Characteristics of Angiotensin I-converting enzyme 2, type II transmembrane serine protease 2 and 4 in tree shrew indicate it as a potential animal model for SARS-CoV-2 infection.

Angiotensin I-converting enzyme 2 (ACE2), type II transmembrane serine protease 2 and 4 (TMPRSS2 and TMPRSS4) are important receptors for SARS-CoV-2 infection. In this study, the full-length tree shrewACE2 gene was cloned and sequenced, and its biological information was analyzed. The expression levels of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs of the tree shrew were detected. The results showed that the full-length ACE2 gene in tree shrews was 2,786 bp, and its CDS was 2,418 bp, encoding 805 amino acids. Phylogenetic analysis based on the CDS of ACE2 revealed that tree shrews were more similar to rabbits (85.93%) and humans (85.47%) but far from mice (82.81%) and rats (82.58%). In silico analysis according to the binding site of SARS-CoV-2 with the ACE2 receptor of different species predicted that tree shrews had potential SARS-CoV-2 infection possibility, which was similar to that of rabbits, cats and dogs but significantly higher than that of mice and rats. In addition, various tissues or organs of tree shrews expressed ACE2, TMPRSS2 and TMPRSS4. Among them, the kidney most highly expressed ACE2, followed by the lung and liver. The esophagus, lung, liver, intestine and kidney had relatively high expression levels of TMPRSS2 and TMPRSS4. In general, we reported for the first time the expression of ACE2, TMPRSS2 and TMPRSS4 in various tissues or organs in tree shrews. Our results revealed that tree shrews could be used as a potential animal model to study the mechanism underlying SARS-CoV-2 infection.

Chinese tree shrew: a permissive model for in vitro and in vivo replication of human adenovirus species B.

Human adenovirus (HAdV) species B can cause severe acute respiratory diseases. However, the researches to combat this infection have been hampered by the lack of an animal model permissive to the virus. Here, we report in vitro and in vivo HAdV species B infections of tree shrews, the closest relative of primates. HAdV-3, -7, -14, and -55 efficiently replicated in primary cell cultures. After intranasal inoculation of tree shrews with HAdV-55, the viral replication in the oropharyngeal region remained high until day 5 post-infection and was still detected until day 12. HAdV-55 in the lung or turbinate bone tissues reached the highest levels between days 3 and 5 post-infection, which indicated viral replication in the upper and lower respiratory tracts. HAdV-55 infection caused severe interstitial pneumonia in the animal. IL-8, IL-10, IL-17A, and IFN-γ expression in the peripheral blood mononuclear cells from infected animals was up-regulated. The pre-vaccination with HAdV-55 cleared the virus faster in the respiratory tract, mitigated lung pathological changes. Finally, HAdV-55 infection was propagated among tree shrews. Our study demonstrated that the tree shrew is a permissive animal model for HAdV species B infection and may serve as a valuable platform for testing multiple anti-viral treatments.

Tree Shrew Is a Suitable Animal Model for the Study of Epstein Barr Virus.

Epstein-Barr virus (EBV) is a human herpesvirus that latently infects approximately 95% of adults and is associated with a spectrum of human diseases including Infectious Mononucleosis and a variety of malignancies. However, understanding the pathogenesis, vaccines and antiviral drugs for EBV-associated disease has been hampered by the lack of suitable animal models. Tree shrew is a novel laboratory animal with a close phylogenetic relationship to primates, which is a critical advantage for many animal models for human disease, especially viral infections. Herein, we first identified the key residues in the CR2 receptor that bind the gp350 protein and facilitate viral entry. We found that tree shrew shares 100% sequence identity with humans in these residues, which is much higher than rabbits (50%) and rats (25%). In vitro analysis showed that B lymphocytes of tree shrews are susceptible to EBV infection and replication, as well as EBV-enhanced cell proliferation. Moreover, results of in vivo experiments show that EBV infection in tree shrews resembles EBV infection in humans. The infected animals exhibited transient fever and loss of weight accompanied by neutropenia and high viremia levels during the acute phase of the viral infection. Thereafter, tree shrews acted as asymptomatic carriers of the virus in most cases that EBV-related protein could be detected in blood and tissues. However, a resurgence of EBV infection occurred at 49 dpi. Nanopore transcriptomic sequencing of peripheral blood in EBV-infected animals revealed the dynamic changes in biological processes occurring during EBV primary infection. Importantly, we find that neutrophil function was impaired in tree shrew model as well as human Infectious Mononucleosis datasets (GSE85599 and GSE45918). In addition, retrospective case reviews suggested that neutropenia may play an important role in EBV escaping host innate immune response, leading to long-term latent infection. Our findings demonstrated that tree shrew is a suitable animal model to evaluate the mechanisms of EBV infection, and for developing vaccines and therapeutic drugs against EBV.

