Tree Shrew Cells Transduced with Human CD4 and CCR5 Support Early Steps of HIV-1 Replication, but Viral Infectivity Is Restricted by APOBEC3.

The host range of human immunodeficiency virus type 1 (HIV-1) is narrow. Therefore, using ordinary animal models to study HIV-1 replication, pathogenesis, and therapy is impractical. The lack of applicable animal models for HIV-1 research spurred our investigation on whether tree shrews (Tupaia belangeri chinensis), which are susceptible to many types of human viruses, can act as an animal model for HIV-1. Here, we report that tree shrew primary cells are refractory to wild-type HIV-1 but support the early replication steps of HIV-1 pseudotyped with the vesicular stomatitis virus glycoprotein envelope (VSV-G), which can bypass entry receptors. The exogenous expression of human CD4 renders the tree shrew cell line infectible to X4-tropic HIV-1IIIB, suggesting that tree shrew CXCR4 is a functional HIV-1 coreceptor. However, tree shrew cells did not produce infectious HIV-1 progeny virions, even with the human CD4 receptor. Subsequently, we identified tree shrew (ts) apolipoprotein B editing catalytic polypeptide 3 (tsAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity, with virus infectivity reduced 10- to 1,000-fold. Unlike human APOBEC3G, the tsA3Z2c-Z1b protein was not degraded by the HIV-1 viral infectivity factor (Vif) but markedly restricted HIV-1 replication through mutagenicity and reverse transcription inhibition. The pooled knockout of tsA3Z2c-Z1b partially restored the infectivity of the HIV-1 progeny. This work suggests that tsAPOBEC3 proteins serve as an additional barrier to the development of HIV-1 tree shrew models, even when virus entry is overcome by exogenous expression of human CD4. IMPORTANCE The development of animal models is critical for studying human diseases and their pathogenesis and for evaluating drug and vaccine efficacy. For improved AIDS research, the ideal animal model of HIV-1 infection should be a small laboratory mammal that closely mimics virus replication in humans. Tree shrews exhibit considerable potential as animal models for the study of human diseases and therapeutic responses. Here, we report that human CD4-expressing tree shrew cells support the early steps of HIV-1 replication and that tree shrew CXCR4 is a functional coreceptor of HIV-1. However, tree shrew cells harbor additional restrictions that lead to the production of HIV-1 virions with low infectivity. Thus, the tsAPOBEC3 proteins are partial barriers to developing tree shrews as an HIV-1 model. Our results provide insight into the genetic basis of HIV inhibition in tree shrews and build a foundation for the establishment of gene-edited tree shrew HIV-1-infected models.

Coinfection with koala retrovirus subtypes A and B and its impact on captive koalas in Japanese zoos.

Koala retrovirus (KoRV) is unique among endogenous retroviruses because its endogenization is still active. Two major KoRV subtypes, KoRV-A and B, have been described, and KoRV-B is associated with disease and poses a health threat to koalas. Here, we investigated the co-prevalence of KoRV-A and KoRV-B, detected by type-specific PCR and sequencing, and their impact on the health of koalas in three Japanese zoos. We also investigated KoRV proviral loads and found varying amounts of genomic DNA (gDNA) in peripheral blood mononuclear cells (PBMCs). We found that 100% of the koalas examined were infected with KoRV-A and 60% (12/20) were coinfected with KoRV-B. The KoRV-A sequence was highly conserved, whereas the KoRV-B sequence varied among individuals. Interestingly, we observed possible vertical transmission of KoRV-B in one offspring in which the KoRV-B sequence was similar to that of the father but not the mother. Moreover, we characterized the KoRV growth patterns in concanavalin-A-stimulated PBMCs isolated from KoRV-B-coinfected or KoRV-B-uninfected koalas. We quantified the KoRV provirus in gDNA and the KoRV RNA copy numbers in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-seeding. As the study population is housed in captivity, a longitudinal study of these koalas may provide an opportunity to study the transmission mode of KoRV-B. In addition, we characterized KoRV isolates by infecting tupaia cells. The results suggested that tupaia may be used as an infection model for KoRV. Thus, this study may enhance our understanding of KoRV-B coinfection and transmission in the captive koalas.

Construction of complete Tupaia belangeri transcriptome database by whole-genome and comprehensive RNA sequencing.

