Bamboo Shark as a Small Animal Model for Single Domain Antibody Production

The development of shark single domain antibodies (sdAbs) is hindered by the high cost and tediousness of large-sized shark farming. Here, we demonstrated white-spotted bamboo sharks (Chiloscyllium plagiosum) being cultivated commercially as a promising small animal model to produce sdAbs. We found that immunoglobulin new antigen receptor (IgNAR) presented in bamboo shark genome, transcriptome, and plasma. Four complete IgNAR clusters including variable domains (vNARs) were discovered in the germline, and the Variable–Joining pair from IgNAR1 cluster was dominant from immune repertoires in blood. Bamboo sharks developed effective immune responses upon green fluorescent protein (GFP), near-infrared fluorescent protein iRFP713, and Freund’s adjuvant immunization revealed by elevated lymphocyte counts and antigen specific IgNAR. Before and after immunization, the complementarity determining region 3 (CDR3) of IgNAR were the major determinant of IgNAR diversity revealed by 400-bp deep sequencing. To prove that bamboo sharks could produce high-affinity IgNAR, we isolated anti-GFP and anti-iRFP713 vNARs with up to 0.3 and 3.8 nM affinities, respectively, from immunized sharks. Moreover, we constructed biparatopic vNARs with the highest known affinities (20.7 pM) to GFP and validated the functions of anti-GFP vNARs as intrabodies in mammalian cells. Taken together, our study will accelerate the discovery and development of bamboo shark sdAbs for biomedical industry at low cost and easy operation.

Teaching a new mouse old tricks: Humanized mice as an infection model for Variola virus

Smallpox, caused by the solely human pathogen Variola virus (VARV), was declared eradicated in 1980. While known VARV stocks are secure, smallpox remains a bioterrorist threat agent. Recent U.S. Food and Drug Administration approval of the first smallpox anti-viral (tecovirimat) therapeutic was a successful step forward in smallpox preparedness; however, orthopoxviruses can become resistant to treatment, suggesting a multi-therapeutic approach is necessary. Animal models are required for testing medical countermeasures (MCMs) and ideally MCMs are tested directly against the pathogen of interest. Since VARV only infects humans, a representative animal model for testing therapeutics directly against VARV remains a challenge. Here we show that three different humanized mice strains are highly susceptible to VARV infection, establishing the first small animal model using VARV. In comparison, the non-humanized, immunosuppressed background mouse was not susceptible to systemic VARV infection. Following an intranasal VARV challenge that mimics the natural route for human smallpox transmission, the virus spread systemically within the humanized mouse before mortality (~ 13 days post infection), similar to the time from exposure to symptom onset for ordinary human smallpox. Our identification of a permissive/representative VARV animal model can facilitate testing of MCMs in a manner consistent with their intended use.

Establishment of Tree Shrew Animal Model for Kaposi’s Sarcoma-Associated Herpesvirus (HHV-8) Infection

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the most common cause of Kaposi’s sarcoma (KS) and other malignant growths in humans. However, the lack of a KSHV-infected small animal model has hampered understanding of the mechanisms of KSHV infection, virus replication, pathogenesis, and persistence. This study was designed to explore the susceptibility of tree shrews as a possible KSHV-infected small animal model. A recombinant GFP (latent)/RFP (lytic)-positive rKSHV.219 strain was used to infect primary cells cultured from different tissues of tree shrews as an in vitro model and adult tree shrews as an in vivo model. KSHV latent nuclear antigen (LANA) and DNA were successfully detected in primary cells of tree shrews. Among them, tree shrew kidney epithelial cells (TSKEC) were the most susceptible cells to KSHV infection compared to other cells. KSHV genomic DNA, mRNA, and KSHV-specific proteins were readily detected in the TSKEC cultured up to 32 dpi. Moreover, KSHV DNA and mRNA transcription were also readily detected in the peripheral blood mononuclear cells (PBMCs) and various tissues of tree shrews infected with KSHV. Haematoxylin and eosin (HE) staining showed lymphocyte infiltration, lymphoid tissue focal aggregation, alveolar wall thickening, hepatocyte edema, hepatic necrosis in the spleen, lung, and liver of KSHV-infected animals. Additionally, immune-histochemical (IHC) staining showed that LANA or ORF62-positive cells were present in the spleen, lung, liver, and kidney of KSHV-infected tree shrews. Here, we have successfully established in vitro and in vivo KSHV latent infection in tree shrews. This small animal model is not only useful for studying the pathogenesis of KSHV in vivo but can also be a useful model to study transmission routes of viral infection and a useful platform to characterize the novel therapeutics against KSHV.

