Maladaptation after a virus host switch leads to increased activation of the pro-inflammatory NF-κB pathway.

Myxoma virus (MYXV) causes localized cutaneous fibromas in its natural hosts, tapeti and brush rabbits; however, in the European rabbit, MYXV causes the lethal disease myxomatosis. Currently, the molecular mechanisms underlying this increased virulence after cross-species transmission are poorly understood. In this study, we investigated the interaction between MYXV M156 and the host protein kinase R (PKR) to determine their crosstalk with the proinflammatory nuclear factor kappa B (NF-κB) pathway. Our results demonstrated that MYXV M156 inhibits brush rabbit PKR (bPKR) more strongly than European rabbit PKR (ePKR). This moderate ePKR inhibition could be improved by hyperactive M156 mutants. We hypothesized that the moderate inhibition of ePKR by M156 might incompletely suppress the signal transduction pathways modulated by PKR, such as the NF-κB pathway. Therefore, we analyzed NF-κB pathway activation with a luciferase-based promoter assay. The moderate inhibition of ePKR resulted in significantly higher NF-κB­dependent reporter activity than complete inhibition of bPKR. We also found a stronger induction of the NF-κB target genes TNFα and IL-6 in ePKR-expressing cells than in bPKR-expressing cells in response to M156 in both transfection and infections assays. Furthermore, a hyperactive M156 mutant did not cause ePKR-dependent NF-κB activation. These observations indicate that M156 is maladapted for ePKR inhibition, only incompletely blocking translation in these hosts, resulting in preferential depletion of short­half-life proteins, such as the NF-κB inhibitor IκBα. We speculate that this functional activation of NF-κB induced by the intermediate inhibition of ePKR by M156 may contribute to the increased virulence of MYXV in European rabbits.

Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d strain: genome sequencing, in vivo virus replication kinetics, and viral dose effect.

BACKGROUND: Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d variant (GI.1d/RHDV) was identified in 1990 in France, and until the emergence of the new genotype GI.2, it was the main variant circulating in the country. The early stages of RHDV infection have been described in a few studies of rabbits experimentally infected with earlier strains, but no information was given on the minimum infective dose. We report the genomic and phenotypic characterisation of a GI.1d/RHDV strain collected in 2000 in France (GI.1d/00-21). RESULTS: We performed in vivo assays in rabbits to study virus replication kinetics in several tissues at the early stage of infection, and to estimate the minimum infective dose. Four tested doses, negligible (10- 1 viral genome copies), low (104), high (107) and very high (1011) were quantified using a method combining density gradient centrifugation of the viral particles and an RT-qPCR technique developed to quantify genomic RNA (gRNA). The GI.1d/00-21 genome showed the same genomic organisation as other lagoviruses; however, a substitution in the 5' untranslated region and a change in the potential p23/2C-like helicase cleavage site were observed. We showed that the liver of one of the two rabbits inoculated via the oral route was infected at 16 h post-infection and all tissues at 39 h post-infection. GI.1d/00-21 induced classical RHD signs (depression) and lesions (haemorrhage and splenomegaly). Although infective dose estimation should be interpreted with caution, the minimum infective dose that infected an inoculated rabbit was lower or equal to 104 gRNA copies, whereas between 104 and 107 gRNA copies were required to also induce mortality. CONCLUSIONS: These results provide a better understanding of GI.1d/RHDV infection in rabbits. The genome analysis showed a newly observed mutation in the 5' untranslated region of a lagovirus, whose role remains unknown. The phenotypic analysis showed that the pathogenicity of GI.1d/00-21 and the replication kinetics in infected organs were close to those reported for the original GI.1 strains, and could not alone explain the observed selective advantage of the GI.1d strains. Determining the minimum dose of viral particles required to cause mortality in rabbits is an important input for in vivo studies.

Shell Disorder Analysis Suggests That Pangolins Offered a Window for a Silent Spread of an Attenuated SARS-CoV-2 Precursor among Humans.