The northern tree shrew (Tupaia belangeri) possesses high potential as an animal model of human diseases and biology, given its genetic similarity to primates. Although genetic information on the tree shrew has already been published, some of the entire coding sequences (CDSs) of tree shrew genes remained incomplete, and the reliability of these CDSs remained difficult to determine. To improve the determination of tree shrew CDSs, we performed sequencing of the whole-genome, mRNA, and total RNA and integrated the resulting data. Additionally, we established criteria for the selection of reliable CDSs and annotated these sequences by comparison to the human transcriptome, resulting in the identification of complete CDSs for 12,612 tree shrew genes and yielding a more accurate tree shrew genome database (TupaiaBase: http://tupaiabase.org ). Transcriptome profiles in hepatitis B virus infected tree shrew livers were analyzed for validation. Gene ontology analysis showed enriched transcriptional regulation at 1 day post-infection, namely in the "type I interferon signaling pathway". Moreover, a negative regulator of type I interferon, SOCS3, was induced. This work, which provides a tree shrew CDS database based on genomic DNA and RNA sequencing, is expected to serve as a powerful tool for further development of the tree shrew model.

Detection and genome characterization of two novel papillomaviruses and a novel polyomavirus in tree shrew (Tupaia belangeri chinensis) in China.

BACKGROUND: Papillomaviruses (PVs) and polyomaviruses (PyVs) infect diverse vertebrates including human and cause a broad spectrum of outcomes from asymptomatic infection to severe disease. There has been no PV and only one PyV detected in tree shrews, though the genomic properties of tree shrews are highly similar to those of the primates. METHODS: Swab and organ samples of tree shrews collected in the Yunnan Province of China, were tested by viral metagenomic analysis and random PCR to detect the presence of PVs and PyVs. By PCR amplification using specific primers, cloning, sequencing and assembling, genomes of two PVs and one PyV were identified in the samples. RESULTS: Two novel PVs and a novel PyV, named tree shrew papillomavirus 1 and 2 (TbelPV1 and TbelPV2) and polyomavirus 1 (TbelPyV1) were characterized in the Chinese tree shrew (Tupaia belangeri chinensis). The genomes of TbelPV1, TbelPV2, and TbelPyV1 are 7410 bp, 7526 bp, and 4982 bp in size, respectively. The TbelPV1 genome contains 7 putative open-reading frames (ORFs) coding for viral proteins E1, E2, E4, E6, E7, L1, and L2; the TbelPV2 genome contains 6 ORFs coding for viral proteins E1, E2, E6, E7, L1, and L2; and the TbelPyV1 genome codes for the typical small and large T antigens of PyV, as well as the VP1, VP2, and VP3 capsid proteins. Genomic comparison and phylogenetic analysis indicated that TbelPV1 and TbelPV2 represented 2 novel PV genera of Papillomaviridae, and TbelPyV1 represented a new species of genus Alphapolyomavirus. Our epidemiologic study indicated that TbelPV1 and TbelPV2 were both detected in oral swabs, while TbelPyV1 was detected in oral swabs and spleens. CONCLUSION: Two novel PVs (TbelPV1 and TbelPV2) and a novel PyV (TbelPyV) were discovered in tree shrews and their genomes were characterized. TbelPV1, TbelPV2, and TbelPyV1 have the highest similarity to Human papillomavirus type 63, Ursus maritimus papillomavirus 1, and Human polyomavirus 9, respectively. TbelPV1 and TbelPV2 only showed oral tropism, while TbelPyV1 showed oral and spleen tropism.

Zika Virus Infection in Tupaia belangeri Causes Dermatological Manifestations and Confers Protection against Secondary Infection.

