Comparison of experimental MERS-CoV infection acquired by three individual routes of infection in the common marmoset

Two strains of Middle East Respiratory Syndrome coronavirus (MERS-CoV), England -1 and EMC/2012, were used to challenge common marmosets ( Callithrix jacchus ) by three routes of infection, aerosol, oral and intra-nasal. Animals challenged by the intra-nasal and aerosol routes presented with a mild, transient disease, while those challenged by the oral route presented with a subclinical immunological response. Animals challenged with MERS-CoV strain EMC/2012 by the aerosol route responded with primary and/or secondary pyrexia. Marmosets had minimal to mild multifocal interstitial pneumonia, with the greatest relative severity observed in animals challenged by the aerosol route. Viable virus was isolated from the host in throat swabs and lung tissue. The transient disease described is consistent with a successful host response and was characterised by upregulation of macrophage and neutrophil function observed in all animals at the time of euthanasia. Importance Middle East Respiratory Syndrome is caused by a human Coronavirus, MERS-CoV, similar to SARS-CoV-2. Humans typically exhibit with a fever, cough, shortness of breath, gastrointestinal issues and breathing difficulties which can lead to pneumonia and/or renal complications. This emerging disease resulted in the first human lethal cases in 2012 and has a case fatality rate of approximately 36%. Consequently, there is a need for medical countermeasures and appropriate animal models for their assessment. This work has demonstrated the requirement for higher concentrations of virus to cause overt disease. Challenge by the aerosol, intra-nasal and oral route resulted in no or mild disease, but all animals had an immunological response. This shows that an appropriate early immunological response is able to control the disease.

Safety and Immunogenicity of Four-Segmented Rift Valley Fever Virus in the Common Marmoset

Rift Valley fever virus (RVFV) is an emerging mosquito-borne bunyavirus that is highly pathogenic to wild- and domesticated ruminants, camelids and humans. While animals are exclusively infected via mosquito bites, humans can also be infected via contact with tissues or blood released during the slaughtering of RVFV-infected animals. No human vaccine is available and currently commercialized veterinary vaccines do not optimally combine efficacy with safety. We previously reported the development of two novel live-attenuated RVF vaccines, created by splitting the M genome segment and deleting the major virulence determinant NSs. The vaccine candidates, referred to as the veterinary vaccine vRVFV-4s and the human vaccine hRVFV-4s, were shown to induce protective immunity in multiple species after a single vaccination. Anticipating on accidental exposure of humans to the veterinary vaccine, and to evaluate the safety of the hRVFV-4s candidate vaccine for humans, the safety of each vaccine was evaluated in the most susceptible nonhuman primate model, the common marmoset (Callithrix jacchus). Marmosets were inoculated with high doses of each vaccine and were monitored for clinical signs as well as for vaccine virus dissemination, shedding and spreading to the environment. To accurately assess the attenuation of both vaccine viruses, separate groups of marmosets were inoculated with the parent wild-type RVFV strains. Both wild-type strains induced high viremia and disseminated to primary target organs, associated with mild- to severe morbidity, while both vaccines were well tolerated with absence of dissemination and shedding, while inducing potent neutralizing antibody responses. The results of the studies support the unprecedented safety profile of both vaccines for animals and humans.

The Common Marmoset: A Highly Translatable Small Nonhuman Primate Model of Aging

The common marmoset (Callithrix jacchus) has been used in biomedical research for many years, but within the last decade its popularity has increased dramatically prompted to a large degree by their realized utility for neuroscience and aging research. Many factors make the marmoset an attractive model system including their genetic and physiological similarity to humans, relatively short lifespan (average of ~13 years, maximum of ~20 years), high fertility (highest of any primate, routine production of 2-3 offspring every 5-6 months), rapid development (reproductively competent by ~1.5 years of age, aged by 7-8 years of age), small size (~400 grams), human-like social structure consisting of cooperative breeding with shared parenting responsibilities, and lack of zoonotic diseases of concern to humans. Marmosets share ~93% sequence identity with the human genome and they develop similar age-related conditions as humans. Marmosets may strike the perfect balance between similarity to humans and abbreviated aging course.

Conservation of the direct and indirect pathways dichotomy in mouse caudal striatum with uneven distribution of dopamine receptor D1- and D2-expressing neurons

The striatum is one of the key nuclei for adequate control of voluntary behaviors and reinforcement learning. Two striatal projection neuron types, expressing either dopamine receptor D1 (D1R) or dopamine receptor D2 (D2R) constitute two independent output routes: the direct or indirect pathways, respectively. These pathways co-work in balance to achieve coordinated behavior. Two projection neuron types are equivalently intermingled in most striatal space. However, recent studies revealed two atypical zones in the caudal striatum: the zone in which D1R-neurons are the minor population (D1R-poor zone) and that in which D2R-neurons are the minority (D2R-poor zone). It remains obscure as to whether these imbalanced zones have similar properties on axonal projections and electrophysiology to other striatal regions. Based on morphological experiments in mice using immunofluorescence, in situ hybridization, and neural tracing, here, we revealed the poor zones densely projected to the globus pallidus and substantia nigra pars lateralis, with a few collaterals in substantia nigra pars reticulata and compacta. As other striatal regions, D1R-neurons were the direct pathway neurons, while projection neurons in the poor zones possessed similar electrophysiological membrane properties to those in the conventional striatum using in vitro electrophysiological recording. In addition, the poor zones existed irrespective of the age of mice. We also identified the poor zones in the common marmoset as well as other rodents. These results suggest that the poor zones in the caudal striatum follow the conventional projection patterns irrespective of imbalanced distribution of projection neurons. The poor zones could be an innate structure and common in mammals and relate to specific functions via highly restricted projections.

