Evolutionary study of leporid CD4 reveals a hotspot of genetic variability within the D2 domain.

CD4 is the major receptor on T helper cells involved in the uptake of human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) into their host cells. Evolutionary studies of CD4 in primates revealed signatures of positive selection in the D1 domain that interacts with primate exogenous lentivirus gp120 proteins. Here, we studied the evolution of CD4 in lagomorphs by comparing sequences obtained for the genera Oryctolagus, Sylvilagus, Lepus, and Ochotona. Our results reveal an overall higher divergence in lagomorphs compared to primates with highest divergence in the D2 domain. A detailed analysis of a small fragment of 33 nucleotides coding for amino acids 169 to 179 in the D2 domain showed dramatic amino acid alterations with a dN/dS value of 3.2 for lagomorphs, suggesting that CD4 is under strong positive selection in this particular region. Within each leporid genus, no significant amino acid changes were observed for the D2 domain which indicates that the genetic differentiation occurred in the ancestor of each genus before the species radiation. The rabbit endogenous lentivirus type K (RELIK) found in leporids shares high structural similarity with HIV which suggests a possible interaction between RELIK and CD4. The presence of RELIK in the studied leporids, the high structural similarity to modern-day exogenous lentiviruses and the absence of exogenous lentiviruses in leporids, allows us to hypothesize that this endogenous retrovirus, that was most probably exogenous in the past, drove the divergent evolution of leporid CD4.

The IgA of hares (Lepus sp.) and rabbit confirms that the leporids IgA explosion is old and reveals a new case of trans-species polymorphism.

Background: Immunoglobulin A (IgA) is the mammalian mucosal antibody, providing an important line of defense against pathogens. With 15 IgA subclasses, the European rabbit has an extremely complex IgA system, strikingly more complex than most other mammals, which have only one IgA or, in the case of hominoids, two IgA subclasses. Similar to the two hominoid primate IGHA genes, the expansion of the rabbit IGHA genes appears to have begun in an ancestral lagomorph since multiple IgA copies were found by Southern blot analysis for the genera Sylvilagus, Lepus, and Ochotona. Results: To gain a better insight into the extraordinary lagomorph IgA evolution, we sequenced, for the first time, expressed IgA genes for two Lepus species, L. europaeus and L. granatensis. These were aligned with the 15 rabbit IgA isotypes, and evolutionary analyses were conducted. The obtained phylogenetic tree shows that the Lepus IgA sequences cluster with and among the rabbit IgA isotypes, and the interspecies and intraspecies nucleotide genetic distances are similar. A comparison of the amino acid sequences of the Lepus and rabbit IgA confirms that there are two trans-species polymorphisms and that the rabbit and Lepus sequences share a common genetic pool. In fact, the main differences between the studied leporids IgAs reside in the characteristics of the hinge region. Conclusion: The Lepus IgA sequences we have obtained strongly suggest that the great expansion of the leporid IGHA genes occurred in a common ancestral species and was then maintained in the descendants. A strong selective pressure caused the extraordinary expansion of the IGHA genes but then subsided, leading to the maintenance of the acquired polymorphisms in the descendants, with little subsequent divergence. This is a unique evolutionary pattern in which an ancient gene expansion has been maintained for approximately 18 million years.

Identification of a Novel Myxoma Virus C7-Like Host Range Factor That Enabled a Species Leap from Rabbits to Hares.

