Animal and translational models of SARS-CoV-2 infection and COVID-19.

COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.

The genetic basis for susceptibility to Rift Valley fever disease in MBT/Pas mice.

The large variation in individual response to infection with Rift Valley fever virus (RVFV) suggests that host genetic determinants play a role in determining virus-induced disease outcomes. These genetic factors are still unknown. The systemic inoculation of mice with RVFV reproduces major pathological features of severe human disease, notably the hepatitis and encephalitis. A genome scan performed on 546 (BALB/c × MBT) F2 progeny identified three quantitative trait loci (QTLs), denoted Rvfs-1 to Rvfs-3, that were associated with disease susceptibility in MBT/Pas mice. Non-parametric interval-mapping revealed one significant and two suggestive linkages with survival time on chromosomes 2 (Rvfs-1), 5 (Rvfs-3) and 11 (Rvfs-2) with respective logarithm of odds (LOD) scores of 4.58, 2.95 and 2.99. The two-part model, combining survival time and survival/death, identified one significant linkage to Rvfs-2 and one suggestive linkage to Rvfs-1 with respective LOD scores of 5.12 and 4.55. Under a multiple model, with additive effects and sex as a covariate, the three QTLs explained 8.3% of the phenotypic variance. Sex had the strongest influence on susceptibility. The contribution of Rvfs-1, Rvfs-2 and Rvfs-3 to survival time of RVFV-infected mice was further confirmed in congenic mice.

Resistance to plague of Mus spretus SEG/Pas mice requires the combined action of at least four genetic factors.

We have previously described SEG/Pas as the first mouse inbred strain able to survive subcutaneous injection of virulent Yersinia pestis, the agent of plague, and we identified Yprl1, Yprl2 and Yprl3 as three quantitative trait loci (QTLs) controlling this exceptional phenotype in females from a backcross between SEG/Pas and C57BL/6 strains. We have now developed congenic strains to further characterize the extent and effect of these genomic regions. In this study, we confirm the importance of two of these regions, both in males and females, while the third one may well be a spurious association. We show that no genomic region alone is able to increase the survival of C57BL/6 mice, but that C57BL/6 mice carrying both Yprl2 and Yprl3 exhibit intermediate resistance. Each of these two QTLs contains at least two subregions, which are required to increase survival. Finally, through the analysis of congenic strains in an F1 background, we establish the mode of inheritance of the SEG-derived resistance alleles. Altogether, this study has clarified and enhanced our understanding of the genetic architecture of resistance to plague in SEG/Pas mice.

Mus spretus SEG/Pas mice resist virulent Yersinia pestis, under multigenic control.

Laboratory mice are well known to be highly susceptible to virulent strains of Yersinia pestis in experimental models of bubonic plague. We have found that Mus spretus-derived SEG/Pas (SEG) mice are exceptionally resistant to virulent CO92 and 6/69 wild type strains. Upon subcutaneous injection of 10(2) colony-forming units (CFU), 90% of females and 68% of males survived, compared with only an 8% survival rate for both male and female C57BL/6 mice. Furthermore, half of the SEG mice survived a challenge of up to 10(7) CFU. The time required for mortality was similar between B6 and SEG, suggesting that survival is dependent on early rather than late processes. The analysis of 322 backcross mice identified three significant quantitative trait loci (QTLs) on chromosomes 3, 4 and 6, with dominant SEG protective alleles. Each QTL increased the survival rate by approximately 20%. The three QTLs function additively, thereby accounting for 67% of the difference between the parental phenotypes. Mice heterozygous for the three QTLs were just as resistant as SEG mice to Y. pestis challenge. The SEG strain therefore offers an invaluable opportunity to unravel mechanisms and underlying genetic factors of resistance against Y. pestis infection.

The stromal gene encoding the CD274 antigen as a genetic modifier controlling survival of mice with γ-radiation-induced T-cell lymphoblastic lymphomas.

Using an inter-specific subcongenic strain, Nested Recombinant Haplotype 3 (NRH3), generated between two mouse strains showing extreme differences in γ-radiation-induced thymic lymphoma susceptibility (SEG/Pas and C57BL/6J), we have identified a critical region on chromosome 19 that regulates survival of mice suffering from T-cell lymphoblastic lymphomas. Mapped on this region, the gene encoding the Cd274 ligand is able to trigger an inhibitory effect that modulates T-cell receptor (TCR) signalling and affects thymocyte maturation. Interestingly, this gene shows differential expression between thymic stromal cells from both strains in early response to a single sublethal γ-ray dose, but is inhibited in T-cell lymphoblastic lymphomas. Furthermore, we have identified several polymorphisms in the complementary DNA sequence of this gene that affect the affinity for its Cd279 receptor and are able to induce a differential rate of thymocyte apoptosis. Taken together, our data are consistent with Cd274 acting as a genetic modifier that influences the survival of γ-radiation-induced T-cell lymphoma-bearing mice. The data similarly support the idea of a co-evolution of tumour cells and associated stromal cells to generate a favourable microenvironment for T-cell lymphoma growth.