Susceptibility of tree shrew to SARS-CoV-2 infection.

Since severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and Coronavirus disease (COVID-19) were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature particularly in female animals. Low levels of virus shedding and replication in tissues occurred in all three age groups. Notably, young tree shrews (6 months to 12 months) showed virus shedding at the earlier stage of infection than adult (2 years to 4 years) and old (5 years to 7 years) animals that had longer duration of virus shedding comparatively. Histopathological examine revealed that pulmonary abnormalities were the main changes but mild although slight lesions were also observed in other tissues. In summary, tree shrew is less susceptible to SARS-CoV-2 infection compared with the reported animal models and may not be a suitable animal for COVID-19 related researches. However, tree shrew may be a potential intermediate host of SARS-CoV-2 as an asymptomatic carrier.

COVID-19-like symptoms observed in Chinese tree shrews infected with SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic continues to pose a global threat to the human population. Identifying animal species susceptible to infection with the SARS-CoV-2/ HCoV-19 pathogen is essential for controlling the outbreak and for testing valid prophylactics or therapeutics based on animal model studies. Here, different aged Chinese tree shrews (adult group, 1 year old; old group, 5-6 years old), which are close relatives to primates, were infected with SARS-CoV-2. X-ray, viral shedding, laboratory, and histological analyses were performed on different days post-inoculation (dpi). Results showed that Chinese tree shrews could be infected by SARS-CoV-2. Lung infiltrates were visible in X-ray radiographs in most infected animals. Viral RNA was consistently detected in lung tissues from infected animals at 3, 5, and 7 dpi, along with alterations in related parameters from routine blood tests and serum biochemistry, including increased levels of aspartate aminotransferase (AST) and blood urea nitrogen (BUN). Histological analysis of lung tissues from animals at 3 dpi (adult group) and 7 dpi (old group) showed thickened alveolar septa and interstitial hemorrhage. Several differences were found between the two different aged groups in regard to viral shedding peak. Our results indicate that Chinese tree shrews have the potential to be used as animal models for SARS-CoV-2 infection.

Longitudinal transcriptomic characterization of viral genes in HSV-1 infected tree shrew trigeminal ganglia.

BACKGROUND: Following acute infection, Herpes Simplex virus-1 (HSV-1) establishes lifelong latency and recurrent reactivation in the sensory neurons of trigeminal ganglia (TG). Infected tree shrew differs from mouse and show characteristics similar to human infection. A detailed transcriptomic analysis of the tree shrew model could provide mechanistic insights into HSV-1 infection in humans. METHODS: We sequenced the transcriptome of infected TGs from tree shrews and mice, and 4 human donors, then examined viral genes expression up to 58 days in infected TGs from mouse and tree shrew, and compare the latency data with that in human TGs. RESULTS: Here, we found that all HSV-1 genes could be detected in mouse TGs during acute infection, but 22 viral genes necessary for viral transcription, replication and viral maturation were not expressed in tree shrew TGs during this stage. Importantly, during latency, we found that LAT could be detected both in mouse and tree shrew, but the latter also has an ICP0 transcript signal absent in mouse but present in human samples. Importantly, we observed that infected human and tree shrew TGs have a more similar LAT region transcription peak. More importantly, we observed that HSV-1 spontaneously reactivates from latently infected tree shrews with relatively high efficiency. CONCLUSIONS: These results represent the first longitudinal transcriptomic characterization of HSV-1 infection in during acute, latency and recurrent phases, and revealed that tree shrew infection has important similar features with human infection.