Myxoma virus (MYXV) is naturally found in rabbit Sylvilagus species and is known to cause lethal myxomatosis in European rabbits (Oryctolagus cuniculus). In 2019, an MYXV strain (MYXV strain Toledo [MYXV-Tol]) causing myxomatosis-like disease in Iberian hares (Lepus granatensis) was identified. MYXV-Tol acquired a recombinant region of ∼2.8 kb harboring several new genes, including a novel host range gene (M159) that we show to be an orthologous member of the vaccinia virus C7 host range family. Here, to test whether M159 alone has enabled MYXV to alter its host range to Iberian hares, several recombinant viruses were generated, including an MYXV-Tol ΔM159 (knockout) strain. While MYXV-Tol underwent fully productive infection in hare HN-R cells, neither the wild-type MYXV-Lau strain (lacking M159) nor vMyxTol-ΔM159 (deleted for M159) was able to infect and replicate, showing that the ability of MYXV-Tol to infect these cells and replicate depends on the presence of M159. Similar to other C7L family members, M159 was shown to be expressed as an early/late gene but was translocated into the nucleus at later time points, indicating that further studies are needed to elucidate its role in the nucleus. Finally, in rabbit cells, the M159 protein did not contribute to increased replication but was able to upregulate the replication levels of MYXV in nonpermissive and semipermissive human cancer cells, suggesting that the M159-targeted pathway is conserved across mammalian species. Altogether, these observations demonstrate that the M159 protein plays a critical role in determining the host specificity of MYXV-Tol in hare and human cells by imparting new host range functions. IMPORTANCE The coevolution of European rabbit populations and MYXV is a textbook example of an arms race between a pathogen and a host. Recently, a recombinant MYXV (MYXV-Tol) crossed the species barrier by jumping from leporid species to another species, causing lethal myxomatosis-like disease. Given the highly pathogenic nature of this new virus in hares and the incidences of other poxvirus cross-species spillovers into other animals, including humans, it is important to understand how and why MYXV-Tol was able to become virulent in a new host species. The results presented clearly demonstrate that M159 is the key factor allowing MYXV-Tol replication in hare cells by imparting new host range functions. These results have the potential to improve current knowledge about the virulence of poxviruses and provide a platform to better understand the new MYXV-Tol, rendering the virus capable of leaping into a new host species.

Sex and Age-Specific Hematology and Biochemistry Reference Intervals of Live Iberian Hares (Lepus granatensis) and Comparison with Postmortem Sampling.

Hematology and serum biochemistry reference intervals were estimated for the Iberian hare (Lepus granatensis). Most parameters differed significantly between hunted and livetrapped Iberian hares. Significant differences were found for sex (red blood cell count, mean corpuscular volume, total protein, albumin, uric acid, triglycerides, cholesterol, chloride) and age classes (red blood cell count, hematocrit, mean corpuscular hemoglobin, glucose, calcium, and sodium). Sex- and age-specific reference intervals were estimated for these parameters. Red blood cell count, hemoglobin concentration, mean corpuscular hemoglobin, urea, and potassium show seasonal variations, with the lowest values in summer and the highest in winter. Creatinine, calcium, sodium, and phosphorus achieve their highest values in summer and stable baseline values throughout the rest of the year. These reference intervals can be used as baseline to monitor health, physiology, ecology, and nutrition of Iberian hare populations.

An Annotated Draft Genome of the Mountain Hare (Lepus timidus).

Hares (genus Lepus) provide clear examples of repeated and often massive introgressive hybridization and striking local adaptations. Genomic studies on this group have so far relied on comparisons to the European rabbit (Oryctolagus cuniculus) reference genome. Here, we report the first de novo draft reference genome for a hare species, the mountain hare (Lepus timidus), and evaluate the efficacy of whole-genome re-sequencing analyses using the new reference versus using the rabbit reference genome. The genome was assembled using the ALLPATHS-LG protocol with a combination of overlapping pair and mate-pair Illumina sequencing (77x coverage). The assembly contained 32,294 scaffolds with a total length of 2.7 Gb and a scaffold N50 of 3.4 Mb. Re-scaffolding based on the rabbit reference reduced the total number of scaffolds to 4,205 with a scaffold N50 of 194 Mb. A correspondence was found between 22 of these hare scaffolds and the rabbit chromosomes, based on gene content and direct alignment. We annotated 24,578 protein coding genes by combining ab-initio predictions, homology search, and transcriptome data, of which 683 were solely derived from hare-specific transcriptome data. The hare reference genome is therefore a new resource to discover and investigate hare-specific variation. Similar estimates of heterozygosity and inferred demographic history profiles were obtained when mapping hare whole-genome re-sequencing data to the new hare draft genome or to alternative references based on the rabbit genome. Our results validate previous reference-based strategies and suggest that the chromosome-scale hare draft genome should enable chromosome-wide analyses and genome scans on hares.

Evolutionary studies on the betaretrovirus RERV-H in the Leporidae family reveal an endogenization in the ancestor of Oryctolagus, Bunolagus and Pentalagus at 9 million years ago.