A model to predict the relative levels of respiratory and fecal-oral transmission potentials of coronaviruses (CoVs) by measuring the percentage of protein intrinsic disorder (PID) of the M (Membrane) and N (Nucleoprotein) proteins in their outer and inner shells, respectively, was built before the MERS-CoV outbreak. With MPID = 8.6% and NPID = 50.2%, the 2003 SARS-CoV falls into group B, which consists of CoVs with intermediate levels of both fecal-oral and respiratory transmission potentials. Further validation of the model came with MERS-CoV (MPID = 9%, NPID = 44%) and SARS-CoV-2 (MPID = 5.5%, NPID = 48%) falling into the groups C and B, respectively. Group C contains CoVs with higher fecal-oral but lower respiratory transmission potentials. Unlike SARS-CoV, SARS-CoV-2 with MPID = 5.5% has one of the hardest outer shells among CoVs. Because the hard shell is able to resist the antimicrobial enzymes in body fluids, the infected person is able to shed large quantities of viral particles via saliva and mucus, which could account for the higher contagiousness of SARS-COV-2. Further searches have found that high rigidity of the outer shell is characteristic for the CoVs of burrowing animals, such as rabbits (MPID = 5.6%) and pangolins (MPID = 5-6%), which are in contact with the buried feces. A closer inspection of pangolin-CoVs from 2017 to 2019 reveals that pangolins provided a unique window of opportunity for the entry of an attenuated SARS-CoV-2 precursor into the human population in 2017 or earlier, with the subsequent slow and silent spread as a mild cold that followed by its mutations into the current more virulent form. Evidence of this lies in both the genetic proximity of the pangolin-CoVs to SARS-CoV-2 (∼90%) and differences in N disorder. A 2017 pangolin-CoV strain shows evidence of higher levels of attenuation and higher fecal-oral transmission associated with lower human infectivity via having lower NPID (44.8%). Our shell disorder model predicts this to be a SARS-CoV-2 vaccine strain, as lower inner shell disorder is associated with the lesser virulence in a variety of viruses.

Fecal, oral, blood and skin virome of laboratory rabbits.

Here, we investigated the fecal, oral, blood, and skin virome of 10 laboratory rabbits using a viral metagenomic method. In the oral samples, we detected a novel polyomavirus (RabPyV), and phylogenetic analysis based on the large T antigen, VP1 and VP2 regions indicated that the novel strain might have undergone a recombination event. Recombination analysis based on related genomes confirmed that RabPyV is a multiple recombinant between rodent-like and avian-like polyomaviruses. In fecal samples, three partial or complete genome sequences of viruses belonging to the families Picobirnaviridae, Parvoviridae, Microviridae and Coronaviridae were characterized, and phylogenetic trees were constructed based on the predicted amino acid sequences of viral proteins. This study increases the amount of genetic information on viruses present in laboratory rabbits.

Next step in the ongoing arms race between myxoma virus and wild rabbits in Australia is a novel disease phenotype.

In host-pathogen arms races, increases in host resistance prompt counteradaptation by pathogens, but the nature of that counteradaptation is seldom directly observed outside of laboratory models. The best-documented field example is the coevolution of myxoma virus (MYXV) in European rabbits. To understand how MYXV in Australia has continued to evolve in wild rabbits under intense selection for genetic resistance to myxomatosis, we compared the phenotypes of the progenitor MYXV and viral isolates from the 1950s and the 1990s in laboratory rabbits with no resistance. Strikingly, and unlike their 1950s counterparts, most virus isolates from the 1990s induced a highly lethal immune collapse syndrome similar to septic shock. Thus, the next step in this canonical case of coevolution after a species jump has been further escalation by the virus in the face of widespread host resistance.

GI.1b/GI.1b/GI.2 recombinant rabbit hemorrhagic disease virus 2 (Lagovirus europaeus/GI.2) in Morocco, Africa.