FELASA guidelines for the production and nomenclature of transgenic rodents.

The standardized nomenclature of rodent strains, genes and mutations has long been the focus of careful attention. Its aim is to provide proper designation of laboratory animals used in research projects and to convey as much information on each strain as possible. Since the development of different techniques to mutate the genome of laboratory rodents on a large scale, the correct application of current nomenclature systems is of increased significance. It facilitates not only the accurate communication of scientific results but is indispensable in controlling the dramatically increased number of transgenic animal models in experimental units, archives and databases. It is regrettable that many publications, especially on transgenic rodents, use vague and inappropriate strain designation. This situation should definitely be improved, particularly considering the increasingly emphasized importance of genetic background on the phenotype of mutations. The aim of these guidelines is to raise awareness about specific features of production and of the current nomenclature system used for transgenic rodents.

Ursodesoxycholic acid and heme-arginate are unable to improve hematopoiesis and liver injury in an erythropoietic protoporphyria mouse model.

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by partial ferrochelatase deficiency, resulting in protoporphyrin overproduction which is responsible for painful skin photosensitivity. Chronic liver disease is the most severe complication of EPP, requiring liver transplantation in some patients. Data from a mouse model suggest that cytotoxic bile formation with high concentrations of bile salts and protoporphyrin may cause biliary fibrosis by damaging bile duct epithelium. In humans, cholestasis is a result of intracellular and canalicular precipitation of protoporphyrin. To limit liver damage two strategies may be considered: the first is to reduce protoporphyrin production and the second is to enhance protoporphyrin excretion. Bile salts are known to increase protoporphyrin excretion via the bile, while heme arginate is used to decrease the production of porphyrins in acute attacks of hepatic porphyrias. The Griseofulvin-induced protoporphyria mouse model has been used to study several aspects of human protoporphyria including the effects of bile salts. However, the best EPP animal model is an ethylnitrosourea-induced point mutation with fully recessive transmission, named ferrochelatase deficiency (Fech(m1Pas)). Here we investigate the effect of early ursodesoxycholic acid (UDCA) administration and heme-arginate injections on the ferrochelatase deficient EPP mouse model. In this model UDCA administration and heme-arginate injections do not improve the protoporphyric condition of Fech(m1Pas)/Fech(m1Pas) mice.

Effect of the genetic background on the phenotype of mouse mutations.

An increasing number of scientific articles report that the phenotype of a given single gene mutation in mice is modulated by the genetic background of the inbred strain in which the mutation is maintained. This effect is attributable to so-called modifier genes, which act in combination with the causative gene. The modulation of the phenotype can be major, as exemplified in the case of several mouse models of polycystic kidney disease. Because of the existence of inbred strains and the possibility of developing congenic strains, the effect of the genetic background can be analyzed in mice, including the identification of major modifier genes. Furthermore, by transferring a given mutation into different genetic backgrounds, mouse models can be manipulated with the aim of more accurately mimicking specific features of human diseases.

Lanceolate hair-J (lahJ): a mouse model for human hair disorders.

Lanceolate hair-J (lahJ) arose spontaneously in 1994 on the DBA/1LacJ inbred background at The Jackson Laboratory. Mutant mice were runted, alopecic, and lacked vibrissae. As they aged, their skin wrinkled. Affected mice developed a noninflammatory, proliferative skin disease with follicular dystrophy. Hair fibers developed a number of abnormalities including periodic nodules along the shaft (trichorrhexis nodosa), compaction resembling trichorrhexis invaginata, spiral fractures, broken tips, and lance-shaped tips. This mutation exhibits some characteristics that resemble an autosomal recessive ichthyosiform disease that occurs in humans characterized in part by peculiar, invaginating, multinodal, hair shaft abnormalities known as Netherton's syndrome. Periodic nodules also resemble the human genetic based disease monilethrix. This autosomal recessive mouse mutation, allelic with lanceolate hair (lah), based on breeding studies, is located on mouse Chromosome 18, within a cluster of genes coding for adhesion molecules. Homozygotes for either of these allelic mouse mutations have elevated serum IgE levels, a feature also common with human Netherton's syndrome.