Isolation and identification of two new strains of mammalian orthoreovirus from Chinese tree shrews.

Chinese tree shrews have been used extensively in studies of different types of cancer and for the modeling of viral infections. In the present study, we report the isolation and characterization of two strains of mammalian orthoreovirus (MRV), MRV1/TS/2011 and MRV3/TS/2012, which were isolated from the feces of tree shrews in Yunnan, China. These two strains of MRV were isolated and cultured in both primary tree shrew intestinal epithelial cells (pTIECs) and primary tree shrew alveolar epithelial cells (pTAECs). A neutralization test using immunofluorescence was employed to determine the subtype of each isolate. Viral RNA was extracted and analyzed by polyacrylamide gel electrophoresis (PAGE), and the sequence was determined by next-generation sequencing for construction of a phylogenetic tree and analysis of gene polymorphism. Electron microscopy examination revealed the presence of virus particles with the typical morphological characteristics of MRV. Serotype analysis showed that strain MRV1/TS/2011 was of type I and strain MRV3/TS/2012 was of type III. A sequence comparison showed that the isolates were 25.4% identical in the S1 gene.

The tree shrew is a promising model for the study of influenza B virus infection.

BACKGROUND: Influenza B virus is a main causative pathogen of annual influenza epidemics, however, research on influenza B virus in general lags behind that on influenza A viruses, one of the important reasons is studies on influenza B viruses in animal models are limited. Here we investigated the tree shrew as a potential model for influenza B virus studies. METHODS: Tree shrews and ferrets were inoculated with either a Yamagata or Victoria lineage influenza B virus. Symptoms including nasal discharge and weight loss were observed. Nasal wash and respiratory tissues were collected at 2, 4 and 6 days post inoculation (DPI). Viral titers were measured in nasal washes and tissues were used for pathological examination and extraction of mRNA for measurement of cytokine expression. RESULTS: Clinical signs and pathological changes were also evident in the respiratory tracts of tree shrews and ferrets. Although nasal symptoms including sneezing and rhinorrhea were evident in ferrets infected with influenza B virus, tree shrews showed no significant respiratory symptoms, only milder nasal secretions appeared. Weight loss was observed in tree shrews but not ferrets. V0215 and Y12 replicated in all three animal (ferrets, tree shrews and mice) models with peak titers evident on 2DPI. There were no significant differences in peak viral titers in ferrets and tree shrews inoculated with Y12 at 2 and 4DPI, but viral titers were detected at 6DPI in tree shrews. Tree shrews infected with influenza B virus showed similar seroconversion and respiratory tract pathology to ferrets. Elevated levels of cytokines were detected in the tissues isolated from the respiratory tract after infection with either V0215 or Y12 compared to the levels in the uninfected control in both animals. Overall, the tree shrew was sensitive to infection and disease by influenza B virus. CONCLUSION: The tree shrew to be a promising model for influenza B virus research.

Infectivity of Zika virus on primary cells support tree shrew as animal model.

Zika virus (ZIKV) is a mosquito-borne flavivirus that caused the public health emergency. Recently, we have proved a novel small animal tree shrew was susceptive to ZIKV infection and presented the most common rash symptoms as ZIKV patients. Here we further cultured the primary cells from different tissues of this animal to determine the tissue tropism of ZIKV infection in vitro. The results showed that the primary cells from tree shrew kidney, lung, liver, skin and aorta were permissive to ZIKV infection and could support viral replication by the detection of viral specific RNA intra- and extra-cells. In comparing, the skin fibroblast and vascular endothelial cells were highly permissive to ZIKV infection with high releasing of active virus particles in supernatants proved by its infectivity in established neonatal mouse model. The expressions of ZIKV envelop and nonstructural protein-1, and the effects and strong immune response of primary tree shrew cells were also detected followed by ZIKV infection. These findings provide powerful in vitro cell-level evidence to support tree shrew as animal model of ZIKV infection and may help to explain the rash manifestations in vivo.