Development of a metatranscriptomic analysis method for multiple intestinal sites and its application to the common marmoset intestine

Background: The intestinal microbiome is closely related to host health, and metatranscriptomic analysis can assess the functional activity of microbiomes by quantifying the bacterial gene expression level, which helps to elucidate the interaction between the microbiome and the environment. However, functional changes in the microbiome along the host intestinal tract remain unknown, and previous analytical methods have limitations, such as potentially overlooking unknown genes due to dependence on existing databases and being unable to take full advantage of metatranscriptome to reveal the functional change among multiple environments. Result: To close these gaps, we develop a novel method that integrates metagenome and metatranscriptome to analyze the functional activity of microbiomes between intestinal sites. This method reconstructs a reference metagenomic sequence across multiple intestinal sites, allowing the gene expression levels of microbiome including unknown bacterial genes to be compared between multiple sites. As a result of applying this method to metatranscriptomic analysis in the intestinal tract of common marmoset, the reconstructed metagenome covered most of the expressed genes and it revealed that the changes in bacterial gene expressions among the caecum, transverse colon, and faeces were more dynamic and sensitive to environmental shifts than its abundance. In typical, the coenzyme synthesis gene and antibacterial resistance gene were more highly expressed in the caecum and transverse colon than in faeces, while there was no significant change in abundance of these genes. Conclusion: Our findings demonstrate that an analytical method that integrates metagenome and metatranscriptome in multiple intestinal sites captures functional changes in the microbiomes at the gene resolution level.

Efficient marmoset genome engineering by autologous embryo transfer and CRISPR/Cas9 technology

Genetic engineering of non-human primates, which are most closely related to humans, has been expected to generate ideal animal models for human genetic diseases. The common marmoset (Callithrix jacchus) is a non-human primate species adequate for the production of genetically modified animals because of their small body size and high reproductive capacity. Autologous embryo transfer (AET) is routinely utilized in assisted reproductive technologies for humans but not for experimental animals. This study has developed a novel method for efficiently producing mutant marmosets using AET and CRISPR/Cas9 systems. The embryos were recovered from oviducts of naturally mated females, injected with Cas9/guide RNA, and transferred into the oviducts of the donors. This AET method can reduce the time for in vitro culture of embryos to less than 30 min. This method uses an embryo donor as the recipient, thus reducing the number of animals and allowing for “Reduction” in the 3R principles of humane experimental technique. Furthermore, this method can utilize nulliparous females as well as parous females. We applied our novel method and generated the 6 marmosets carrying mutations in the fragile X mental retardation 1 (FMR1) gene using only 18 females including 14 nulliparous females.

Nipah virus Bangladesh infection elicits organ-specific innate and inflammatory responses in the marmoset model

The common marmoset (Callithrix jacchus) has, in recent years, received more recognition as an ideal non-human primate (NHP) model for studies at high-biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets. Four marmosets were infected via the intranasal and intratracheal route and monitored for disease development. All four subjects developed fatal disease between days 8 and 11 post infection, with three animals showing severe respiratory disease and one marmoset recapitulating the neurologic clinical symptoms seen in humans, as well as cardiomyopathy on gross pathology. We obtained histopathological data, quantified genome copies on >25 tissue-types, and performed RNA-seq on six different organs from all infected and control marmosets. Three out of four marmosets showed pulmonary edema and hemorrhage as well as multi-focal hemorrhagic lymphadenopathy. In all animals, syncytia were evident in endothelial cells in pulmonary vessels, cells in alveolar septum, and in splenic follicles or red pulp. To define the organ-specific innate and inflammatory responses, we performed RNA-seq on six different organs from all infected and compared to naive non-infected marmoset tissues. RNA-seq gave 17.4 million reads per sample on average, with the most highly infected tissue sample, the lung of one marmoset, containing >180,000 virus-specific reads. NiV V and W transcripts comprised ~40% of all phosphoprotein-derived transcripts with V and W being very close to each other proportionally. Principal component analysis showed that in brain stem, the marmoset exhibiting neurological symptoms displayed a unique RNA transcriptome relative even to other infected marmosets. Upregulated genes in the various tissues belonged to distinct GO pathways. Additionally, one male and female animal had detectable viral reads in their ovaries (27,120) and testes (858). Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.