Rabbit hemorrhagic disease virus (RHDV) is highly lethal to the European rabbit (Oryctolagus cuniculus). It was first reported in 1984 in China, but in 2010, a new variant of the virus was detected (GI.2) in France. Several recombination events with pathogenic and non-pathogenic strains have been described. Here, we report the first sequences of RHDV in Africa, isolated from Moroccan rabbits, and these resemble GI.1b/GI.1b/GI.2 recombinants found in the Iberian Peninsula. Monitoring and characterization of strains from future outbreaks are advised to guarantee the success of current programs on small-rabbit production for poverty alleviation in African countries.

Genetic characterization and phylogenetic analysis of rabbit hemorrhagic disease virus isolated in Tunisia from 2015 to 2018.

Two distinct genotypes responsible for rabbit hemorrhagic disease (RHD) are reported, GI.1 (RHDV) and GI.2 (RHDV2). Vaccines based on these two genotypes are only partially cross-protective. Hence, knowing which genotype is circulating is important for appropriate control measures. We have investigated 25 field samples isolated between 2015 and 2018 from rabbits with clinical signs of RHD. Only GI.2 (RHDV2) is currently circulating in Tunisia. All Tunisian samples were grouped together with typical genotypic and phenotypic mutations. Therefore, we recommend initiating an extensive preventive vaccination program based on GI.2 vaccines in addition to a regular monitoring of the circulating lagoviruses.

Detection and Circulation of a Novel Rabbit Hemorrhagic Disease Virus in Australia.

The highly virulent rabbit hemorrhagic disease virus (RHDV) has been widely used in Australia and New Zealand since the mid-1990s to control wild rabbits, an invasive vertebrate pest in these countries. In January 2014, an exotic RHDV was detected in Australia, and 8 additional outbreaks were reported in both domestic and wild rabbits in the 15 months following its detection. Full-length genomic analysis revealed that this virus is a recombinant containing an RHDVa capsid gene and nonstructural genes most closely related to nonpathogenic rabbit caliciviruses. Nationwide monitoring efforts need to be expanded to assess if the increasing number of different RHDV variants circulating in the Australian environment will affect biological control of rabbits. At the same time, updated vaccines and vaccination protocols are urgently needed to protect pet and farmed rabbits from these novel rabbit caliciviruses.

Cottontail rabbits shed clade 2.3.4.4 H5 highly pathogenic avian influenza A viruses.

During 2014-2015, clade 2.3.4.4 H5Nx highly pathogenic (HP) avian influenza A viruses (IAV) were first detected in North America and subsequently caused one of the largest agricultural emergencies in U.S. HISTORY: Recent evidence has suggested that cottontail rabbits can shed multiple IAV subtypes. We experimentally infected cottontail rabbits with three HP H5Nx IAVs. All rabbits tested shed virus on at least one day by at least one route. Cottontail rabbits appear to be an exception to the limited capacity for replication that has been previously reported for certain other mammalian species inoculated with clade 2.3.4.4 HP H5Nx avian influenza A viruses.

Rapid detection of three rabbit pathogens by use of the Luminex x-TAG assay.

BACKGROUND: Domestic rabbits especially New Zealand white rabbits play an important role in biological research. The disease surveillance and quality control are essential to guarantee the results of animal experiments performed on rabbits. Rabbit hemorrhagic disease virus, rabbit rotavirus and Sendai virus are the important pathogens that needed to be eliminated. Rapid and sensitive method focus on these three viruses should be established for routine monitoring. The Luminex x-TAG assay based on multiplex PCR and fluorescent microsphere is a fast developing technology applied in high throughput detection. Specific primers modified with oligonucleotide sequence/biotin were used to amplify target fragments. The conjugation between oligonucleotide sequence of the PCR products and the MagPlex-TAG microspheres was specific without any cross-reaction, and the hybridization products could be analyzed using the Luminex 200 analyzer instrument. Recombinant plasmids were constructed to estimate the detection limit of the viruses. Furthermore, 40 clinical samples were used to evaluate the efficiency of this multiplex PCR based Luminex x-TAG assay. RESULTS: According to the results, this new method showed high specificity and good stability. Assessed by the recombinant plasmids, the detection limit of three viruses was 100copies/µl. Among 40 clinical specimens, 18 specimens were found positive, which was completely concordant with the conventional PCR method. CONCLUSIONS: The new developed Luminex x-TAG assay is an accurate, high throughput method for rapid detection of three important viruses of rabbits.