Characteristics of the tree shrew gut virome.

The tree shrew (Tupaia belangeri) has been proposed as an alternative laboratory animal to primates in biomedical research in recent years. However, characteristics of the tree shrew gut virome remain unclear. In this study, the metagenomic analysis method was used to identify the features of gut virome from fecal samples of this animal. Results showed that 5.80% of sequence reads in the libraries exhibited significant similarity to sequences deposited in the viral reference database (NCBI non-redundant nucleotide databases, viral protein databases and ACLAME database), and these reads were further classified into three major orders: Caudovirales (58.0%), Picornavirales (16.0%), and Herpesvirales (6.0%). Siphoviridae (46.0%), Myoviridae (45.0%), and Podoviridae (8.0%) comprised most Caudovirales. Picornaviridae (99.9%) and Herpesviridae (99.0%) were the primary families of Picornavirales and Herpesvirales, respectively. According to the host types and nucleic acid classifications, all of the related viruses in this study were divided into bacterial phage (61.83%), animal-specific virus (34.50%), plant-specific virus (0.09%), insect-specific virus (0.08%) and other viruses (3.50%). The dsDNA virus accounted for 51.13% of the total, followed by ssRNA (33.51%) and ssDNA virus (15.36%). This study provides an initial understanding of the community structure of the gut virome of tree shrew and a baseline for future tree shrew virus investigation.

Tree shrew as a new animal model to study the pathogenesis of avian influenza (H9N2) virus infection.

Outbreaks of avian influenza virus continue to pose threats to human health. Animal models such as the mouse, ferret, and macaque are used to understand the pathogenesis of avian influenza virus infection in humans. We previously reported that the tree shrew (Tupaia belangeri, family Tupaiidae), which is regarded as a "low-level primate", has α2,3- and α2,6-linked sialic acid receptor distributions similar to those of humans and is potentially a useful mammalian model for studying mild human influenza (H1N1) virus infection. In this study, we used the tree shrew experimental model to investigate the pathogenesis of avian influenza A (H9N2) virus infection and the effect of the E627K mutation in the PB2 gene, an adaptation to mammalian hosts. Evidence of disease, virus titers in the upper and lower respiratory tract, histopathology and induction of proinflammatory cytokines are described. We also established ex vivo culture models of tree shrew respiratory tissues to study the tropism and replication of the H9N2 virus. Our results demonstrated that the tree shrew is a viable new in vivo experimental model for avian influenza research that provides results comparable to those observed in ferrets. The disease spectrum and pathogenesis in tree shrews correlate well with what is observed in humans.

A study of Epstein-Barr virus infection in the Chinese tree shrew(Tupaia belangeri chinensis).

BACKGROUND: Epstein-Barr virus (EBV) is closely associated with many human diseases, including a variety of deadly human malignant tumours. However, due to the lack of ideal animal models,the biological characteristics of EBV, particularly its function in tumourigenesis, have not been determined. Chinese tree shrews (Tupaia belangeri chinensis), which are similar to primates, have been used to establish a variety of animal models and have recently received much attention. Here, we established tree shrews as a model for EBV infection by intravenous injection. METHODS: Ten tree shrews were inoculated with EBV by intravenous injection,and blood was collected at regular intervals thereafter from the femoral artery or vein to detect EBV markers. RESULTS: Eight of 10 tree shrews showed evidence of EBV infection. In the 8 EBV-infected tree shrews, EBV copy number increased intermittently or transiently, EBV-related gene expression was detected, and anti-EBV antibodies increased to varying degrees. Macroscopic hepatomegaly was observed in 1 tree shrew, splenomegaly was observed in 4 tree shrews, and enlarged mesenteric lymph nodes were observed in 3 tree shrews. Haematoxylin and eosin (HE) staining showed splenic corpuscle hyperplasia in the spleens of 4 tree shrews and inflammatory cell infiltration of the liver of 1 tree shrew and of the mesenteric lymph nodes of 3 tree shrews. EBER in situ hybridization(ISH) and immunohistochemical (IHC) staining showed that EBER-, LMP1- and EBNA2- positive cells were present in the spleens and mesenteric lymph nodes of some tree shrews. Western blotting (WB) revealed EBNA1-positive cells in the spleens of 4 tree shrews. EBV markers were not detected by HE, EBER-ISH or IHC in the lung or nasopharynx. CONCLUSIONS: These findings suggest that EBV can infect tree shrews via intravenous injection. The presented model offers some advantages for exploring the pathophysiology of EBV infection in humans.