Tree Shrew as an Emerging Small Animal Model for Human Viral Infection: A Recent Overview

Viral infection is a global public health threat causing millions of deaths. A suitable small animal model is essential for viral pathogenesis and host response studies that could be used in antiviral and vaccine development. The tree shrew (Tupaia belangeri or Tupaia belangeri chinenesis), a squirrel-like non-primate small mammal in the Tupaiidae family, has been reported to be susceptible to important human viral pathogens, including hepatitis viruses (e.g., HBV, HCV), respiratory viruses (influenza viruses, SARS-CoV-2, human adenovirus B), arboviruses (Zika virus and dengue virus), and other viruses (e.g., herpes simplex virus, etc.). The pathogenesis of these viruses is not fully understood due to the lack of an economically feasible suitable small animal model mimicking natural infection of human diseases. The tree shrew model significantly contributes towards a better understanding of the infection and pathogenesis of these important human pathogens, highlighting its potential to be used as a viable viral infection model of human viruses. Therefore, in this review, we summarize updates regarding human viral infection in the tree shrew model, which highlights the potential of the tree shrew to be utilized for human viral infection and pathogenesis studies.

STAT-1 Knockout Mice as a Model for Wild-Type Sudan Virus (SUDV)

Currently there is no FDA-licensed vaccine or therapeutic against Sudan ebolavirus (SUDV) infections. The largest ever reported 2014–2016 West Africa outbreak, as well as the 2021 outbreak in the Democratic Republic of Congo, highlight the critical need for countermeasures against filovirus infections. A well-characterized small animal model that is susceptible to wild-type filoviruses would greatly add to the screening of antivirals and vaccines. Here, we infected signal transducer and activator of transcription-1 knock out (STAT-1 KO) mice with five different wildtype filoviruses to determine susceptibility. SUDV and Marburg virus (MARV) were the most virulent, and caused 100% or 80% lethality, respectively. Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Taï Forest ebolavirus (TAFV) caused 40%, 20%, and no mortality, respectively. Further characterization of SUDV in STAT-1 KO mice demonstrated lethality down to 3.1 × 101 pfu. Viral genomic material was detectable in serum as early as 1 to 2 days post-challenge. The onset of viremia was closely followed by significant changes in total white blood cells and proportion of neutrophils and lymphocytes, as well as by an influx of neutrophils in the liver and spleen. Concomitant significant fluctuations in blood glucose, albumin, globulin, and alanine aminotransferase were also noted, altogether consistent with other models of filovirus infection. Finally, favipiravir treatment fully protected STAT-1 KO mice from lethal SUDV challenge, suggesting that this may be an appropriate small animal model to screen anti-SUDV countermeasures.

Rat model of smoke inhalation-induced acute lung injury

BackgroundAcute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a lethal disease with limited therapeutic options and an unacceptably high mortality rate. Understanding the complex pathophysiological processes involved in the development of ALI/ARDS is critical for developing novel therapeutic strategies. Smoke inhalation (SI) injury is the leading cause of morbidity and mortality in patients with burn-associated ALI/ARDS; however, to our knowledge few reliable, reproducible models are available for pure SI animal model to investigate therapeutic options for ALI/ARDS without the confounding variables introduced by cutaneous burn or other pathology.ObjectiveTo develop a small animal model of pure SI-induced ALI and to use this model for eventual testing of novel therapeutics for ALI.MethodsRats were exposed to smoke using a custom-made smoke generator. Peripheral oxygen saturation (SpO2), heart rate, arterial blood gas, and chest X-ray (CXR) were measured before and after SI. Wet/dry weight (W/D) ratio, lung injury score and immunohistochemical staining of cleaved caspase 3 were performed on harvested lung tissues of healthy and SI animals.ResultsThe current study demonstrates the induction of ALI in rats after SI as reflected by a significant, sustained decrease in SpO2 and the development of diffuse bilateral pulmonary infiltrates on CXR. Lung tissue of animals exposed to SI showed increased inflammation, oedema and apoptosis as reflected by the increase in W/D ratio, injury score and cleaved caspase 3 level of the harvested tissues compared with healthy animals.ConclusionWe have successfully developed a small animal model of pure SI-induced ALI. This model is offered to the scientific community as a reliable model of isolated pulmonary SI-induced injury without the confounding variables of cutaneous injury or other systemic pathology to be used for study of novel therapeutics or other investigation.