Reversion of hepatobiliary alterations By bone marrow transplantation in a murine model of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is characterized clinically by cutaneous photosensitivity and biochemically by the accumulation of excessive amounts of protoporphyrin in erythrocytes, plasma, feces, and other tissues, such as the liver. The condition is inherited as an autosomal dominant or recessive trait, with a deficiency of ferrochelatase activity. A major concern in EPP patients is the development of cholestasis with accumulation of protoporphyrin in hepatobiliary structures and progressive cellular damage, which can rapidly lead to fatal hepatic failure. The availability of a mouse model for the disease, the Fech(m1Pas)/Fech(m1Pas) mutant mouse, allowed us to test a cellular therapy protocol to correct the porphyric phenotype. When Fech/Fech mice received bone marrow cells from normal animals, the accumulation of protoporphyrin in red blood cells and plasma was reduced 10-fold but still remained 2.5 times above normal levels. Interestingly, in very young animals, bone marrow transplantation can prevent hepatobiliary complications as well as hepatocyte alterations and partially reverse protoporphyrin accumulation in the liver. Bone marrow transplantation may be an option for EPP patients who are at risk of developing hepatic complications.

Identification of the mutation in the alkaptonuria mouse model. Mutations in brief no. 216. Online.

Alkaptonuria (aku), an inborn error of metabolism caused by the loss of homogentisate 1,2-dioxygenase (HGD), has been described in a mouse model created by ethylnitrosourea mutagenesis but the mutation in these mice has not previously been identified. We used RT-PCR to amplify the Hgd cDNA from Hgd(aku)/Hgd(aku) mice. Two products shorter than the wild-type product were amplified. Restriction mapping and DNA sequencing were then used to identify the Hgd(aku) mouse mutation, found to be a single base change in a splice donor consensus sequence, causing exon skipping and frame-shifted products. This base change allowed us to create a non-radioactive genotyping assay for this allele.

Linkage of a major quantitative trait locus to Yaa gene-induced lupus-like nephritis in (NZW x C57BL/6)F1 mice.

In the present study, we mapped the major quantitative trait loci (QTL) differing between the NZW and C57BL/6 inbred strains of mice by making use of (NZW x C57BL/6.Yaa)F1 mice, a model in which the lupus-like autoimmune syndrome observed in male mice is associated with the presence of an as yet unidentified Y chromosome-linked autoimmune acceleration gene, Yaa. Linkage analysis of 126 C57BL/6 x (NZW x C57BL/6.Yaa)F1 backcross males provided evidence for a major QTL on chromosome 7 controlling both the severity of glomerulonephritis and the production of IgG anti-DNA autoantibody and retroviral gp70-anti-gp70 immune complexes. Two additional QTL of C57BL/6 origin on chromosome 17 had no apparent individual effects, but showed strong epistatic interaction with chromosome 7 QTL for disease severity and anti-DNA autoantibody production. Our data also identified on chromosome 13 a QTL of NZW origin with a major effect on the level of gp70, and showing an additive effect with the chromosome 7 QTL on the level of gp70 immune complexes. Our study thus provides a model to dissect the complex genetic interactions that result in manifestations of murine lupus-like disease.

Maintenance of granulocyte numbers during acute peritonitis is defective in galectin-3-null mutant mice.

Galectin-3, also known as the macrophage marker Mac-2, is a member of a family of structurally related animal lectins that exhibit specificity for beta-galactosides. In order to investigate the role of galectin-3 in acute inflammation, we have compared the number of leucocytes present in the peritoneal cavity of wild type and galectin-3 null mutant mice after intraperitoneal (i.p.) injection of thioglycolate broth. At day 1 after injection, we found no difference in the recruitment of mononuclear phagocytes and granulocytes to the peritoneal cavity. However, 4 days after thioglycolate injection, galectin-3 mutant mice exhibited a significantly reduced number of recoverable granulocytes compared to wild-type animals. As mutant granulocytes did not exhibit an accelerated rate of apoptosis and their uptake by macrophages appeared to be unaffected by the mutation, the phenotype described here suggests that galectin-3 participates in an additional level of control during the resolution of acute inflammation.

A high-resolution microsatellite map of the mouse genome.

The European Collaborative Interspecific Backcross (EUCIB) resource was constructed for the purposes of high-resolution genetic mapping of the mouse genome (). The large Mus spretus/C57BL/6 backcross of 982 progeny has a genetic resolution of 0.3 cM at the 95% confidence level ( approximately 500 kb in the mouse genome). We have used the EUCIB mapping resource to develop a genome-wide high-resolution genetic map incorporating 3368 microsatellites. The microsatellites are distributed among 2302 genetically separated bins with 1.46 markers per bin on average. Average bin separation is 0.61 cM. This high-resolution genetic map will aid the construction of a robust physical map of the mouse genome.