RERV-H was first identified in human tissues and mistaken for a human exogenous retrovirus. However, the integration sites carried by this virus showed that it was instead a European rabbit (Oryctolagus cuniculus) endogenous retrovirus. The first clones retrieved from European rabbit samples represented defective proviruses, although estimation of proviral copy numbers found in the European rabbit genome ranged from hundreds to thousands. Screening for the presence of RERV-H showed the absence of the virus in two other lagomorphs, pika (Ochotona) and hares (Lepus), which diverged from rabbits about 35 and 12 million years ago, respectively. Using a PCR-based approach, samples of seven different Lagomorph genera were tested for the presence of RERV-H. It was possible to amplify a proviral fragment corresponding to RNaseH from Oryctolagus, Bunolagus and Pentalagus genomic samples. The amplification of proviral DNA in species other than Oryctolagus revealed that this virus was endogenized in their common ancestor, roughly 9 million years ago. Using the European rabbit genome sequence OryCun2.0, it was possible to find multiple copies spread throughout the genome and several complete proviral genomes were retrieved. Some copies contained full open reading frames for all viral components. The lack of a complete genome in the other Lagomorph species did not allow further analyses of the provirus, although more deleterious mutations were found in Bunolagus and Pentalagus than in Oryctolagus RNaseH-amplified sequences. To what extent RERV-H and other endogenous viruses might have had an impact on the rabbit genome and its immune system remains elusive.

Sequencing of VDJ genes in Lepus americanus confirms a correlation between VHn expression and the leporid species continent of origin.

Leporid VH genes used in the generation of their primary antibody repertoire exhibit highly divergent lineages. For the European rabbit (Oryctolagus cuniculus) four VHa lineages have been described, the a1, a2, a3 and a4. Hares (Lepus spp.) and cottontail (Sylvilagus floridanus) express one VHa lineage each, the a2L and the a5, respectively, along with a more ancient lineage, the Lepus spp. sL and S. floridanus sS. Both the European rabbit and the Lepus europaeus use a third lineage, VHn, in a low proportion of their VDJ rearrangements. The VHn genes are a conserved ancestral polymorphism that is being maintained in the leporid genome.Their usage in a low proportion of VDJ rearrangements by both European rabbit and L. europaeus but not S. floridanus has been argued to be a remnant of an ancient European leporid immunologic response to pathogens. To address this hypothesis, in this study we sequenced VDJ rearranged genes for another North American leporid, L. americanus. Our results show that L. americanus expressed these genes less frequently and in a highly modified fashion compared to the European Lepus species. Our results suggest that the American leporid species use a different VH repertoire than the European species which may be related with an immune adaptation to different environmental conditions, such as different pathogenic agents.

Genetic Diversity of IGHM and IGHE in the Leporids Revealed Different Patterns of Diversity in the Two European Rabbit Subspecies (O. cuniculus algirus and O. c. cuniculus).

The European rabbit (Oryctolagus cuniculus) has been an important model for immunological studies but the study of its immunoglobulins (Ig) has been restricted to its unique IgA and IgG. Here, we studied the genetic diversity of IgM and IgE in several species of leporids and performed population genetics studies on European rabbit wild populations and domestic breeds. The leporids sequencing showed that these Ig are well conserved (98% sequence similarity among leporids), For IgM the Cµ1 and Cµ4 were the most diverse and most conserved domains, respectively, while for IgE the Cε1 was the most diverse domain and Cε2 and Cε3 the most conserved domains. The differences in the pattern of most conserved and most diverse domain between the Ig isotypes are most likely related to each isotype function. The genetic population data showed contrasting results for IgM and IgE. For both Ig, as expected, a greater diversity was observed in the original species range, the Iberian Peninsula. However, unexpectedly the genetic diversity found for IgE in the domestic animals is higher than that for the French wild populations. These results will increase knowledge of the genetic diversity of leporids and wild and domestic rabbit populations and are important tools for the management of wild populations and rabbitries.

Development and validation of a real time PCR for the detection of myxoma virus based on the diploid gene M000.5L/R.

