-2 genetic variants emphasized the need to enlarge the repertoire of antibodies that target diverse epitopes, the combination of which may improve immune-diagnostics, augment the efficiency of the immunotherapy and prevent selection of escape-mutants. Antigen-specific controlled immunization of experimental animals may elicit antibody repertoires that significantly differ from those generated in the context of the immune response mounted in the course of disease. Accordingly, rabbits were immunized by several recombinant antigens representing distinct domains of the viral spike protein and monoclonal antibodies were isolated from single cells obtained by cell sorting. Characterization of a panel of successfully isolated anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies demonstrated that they exhibit high specificity and affinity profiles. Anti-RBD antibodies revealing significant neutralizing potency against SARS-CoV-2 in vitro were found to target at least three distinct epitopes. Epitope mapping established that two of these antibodies recognized a novel epitope located on the surface of the RBD. We suggest that the antibodies isolated in this study are useful for designing SARS-CoV-2 diagnosis and therapy approaches.

A Potential Atypical Case of Rabbit Haemorrhagic Disease in a Dwarf Rabbit.

Rabbit haemorrhagic disease (RHD) is a highly contagious infectious disease of European wild and domestic rabbits. Rabbit haemorrhagic disease virus (RHDV, GI.1) emerged in 1986 in Europe, rapidly spreading all over the world. Several genotypes of RHDV have been recognised over time, but in 2010, a new virus (RHDV2/RHDVb, GI.2) emerged and progressively replaced the previous RHDV strains, due to the lack of cross-immunity conferred between RHDV and RHDV2. RHDV2 has a high mutation rate, similarly to the other calivirus and recombines with strains of RHDV and non-pathogenic calicivirus (GI.4), ensuring the continuous emergence of new field strains. Although this poses a threat to the already endangered European rabbit species, the available vaccines against RHDV2 and the compliance of biosafety measures seem to be controlling the infection in the rabbit industry Pet rabbits, especially when kept indoor, are considered at lower risk of infections, although RHDV2 and myxoma virus (MYXV) constitute a permanent threat due to transmission via insects. Vaccination against these viruses is therefore recommended every 6 months (myxomatosis) or annually (rabbit haemorrhagic disease). The combined immunization for myxomatosis and RHDV through a commercially available bivalent vaccine with RHDV antigen has been extensively used (Nobivac® Myxo-RHD, MSD, Kenilworth, NJ, USA). This vaccine however does not confer proper protection against the RHDV2, thus the need for a rabbit clinical vaccination protocol update. Here we report a clinical case of hepatitis and alteration of coagulation in a pet rabbit that had been vaccinated with the commercially available bivalent vaccine against RHDV and tested positive to RHDV2 after death. The animal developed a prolonged and atypical disease, compatible with RHD. The virus was identified to be an RHDV2 recombinant strain, with the structural backbone of RHDV2 (GI.2) and the non-structural genes of non-pathogenic-A1 strains (RCV-A1, GI.4). Although confirmation of the etiological agent was only made after death, the clinical signs and analytic data were very suggestive of RHD.