The recombined cccDNA produced using minicircle technology mimicked HBV genome in structure and function closely.

HBV covalently closed circular DNA (cccDNA) is drug-resistant and responsible for viral persistence. To facilitate the development of anti-cccDNA drugs, we developed a minicircle DNA vector (MC)-based technology to produce large quantity of recombined cccDNA (rcccDNA) resembling closely to its wild-type counterpart both in structure and function. The rcccDNA differed to the wild-type cccDNA (wtcccDNA) only in that it carried an extra 36-bp DNA recombinant product attR upstream of the preC/C gene. Using a procedure similar to standard plasmid production, milligrams of rcccDNA can be generated in common laboratories conveniently. The rcccDNA demonstrated many essential biological features of wtcccDNA, including: (1) undergoing nucleation upon nucleus entry; (2) serving as template for production of all HBV RNAs and proteins; (3) deriving virions capable of infecting tree shrew, and subsequently producing viral mRNAs, proteins, rcccDNA and infectious virions. As an example to develop anti-cccDNA drugs, we used the Crispr/Cas9 system to provide clear-cut evidence that rcccDNA was cleaved by this DNA editing tool in vitro. In summary, we have developed a convenient technology to produce large quantity of rcccDNA as a surrogate of wtcccDNA for investigating HBV biology and developing treatment to eradicate this most wide-spreading virus.

Herpes Simplex Virus 1 Infection of Tree Shrews Differs from That of Mice in the Severity of Acute Infection and Viral Transcription in the Peripheral Nervous System.

UNLABELLED: Studies of herpes simplex virus (HSV) infections of humans are limited by the use of rodent models such as mice, rabbits, and guinea pigs. Tree shrews (Tupaia belangeri chinensis) are small mammals indigenous to southwest Asia. At behavioral, anatomical, genomic, and evolutionary levels, tree shrews are much closer to primates than rodents are, and tree shrews are susceptible to HSV infection. Thus, we have studied herpes simplex virus 1 (HSV-1) infection in the tree shrew trigeminal ganglion (TG) following ocular inoculation. In situ hybridization, PCR, and quantitative reverse transcription-PCR (qRT-PCR) analyses confirm that HSV-1 latently infects neurons of the TG. When explant cocultivation of trigeminal ganglia was performed, the virus was recovered after 5 days of cocultivation with high efficiency. Swabbing the corneas of latently infected tree shrews revealed that tree shrews shed virus spontaneously at low frequencies. However, tree shrews differ significantly from mice in the expression of key HSV-1 genes, including ICP0, ICP4, and latency-associated transcript (LAT). In acutely infected tree shrew TGs, no level of ICP4 was observed, suggesting the absence of infection or a very weak, acute infection compared to that of the mouse. Immunofluorescence staining with ICP4 monoclonal antibody, and immunohistochemistry detection by HSV-1 polyclonal antibodies, showed a lack of viral proteins in tree shrew TGs during both acute and latent phases of infection. Cultivation of supernatant from homogenized, acutely infected TGs with RS1 cells also exhibited an absence of infectious HSV-1 from tree shrew TGs. We conclude that the tree shrew has an undetectable, or a much weaker, acute infection in the TGs. Interestingly, compared to mice, tree shrew TGs express high levels of ICP0 transcript in addition to LAT during latency. However, the ICP0 transcript remained nuclear, and no ICP0 protein could be seen during the course of mouse and tree shrew TG infections. Taken together, these observations suggest that the tree shrew TG infection differs significantly from the existing rodent models. IMPORTANCE: Herpes simplex viruses (HSVs) establish lifelong infection in more than 80% of the human population, and their reactivation leads to oral and genital herpes. Currently, rodent models are the preferred models for latency studies. Rodents are distant from primates and may not fully represent human latency. The tree shrew is a small mammal, a prosimian primate, indigenous to southwest Asia. In an attempt to further develop the tree shrew as a useful model to study herpesvirus infection, we studied the establishment of latency and reactivation of HSV-1 in tree shrews following ocular inoculation. We found that the latent virus, which resides in the sensory neurons of the trigeminal ganglion, could be stress reactivated to produce infectious virus, following explant cocultivation and that spontaneous reactivation could be detected by cell culture of tears. Interestingly, the tree shrew model is quite different from the mouse model of HSV infection, in that the virus exhibited only a mild acute infection following inoculation with no detectable infectious virus from the sensory neurons. The mild infection may be more similar to human infection in that the sensory neurons continue to function after herpes reactivation and the affected skin tissue does not lose sensation. Our findings suggest that the tree shrew is a viable model to study HSV latency.