The myxoma virus (MYXV) causes severe infections in European rabbits that may reach mortality rates up to 100% depending on the viral strain. The typical symptoms and lesions induced by the virus are usually enough to permit the correct clinical diagnosis. However, in peracute forms the infection may be accompanied by unspecific symptoms. Sudden death may also occur without evident clinical signs of myxomatosis. Likewise, a clinical diagnosis of atypical forms of myxomatosis (amyxomatous) is often complicated and delayed due to the scarceness of skin lesions. As the disease control often depends on an early and unequivocal diagnosis of MYXV, laboratorial methods play a relevant role in the confirmation of MYXV infection. This study describes the development and validation of a novel, high accurate real time polymerase chain reaction assay (rtPCR) for the detection of MYXV. Primers were designed to amplify a 125-bp within the gene M000.5L/R, which is duplicated in the termini of the genome and is unique among Leporipoxvirus. The assay was negative for SFV and other poxviruses and was able to detect 2.6 copies of MYXV DNA proving the effectiveness, specificity and sensitivity of this diagnosis tool. The rtPCR has been applied successfully in INIAV laboratory for routine diagnosis of myxomatosis since 2005.

Systemic and neurotoxic effects of epidural meloxicam in rabbits / Efeitos sistêmicos e neurotóxicos do meloxicam por via epidural em coelhos

ABSTRACT: The aim of this study was to assess systemic and neurotoxic changes following an epidural administration of meloxicamin to rabbits. Twelve adult rabbits four males and eight females; average mass, 1.9 ± 0.1kg were randomly divided into two groups: a control group (GC), which received a single dose of 0.9% NaCl epidurally in a volume of 0.3mL kg-1and a meloxicam group (GM), which received 0.2mg kg-1 meloxicam epidurally along with 0.9% NaCl in a total volume of 0.3mL kg-1. Heart rate, respiratory rate, body temperature, and neurological abnormalities were assessed prior to administration of anesthesia (H0), 1, 2, 3, 6, 12, and 24h following epidural puncture (H1, H2, H3, H6, H12, and H24, respectively), and every 24h afterward for 10 days after epidural puncture (D2, D3, D4, D5, D6, D7, D8, D9, and D10). The surface temperature of lumbosacral region was also measured at H0, H1, H6, H12, H24, D5 and D10. Three animals from each group were euthanized on days 15 and 30 after epidural puncture to assess possible spinal injuries. Variances observed in physiological parameters were not suggestive of adverse effects of meloxicam, as all were within the reference standards, and there were no physical or behavioral changes observed. Neurological function was similar between groups, with only difference between baseline values and values 1h after epidural administration in both groups. There were no histopathological changes in the GM group, and only one animal showed discrete lymphocytic infiltrate. Epidural lumbosacral administration of meloxicam at a dose of 0.2mg kg-1 caused no significant systemic or neurotoxic effects in rabbits.

RESUMO: O objetivo desse estudo foi avaliar as alterações sistêmicas e neurotóxicas promovidas pelo meloxicam, administrado por via epidural, em coelhos. Foram utilizados 12 coelhos adultos, quatro machos e oito fêmeas, pensando em média 1,9 ± 0,1kg. Os animais foram divididos equitativa e aleatoriamente em dois grupos, os quais receberam dose única de solução de NaCl 0,9% no volume de 0,3mL kg-1, por via epidural (grupo controle - GC) ou meloxicam (0,2mg kg-1) associado à solução de NaCl 0,9%, compondo um volume total de 0,3mL kg-1 (grupo meloxicam - GM). Avaliaram-se frequências cardíaca e respiratória, temperatura corporal e alterações neurológicas, antes da administração da anestesia (H0), uma, duas, três, seis, 12 e 24 horas após a punção epidural (H1, H2, H3, H6, H12 e H24, respectivamente) e a cada 24 horas após o H24, até o 10º dia após a punção epidural (D2, D3, D4, D5, D6, D7, D8, D9 e D10). Mensurou-se ainda a temperatura superficial da região lombossacra em H0, H1, H6, H12, H24, D5 e D10. Realizou-se eutanásia em três animais de cada grupo no 15o e no 30o dia após o início do experimento, para avaliação das possíveis lesões medulares. As variâncias observadas nos parâmetros fisiológicos não foram sugestivas de efeito adverso do meloxicam, pois estiveram dentro do padrão de referência e não houve alterações físicas ou comportamentais. O exame neurológico se mostrou semelhante entre os grupos, havendo diferença apenas entre a avaliação inicial e uma hora após a epidural em ambos os grupos. Na histopatologia não houve alterações no GM e apenas um animal do GC apresentou discreto infiltrado linfoplasmocitário. A administração epidural lombossacra de meloxicam, na dose de 0,2mg kg-1, não causa efeitos sistêmicos e neurotóxicos significativos, em coelhos.