Detection of rabbit haemorrhagic disease virus 2 (GI.2) in Poland.

In this paper we present the first cases of rabbit haemorrhagic disease virus 2 (RHDV2 - GI.2) in Poland. The virus was detected in liver samples of RHD-suspected rabbits from Lodzkie and west Pomeranian voivodeships. In both cases, the typical clinical symptoms of the disease were observed despite the fact that the rabbits were previously vaccinated against RHD. In order to extend the analysis of the RHDV2 strain infecting the rabbits, the entire VP60 and NSP genes were amplified and sequenced. The results of rRT-PCR assay have shown that tested RHDV samples were positive for the presence of RHDV2. In the phylogenetic analysis of vp60gene the first Polish RHDV isolates (RED 2016 and VMS 2017) clustered together with the reference RHDV2, meaning they represent new evolutionary RHDV linkeages. The first Polish RHDV2 isolates showed about 97% nucleotide sequence identity with the reference RHDV2 strains and approximately 18% difference from classic RHDV and RHDVa variants.

Benign Rabbit Calicivirus in New Zealand.

The Czech v351 strain of rabbit hemorrhagic disease virus (RHDV1) is used in Australia and New Zealand as a biological control agent for rabbits, which are important and damaging introduced vertebrate pests in these countries. However, nonpathogenic rabbit caliciviruses (RCVs) can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with effective rabbit biocontrol. Antibodies that cross-reacted against RHDV antigens were found in wild rabbits before the release of RHDV1 in New Zealand in 1997, suggesting that nonpathogenic RCVs were already present in New Zealand. The aim of this study was to confirm the presence of nonpathogenic RCV in New Zealand and describe its geographical distribution. RCV and RHDV antibody assays were used to screen serum samples from 350 wild rabbits from 14 locations in New Zealand. The serological survey indicated that both RCV and RHDV are widespread in New Zealand wild rabbits, with antibodies detected in 10 out of 14 and 12 out of 14 populations, respectively. Two closely related RCV strains were identified in the duodenal tissue from a New Zealand wild rabbit (RCV Gore-425A and RCV Gore-425B). Both variants are most closely related to Australian RCV strains, but with 88% nucleotide identity, they are genetically distinct. Phylogenetic analysis revealed that the New Zealand RCV strains fall within the genetic diversity of the Australian RCV isolates, indicating a relatively recent movement of RCVs between Australia and New Zealand.IMPORTANCE Wild rabbits are important and damaging introduced vertebrate pests in Australia and New Zealand. Although RHDV1 is used as a biological control agent, some nonpathogenic RCVs can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with its effectiveness for rabbit control. The presence of nonpathogenic RCVs in New Zealand wild rabbits has been long hypothesized, but earlier attempts to isolate a New Zealand RCV strain have been unsuccessful. Therefore, it is important to determine if such nonpathogenic viruses exist in New Zealand rabbits, especially considering the proposed introduction of new RHDV strains into New Zealand as biocontrols.

Prevalence and genetic features of rabbit hepatitis E virus in Korea.

Hepatitis caused by hepatitis E virus (HEV) is a public health concern worldwide. HEV strains have been isolated from several animal species, some of which induce zoonosis. Recently, the isolation of HEV from rabbits was reported. Here, the partial capsid gene (320 bp) of HEV was detected in rabbit feces via reverse transcriptase-polymerase chain reaction (RT-PCR). Rabbit HEV was found in two of six rabbit farms and 17 of 264 rabbit fecal samples (6.4%). A phylogenetic analysis of the partial capsid gene classified the 17 HEV isolates into the putative rabbit HEV clade. A full genomic sequence, KOR-Rb-1, was obtained from one rabbit HEV isolate by 5' and 3' rapid amplification of cDNA ends-PCR and RT-PCR, and comprised 7275 bp excluding the 3' poly(A) tail. It shared 77.5-86.8%, 86.6%, and 80.2-84.3% nucleotide identities with rabbit HEV isolates from China, the US, and France, respectively. It also shared 72.3-73.0%, 71.4%, 76.7-78.3%, 72.8-73.3%, and 47.1-47.2% nucleotide identities with representative strains of HEV-1, HEV-2, HEV-3, HEV-4, and avian HEV, respectively. A full-genome phylogenetic analysis classified KOR-Rb-1 into the provisional rabbit HEV clade. This isolate could be used to study the pathogenesis and zoonotic potential of rabbit HEV.