Recapitulating Zika Virus Infection in Vagina of Tree Shrew (Tupaia belangeri)

Sexual transmission of Zika Virus (ZIKV) elevates the risk of its dissemination in the female reproductive tract and causes a serious threat to the fetus. However, the available animal models are not appropriate to investigate sexual transmission, dynamics of ZIKV infection, replication, and shedding. The use of tree shrew as a small animal model of ZIKV vaginal infection was assessed in this study. A total of 23 sexually mature female tree shrews were infected with ZIKV GZ01 via the intravaginal route. There was no significant difference in change of body weight, and the temperature between ZIKV infected and control animals. Viral RNA loads were detected in blood, saliva, urine, and vaginal douching. ZIKV RNA was readily detected in vaginal lavage of 22 animals (95.65%, 22/23) at 1 dpi, and viral load ranged from 104.46 to 107.35 copies/ml, and the peak of viral load appeared at 1 dpi. The expression of key inflammatory genes, such as IL6, 8, CCL5, TNF-a, and CXCL9, was increased in the spleen of ZIKV infected animals. In the current study, female tree shrews have been successfully infected with ZIKV through the vaginal route for the first time. Interestingly, at first, ZIKV replicates at the local site of infection and then spreads throughout the host body to develop a robust systemic infection and mounted a protective immune response. This small animal model is not only valuable for exploring ZIKV sexual transmission and may also help to explain the cause of debilitating manifestations of the fetus in vivo.

Broadening a SARS-CoV-1 neutralizing antibody for potent SARS-CoV-2 neutralization through directed evolution

The emergence of SARS-CoV-2 underscores the need for strategies to rapidly develop neutralizing monoclonal antibodies that can function as prophylactic and therapeutic agents and to help guide vaccine design. Here, we demonstrate that engineering approaches can be used to refocus an existing neutralizing antibody to a related but resistant virus. Using a rapid affinity maturation strategy, we engineered CR3022, a SARS-CoV-1 neutralizing antibody, to bind SARS-CoV-2 receptor binding domain with >1000-fold improved affinity. The engineered CR3022 neutralized SARS-CoV-2 and provided prophylactic protection from viral challenge in a small animal model of SARS-CoV-2 infection. Deep sequencing throughout the engineering process paired with crystallographic analysis of an enhanced antibody elucidated the molecular mechanisms by which engineered CR3022 can accommodate sequence differences in the epitope between SARS-CoV-1 and SARS-CoV-2. The workflow described provides a blueprint for rapid broadening of neutralization of an antibody from one virus to closely related but resistant viruses.

Mucosal Challenge Ferret Models of Ebola Virus Disease

Recent studies have shown the domestic ferret (Mustela putorius furo) to be a promising small animal model for the study of Ebola virus (EBOV) disease and medical countermeasure evaluation. To date, most studies have focused on traditional challenge routes, predominantly intramuscular and intranasal administration. Here, we present results from a non-clinical pathogenicity study examining oronasal, oral, and ocular mucosal challenge routes in ferrets. Animals were challenged with 1, 10, or 100 plaque forming units EBOV followed by monitoring of disease progression and biosampling. Ferrets administered virus via oronasal and oral routes met euthanasia criteria due to advanced disease 5–10 days post-challenge. Conversely, all ferrets dosed via the ocular route survived until the scheduled study termination 28-day post-challenge. In animals that succumbed to disease, a dose/route response was not observed; increases in disease severity, febrile responses, serum and tissue viral load, alterations in clinical pathology, and gross/histopathology findings were similar between subjects. Disease progression in ferrets challenged via ocular administration was unremarkable throughout the study period. Results from this study further support the ferret as a model for EBOV disease following oral and nasal mucosa exposure.