Animal models of Zika virus (ZIKV) infection have recently been established in mice, guinea pigs, and nonhuman primates. Tree shrews (Tupaia belangeri) are an emerging experimental animal in biomedical applications, but their susceptibility to ZIKV infection has not been explored. In the present study, we show that subcutaneous inoculation of ZIKV led to rapid viremia and viral secretion in saliva, as well as to typical dermatological manifestations characterized by massive diffuse skin rash on the trunk. Global transcriptomic sequencing of peripheral blood mononuclear cells isolated from ZIKV-infected animals revealed systematic gene expression changes related to the inflammatory response and dermatological manifestations. Importantly, ZIKV infection readily triggered the production of high-titer neutralizing antibodies, thus preventing secondary homologous infection in tree shrews. However, neonatal tree shrews succumbed to ZIKV challenge upon intracerebral infection. The tree shrew model described here recapitulates the most common dermatological manifestations observed in ZIKV-infected patients and may greatly facilitate the elucidation of ZIKV pathogenesis and the development of novel vaccines and therapeutics.IMPORTANCE The reemergence of Zika virus (ZIKV) has caused a global public health crisis since 2016, and there are currently no vaccines or antiviral drugs to prevent or treat ZIKV infection. However, considerable advances have been made in understanding the biology and pathogenesis of ZIKV infection. In particular, various animal models have been successfully established to mimic ZIKV infection and its associated neurological diseases and to evaluate potential countermeasures. However, the clinical symptoms in these mouse and nonhuman primate models are different from the common clinical manifestations seen in human ZIKV patients; in particular, dermatological manifestations are rarely recapitulated in these animal models. Here, we developed a new animal model of ZIKV infection in tree shrews, a rat-sized, primate-related mammal. In vitro and in vivo characterization of ZIKV infection in tree shrews established a direct link between ZIKV infection and the immune responses and dermatological manifestations. The tree shrew model described here, as well as other available animal models, provides a valuable platform to study ZIKV pathogenesis and to evaluate vaccines and therapeutics.

Avian H5N1 influenza virus infection causes severe pneumonia in the Northern tree shrew (Tupaia belangeri).

Avian-origin influenza viruses like H5N1 and H7N9 often cause severe symptoms with high mortality in humans. Animal models are useful for clarification of the mechanisms of pathogenicity of these infections. In this study, to expand the potential utility of the Northern tree shrew (Tupaia belangeri) for influenza virus infection, we assessed the pathogenicity of H5N1 and H7N9 avian influenza viruses in tupaia. Infectious virus was detected continuously from nasal, oral, tracheal, and conjunctival swab samples in the animals infected with these viruses. H5N1 influenza virus infection of tupaia caused severe diffuse pneumonia with fever and weight loss. In contrast, H7N9 influenza virus infection caused focal pneumonia. The severity of pneumonia was correlated with proinflammatory cytokine transcript levels. These results indicated that tupaia can be another suitable animal model for avian influenza virus research.

A Tupaia paramyxovirus vector system for targeting and transgene expression.

Viruses from the diverse family of Paramyxoviridae include important pathogens and are applied in gene therapy and for cancer treatment. The Tupaia paramyxovirus (TPMV), isolated from the kidney of a tree shrew, does not infect human cells and neutralizing antibodies against other Paramyxoviridae do not cross-react with TPMV. Here, we present a vector system for de novo generation of infectious TPMV that allows for insertion of additional genes as well as targeting using antibody single-chain variable fragments. We show that the recombinant TPMV specifically infect cells expressing the targeted receptor and replicate in human cells. This vector system provides a valuable tool for both basic research and therapeutic applications.

Tree shrew, a potential animal model for hepatitis C, supports the infection and replication of HCV in vitro and in vivo.

The tree shrew (Tupaia belangeri chinensis), a small animal widely distributed in Southeast Asia and southwest China, has the potential to be developed as an animal model for hepatitis C. To determine the susceptibility of the tree shrew to hepatitis C virus (HCV) infection in vitro and in vivo, a well-established HCV, produced from the J6/JFH1-Huh7.5.1 culture system, was used to infect cultured primary tupaia hepatocytes (PTHs) and tree shrews. The in vitro results showed that HCV genomic RNA and HCV-specific nonstructural protein 5A (NS5A) could be detected in the PTH cell culture from days 3-15 post-infection, although the viral load was lower than that observed in Huh7.5.1 cell culture. The occurrence of five sense mutations [S391A, G397A, L402F and M405T in the hypervariable region 1 (HVR1) of envelope glycoprotein 2 and I2750M in NS5B] suggested that HCV undergoes genetic evolution during culture. Fourteen of the 30 experimental tree shrews (46.7 %) were found to be infected, although the HCV viremia was intermittent in vivo. A positive test for HCV RNA in liver tissue provided stronger evidence for HCV infection and replication in tree shrews. The results of an immunohistochemistry assay also demonstrated the presence of four HCV-specific proteins (Core, E2, NS3/4 and NS5A) in the hepatocytes of infected tree shrews. The pathological changes observed in the liver tissue of infected tree shrews could be considered to be representative symptoms of mild hepatitis. These results revealed that the tree shrew can be used as an animal model supporting the infection and replication of HCV in vitro and in vivo.