Resistance to RHD virus in wild Australian rabbits: Comparison of susceptible and resistant individuals using a genomewide approach.

Deciphering the genes involved in disease resistance is essential if we are to understand host-pathogen coevolutionary processes. The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus). During the first outbreaks of the disease, RHDV caused mortality rates of up to 97%. Recently, however, increased genetic resistance to RHDV has been reported. Here, we have aimed to identify genomic differences between rabbits that survived a natural infection with RHDV and those that died in the field using a genomewide next-generation sequencing (NGS) approach. We detected 72 SNPs corresponding to 133 genes associated with survival of a RHD infection. Most of the identified genes have known functions in virus infections and replication, immune responses or apoptosis, or have previously been found to be regulated during RHD. Some of the genes identified in experimental studies, however, did not seem to play a role under natural selection regimes, highlighting the importance of field studies to complement the genomic background of wildlife diseases. Our study provides a set of candidate markers as a tool for the future scanning of wild rabbits for their resistance to RHDV. This is important both for wild rabbit populations in southern Europe where RHD is regarded as a serious problem decimating the prey of endangered predator species and for assessing the success of currently planned RHDV variant biocontrol releases in Australia.

Low viral doses are sufficient to infect cottontail rabbits with avian influenza A virus.

Influenza A viruses (IAVs) have been reported in wild lagomorphs in environments where they share resources with waterfowl. Recent studies have conclusively shown that a North American lagomorph, cottontail rabbits (Sylvilagus sp.), become infected following exposure to IAVs and can shed significant quantities of virus. However, the minimum infectious dose and the efficiency of various routes of infection have not been evaluated. Thirty-six cottontail rabbits were used in a dose response study assessing both the oral and nasal routes of infection. The nasal route of infection proved to be the most efficient, as all cottontail rabbits shed viral RNA following inoculation with doses as low as 102 EID50. The oral route of infection was less efficient, but still produced infection rates of ≥ 50% at relatively low doses (i.e., 103 and 104 EID50). These results suggest that cottontail rabbits are highly susceptible to IAVs at low exposure doses that have been routinely observed in environments contaminated by waterfowl. Furthermore, this study supports earlier observations that cottontail rabbits may pose a biosecurity risk to poultry operations, as a virus-contaminated water source or contaminated environment, even at low viral titers, could be sufficient to initiate viral replication in cottontail rabbits.

An update on the rabbit hemorrhagic disease virus (RHDV) strains circulating in Portugal in the 1990s: earliest detection of G3-G5 and G6.

Rabbit hemorrhagic disease virus (RHDV) causes devastating effects on European rabbit (Oryctolagus cuniculus) populations in the Iberian Peninsula. According to the information available, only genogroup 1 strains were circulating in Iberian wild rabbits until 2011; the antigenic variant G6 has been sporadically detected in rabbitries since 2007. Here, we show for the first time that G3-G5 strains were already present in mainland Portugal in 1998 and that G6 has been circulating since at least 1999. Moreover, we report a G3-G5 strain from the Azores collected in 1998, which is the likely ancestor of Azorean G3-G5like strains. These observations improve the current knowledge on RHDV epidemiology in the Iberian Peninsula and the Azores.

Detection of rabbit Haemorrhagic disease virus 2 during the wild rabbit (Oryctolagus cuniculus) eradication from the Berlengas archipelago, Portugal.