The tree shrew provides a useful alternative model for the study of influenza H1N1 virus.

BACKGROUND: The influenza pandemics have resulted in significant morbidity and mortality worldwide. Animal models are useful in the study of influenza virus pathogenesis. Because of various limitations in current laboratory animal models, it is essential to develop new alternative animal models for influenza virus research aimed at understanding the viral and host factors that contribute to virus infection in human. METHOD: We investigated the replicative efficiency of influenza H1N1 virus (classic strain (Influenza A/PR/8/34), seasonal influenza isolate (A/Guangzhou/GIRD/02/09) and swine-origin human influenza virus (A/Guangzhou/GIRD/07/09)) at Day1,2,4,6 and 9 p.i. using TCID50 and qPCR assay in tree shrew model. Body temperature was monitored in the morning and evening for 3 days before infection and for 14 days. Seroconversion was detected by determining the neutralizing antibody titers against the challenge viruses in the pre- and exposure serum samples collected before infection and at 14 days p.i., respectively. Lungs and tracheas of tree shews were collected at day 14 post p.i. for histopathological analysis. Lectinhistochemistry analysis was conducted to identify the distribution of SAα2,3 Gal and SAα2,6 Gal receptors in the lung and trachea. RESULTS: The infected tree shrew displayed mild or moderate systemic and respiratory symptoms and pathological changes in respiratory tracts. The human H1N1 influenza virus may replicate in the upper respiratory tract of tree shrews. Analysis of the receptors distribution in the respiratory tract of tree shrews by lectinhistochemistry showed that sialic acid (SA)α2,6-Gal receptors were widely distributed in the trachea and nasal mucosa, whereas (SA)α2,3-Gal receptor was the main receptor in the lung tissue. CONCLUSIONS: Based on these findings, tree shrew seemed to mimic well influenza virus infection in humans. We propose that tree shrews could be a useful alternative mammalian model to study pathogenesis of influenza H1N1 virus.

[Preliminary investigation of viruses to the wild tree shrews (Tupaia belangeri Chinese)].