A locus for radiation-induced gastroschisis on mouse Chromosome 7.

Gastroschisis (abdominal wall defects) occurs with a high frequency in the mouse inbred strain HLG compared with C57BL/6J mice. The risk of gastroschisis increases significantly after exposure to irradiation with X-rays during preimplantation development and follows a recessive mode of inheritance for the HLG susceptibility alleles. We have used a backcross strategy and genome-wide microsatellite typing to chromosomally map this trait. A suggestive linkage for a locus responsible for radiation-induced gastroschisis (Rigs1) was found in a region of mouse Chromosome 7.

Vesicle formation and follicular root sheath separation in mice homozygous for deleterious alleles at the balding (bal) locus.

The balding (bal) mutation of the mouse is an autosomal recessive mutation that causes alopecia and immunologic anomalies. A new allele was identified by allelism testing after using an interspecific backcross to localize the mutation to the centromeric end of mouse chromosome 18. We investigated the skin and hair histologic lesions of two alleles (bal(J) and bal(Pas)) at this locus and analyzed the expression of several keratinocyte markers and the production of autoantibodies by immunofluorescence on frozen skin sections. The lesions observed included separation of the inner and outer root sheath in anagen follicles resulting in the hair fiber being very easily plucked from the follicle. Vesicles on the ventral tongue, mucocutaneous junction of the eyelid, foot pads, and rarely in skin were also evident. Separation occurred between the basal and suprabasilar cells forming an empty cleft, resembling that observed in human pemphigus vulgaris. Immunofluorescence studies did not reveal the presence of tissue-bound or circulating autoantibodies. Expression of keratinocyte markers in hair follicles was normal. Keratin 6-positive cells were found on either side of the follicular separation suggesting a molecular defect in adhesion molecules between the inner layer of the outer root sheath cells to layers on either sides. This hypothesis has been confirmed by another group who demonstrated that the bal(J) mutation is due to the insertion of a thymidine in the desmoglein 3 gene, resulting in a premature stop codon.

[Animal models of hereditary diseases]. / Modèles animaux des maladies héréditaires.

A large number of genetic diseases are found both in humans and in one or several animal species. The mouse is a species of choice since, in addition to hundreds of spontaneous mutations which have been described, it is now possible to produce at will a mutation in any gene. This strategy has been used to generate genetically engineered mice which carry genetic defects found in human molecular pathology. Animal models are an invaluable tool to study the pathophysiology of diseases and to test new therapies. However, they are rarely exact replicates of the human disease. Possible origins for these differences are discussed in detail.

Male sterility caused by sperm cell-specific structural abnormalities in ebouriffé, a new mutation of the house mouse.

We have investigated the male sterility associated with a new recessive mutation of the house mouse: ébouriffé (ebo). All spermatozoa present in the epididymis showed severe malformations, mostly of the tail. Light and electron microscopy showed a drastic decrease of the spermatid population, whereas spermatogonia and spermatocytes seemed moderately affected. This suggests that the mutation affects mostly spermiogenesis. Defects appeared during formation of the acrosome: the acrosomal granule was frequently vacuolated at stages II-III, giving rise first to abnormal acrosomes (stages VI-VII) with dilations and perforations, and then to an abnormal head and acrosome shape (stages IX-XI). However, the most common malformations affected the flagella in elongated spermatids. Sometimes the centriole doublet did not move into the implantation fossa, causing an unattached and isolated flagellum. The major defect occurred in the midpiece region of differentiating spermatozoa: flagellar components were present but highly disorganized, and mitochondria were aggregated in a compact mass. Even though we have no evidence that the ebo gene is a testis structural gene or a regulatory gene that disrupts the complex spermatogenesis process, this mutation may provide an interesting tool for studying the late stages of spermatogenesis. Using an interspecific backcross, we localized the ebo mutation on chromosome 2, with no recombination out of 44 meioses with locus D2Mit32.

Epistatic control of non-Mendelian inheritance in mouse interspecific crosses.

Strong deviation of allele frequencies from Mendelian inheritance favoring Mus spretus-derived alleles has been described previously for X-linked loci in four mouse interspecific crosses. We reanalyzed data for three of these crosses focusing on the location of the gene(s) controlling deviation on the X chromosome and the genetic basis for incomplete deviation. At least two loci control deviation on the X chromosome, one near Xist (the candidate gene controlling X inactivation) and the other more centromerically located. In all three crosses, strong epistasis was found between loci near Xist and marker loci on the central portion of chromosome 2. The mechanism for this deviation from Mendelian expectations is not yet known but it is probably based on lethality of embryos carrying particular combinations of alleles rather than true segregation distortion during oogenesis in F1 hybrid females.