BACKGROUND: In the regular wildlife monitoring action carried out in the summer of the past few years at the Berlenga Island, wild rabbits (Oryctolagus cuniculus) have been repeatedly found dead. However, the origin of those deaths was never investigated. Our aim was to investigate the cause of death of 11 rabbits collected between April and May 2016. RESULTS: While screening samples from rabbit carcasses for the major viral rabbit pathogens, five tested positive to RHDV2 but all were negative for RHDV and myxoma virus (MYXV). For six RHDV2-negative specimens, emaciation and parasitism were considered the most probable cause of death. Lesions identified in the RHDV2-positive rabbits included non-suppurative diffuse hepatic necrosis and pulmonary lesions varying from congestion and oedema of the lungs to interstitial pneumonia. Sequencing analysis of the vp60 gene obtained from two specimens showed identical vp60 sequences. Comparison with other known RHDV2 strains from public databases through BLAST analysis revealed a closer similarity with strains from Alentejo collected during 2013. Maximum Likelihood and Bayesian phylogenetic analysis showed that the 2016 strains from the archipelago have a higher resemblance with a group of strains mostly collected in the South of Portugal between 2013 and 2014. CONCLUSION: The results suggest that RHDV2 may have been introduced on the Berlenga Island a few years ago, having evolved separately from mainland strains due to insularity.

Multicausal etiology of the enteric syndrome in rabbits from Mexico.

Enteropathies in rabbits are difficult to diagnose; their etiology involves pathogens that act synergistically, causing damage to the intestine. The aim of the present study was isolate enteric pathogens from rabbits in Mexico. Using parasitological, bacteriological and molecular analyses, we screened 58 samples of the intestinal content of rabbits having a clinical history of enteric disease from the southeastern part of the State of Mexico. Out of the 58 samples analyzed, a total of 86 identifications were made, Eimeria spp. were found in 77.5%, followed by Aeromonas spp. in 15.5% and Escherichia coli in 8.6%, which were identified as enteropathogenic E. coli (EPEC), and the presence of the following agents was also confirmed: Salmonella spp., Klebsiella spp., Streptococcus spp., Staphylococcus aureus, Enterococcus spp., Mannheimia spp. and Rotavirus. The concurrent presence of Eimeria spp. with Aeromonas was frequent (15.5%); there was statistical significance for the presence of an association between the clinical profiles and Eimeria spp. (p=0.000), Mannheimia spp. (p=0.001), Salmonella spp., Klebsiella spp., Streptococcus spp. and Enterococcus spp. (p=0.006).

Serological survey of hepatitis E virus infection in farmed and pet rabbits in Italy.

The recent identification in rabbits of hepatitis E viruses (HEV) related to viruses infecting humans raises the question of the role of this species as possible HEV reservoir. A serological survey on rabbit HEV infection was conducted in Italy during 2013-2014, including both farmed and pet rabbits. We found an anti-HEV antibody seroprevalence of 3.40 % in 206 farmed rabbits (collected on 7 farms) and 6.56 % in 122 pets. RNA was extracted from IgG-positive sera and analyzed by HEV-specific real-time RT-PCR. None of the samples were positive, confirming that no viremia was present in the presence of IgG. Only one serum sample from a farmed rabbit was positive for IgM, but no HEV RNA was detected in it. Pet rabbit feces were also tested for HEV RNA, with negative results. This finding suggests that HEV is circulating in rabbits in Italy.

Comparative analysis of rabbit hemorrhagic disease virus strains originating from outbreaks in the Russian Federation.

Since the first introduction of rabbit hemorrhagic disease (RHD) in 1986, disease outbreaks have been continuously reported in different regions of Russia. Despite extensive vaccination, sporadic RHD cases are still reported. Here, we examine eleven RHDV strains originating from disease outbreaks occurring between 2003 and 2012 and one widely used vaccine strain. Notable phenotypic and genetic heterogeneity among RHDV strains was observed. The RHDV strains Tambov-2010, Perm-2010, Manihino-09 showed different hemagglutinating activity (HA) at 4 °C and room temperature. While all RHDV field strains were identified as hemagglutinating virulent viruses of the RHDVa variant, the vaccine strain was assigned as a "classical" RHDV. These data indicate that since 2003, RHDVa has become the predominant variant circulating in Russia.