Virological testing and monitoring is a fundamental part of quality control of experimental animals. However, there are few papers regarding the spectrum and status of natural infection in wild tree shrews with human and animal pathogenic viruses. Using enzyme-linked immunosorbent adsorption assay (ELISA), we tested sixty wild tree shrews captured from Qinglong, an outskirt region of Kunming, Yunnan Province, China for eleven viruses, including herpes simplex virus, coxsackie virus, influenza virus, HAV, HBV, HCV, HDV, dengue virus, hemorrhagic fever virus and measles virus. Our results showed that, in the serum samples, 22/60 (36.7%) and 1/60 (1.67%) were antibody positive for herpes simplex virus and coxsackie virus, respectively, and 4/60 (6.7%) were antigen positive for rotavirus in the feces. The remaining species of viruses were negative in these tree shrews. Based on these results, we propose that herpes simplex virus, coxsackie virus and cotavirus should be listed as top priority for routine virological monitoring of tree shrews.

[Progress on establishment of tree shrew (Tupaia) chronic infection with HBV in vivo].

Hepatitis B virus (HBV) infection is one of the important health problems worldwide, especially in China. Feasible and effective animal models of HBV infection in vivo are prerequisite for the HBV-related basic and clinical studies. Located in the highly prevalent region of HBV and hepatocellular carcinoma (HCC), the laboratory of Guangxi Cancer Institute has focused on the cause, pathogenesis and chemoprevention of HCC, and has started the work of establishing tree shrew (Tupaia) models of HBV infection in vivo since the early 1980s. This paper provides an overview of the research process, and highlights the new progress on the chronic infection of tree shrews after inoculated with HBV neonatally in vivo.

APOBEC1 and APOBEC3 cytidine deaminases as restriction factors for hepadnaviral genomes in non-humans in vivo.

Reverse transcription of the hepadnavirus RNA pre-genome means that nascent cDNA may be vulnerable to genetic editing by host cell APOBEC cytidine deaminases that have specificity single-stranded DNA as substrate. Hepatitis B virus (HBV) is particularly vulnerable to editing by APOBEC3G (hA3G) in late-stage disease where up to 35% of genomes can be edited. Yet, the organization of the A3 locus varies considerably among mammals with a single gene for the mouse and seven genes for Old and New World monkeys, which suggests that the outcome may be very variable for other natural hepadnavirus infections. In addition, there is the powerful mouse transgenic model of HBV replication (mHBV) that has proved to be immensely useful in understanding HBV immunopathogenesis. Here, we show that mHBV is edited in vivo by mAPOBEC1 (mA1) and not mAPOBEC3 (mA3), which follows from the fact that unlike humans, the mA1 gene is highly expressed in the liver. For woodchuck hepatitis virus, an mA3 ortholog is probably operative. For HBV-infected tree shrew primary liver cultures, the editing profile more resembles that observed in humans in keeping with fact that this species belongs to the order closest to Primates. There seems to be more genetic editing in liver or cell-associated genomes than serum or culture supernatants, suggesting that too much editing of virion cDNA might impede completion of DNA synthesis.

Efficient infection of tree shrew (Tupaia belangeri) with hepatitis C virus grown in cell culture or from patient plasma.

The generation of a new, cost-effective, non-primate, small-animal model would greatly facilitate research into hepatitis C virus (HCV) pathogenesis and the development of novel therapeutic and preventative technologies to control the increasing HCV threat to public health. Native HCV from patient plasma and HCV grown in cell culture (HCVcc) were used to inoculate adult tree shrews. Each animal was inoculated with one HCV genotype. Alanine aminotransferase (ALT) levels, HCV RNA and viral load were determined in the animals before and after inoculation. For native HCV, 16/18 inoculated tree shrews (89 %) became infected; 12/16 (75 %) of these animals became chronically infected, whilst infection was resolved in the remaining four (25 %). For HCVcc, infection occurred in 10/12 inoculated tree shrews (83 %) and chronic infection was observed in two of these animals. HCVcc from Huh7 cells showed a higher infectivity than that from HeLa cells. The animals inoculated with inadequate amounts of HCV were not infected in either native HCV or HCVcc experiments. Peak viral loads reached 10(3)-10(5) international units ml(-1) in chronically infected animals. ALT level changes reflected the normal fluctuation range in most animals. Thus, tree shrews without immunosuppression can be infected efficiently by native HCV and HCVcc when the animal is inoculated with an adequate amount of single-genotype HCV.