Oxidative Stress and Immune Responses During Hepatitis C Virus Infection in Tupaia belangeri.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. To address the molecular basis of HCV pathogenesis using tupaias (Tupaia belangeri), we characterized host responses upon HCV infection. Adult tupaias were infected with HCV genotypes 1a, 1b, 2a, or 4a. Viral RNA, alanine aminotransferase, anti-HCV core and anti-nonstructural protein NS3 antibody titres, reactive oxygen species (ROS), and anti-3ß-hydroxysterol-Δ24reductase (DHCR24) antibody levels were measured at 2-week intervals from 0 to 41 weeks postinfection. All HCV genotypes established infections and showed intermittent HCV propagation. Moreover, all tupaias produced anti-core and anti-NS3 antibodies. ROS levels in sera and livers were significantly increased, resulting in induction of DHCR24 antibody production. Similarly, lymphocytic infiltration, disturbance of hepatic cords, and initiation of fibrosis were observed in livers from HCV-infected tupaias. Intrahepatic levels of Toll-like receptors 3, 7, and 8 were significantly increased in all HCV-infected tupaias. However, interferon-ß was only significantly upregulated in HCV1a- and HCV2a-infected tupaias, accompanied by downregulation of sodium taurocholate cotransporting polypeptide. Thus, our findings showed that humoral and innate immune responses to HCV infection, ROS induction, and subsequent increases in DHCR24 auto-antibody production occurred in our tupaia model, providing novel insights into understanding HCV pathogenesis.

Susceptibility and initial immune response of Tupaia belangeri cells to dengue virus infection.

Dengue is an emerging disease of great public health significance worldwide. The lack of a suitable infection model has hampered dengue virus (DENV) pathogenesis study, and developing a suitable small animal model has been a long-standing challenge. The aim of this study was to develop a feasible experimental model of DENV infection using Tupaia belangeri. The susceptibility of tupaia to DENV infection and characteristics of its innate immune response were examined in vitro. We found that tupaia fibroblast cells support replication of DENV serotypes 1-4 with a linear increase in viral load 24-96h post-infection in both cells and culture supernatants. DENV-2 resulted in the highest viral growth among all serotypes. To characterize the innate immune response in tupaia cells during the early phase of DENV infection, we first evaluated the evolutionary relationship between tupaia Toll-like receptors (TLR1-9) and those of other mammalian species. Phylogenetic analysis showed that tupaia TLRs are evolutionarily much closer to human than they are to rodent. We next established an innate immune response measurement system by assessing the mRNA expression of TLR1-9 and four cytokines in DENV-infected tupaia cells. All serotypes induced the upregulation of TLR8 mRNA expression in infected tupaia cells. Silencing of TLR8 led to an increase in viral replication, indicating the existence of antiviral response through TLR8 on DENV infection. Although upregulation of IFN-ß and IL-6 expression was only observed in DENV-1 infected cells and a significant suppression of TNF-α was observed in DENV-2 infected cells alone, IL-8 was upregulated in all DENV-1-4. Thus, this study demonstrates for the first time the susceptibility of tupaia cells to DENV infections and the role of TLR8 in the anti-viral response of tupaia cells to DENV. These findings demonstrate the potential utility of tupaia as a model for DENV research in the future.

Property of hepatitis B virus replication in Tupaia belangeri hepatocytes.

The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3-6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10(5) copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10(4)-10(6) copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10(3) copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV.

Chronic hepatitis B virus infection and occurrence of hepatocellular carcinoma in tree shrews (Tupaia belangeri chinensis).

BACKGROUND: Hepatitis B virus (HBV) infection has been believed as a major cause of hepatocellular carcinoma (HCC) for a long time, however, the evidences of which are mostly from clinical and epidemiological investigations while there is no evidence from animal experiments. Tree shrew (Tupaia) is a small animal closely related to primates evolutionarily, with about 8 years of lifespan. Our previous study proved that tree shrews can be chronically HBV-infected after being inoculated neonatally with HBV. The present study reports the further results from the longer-term observation of these animals. METHODS: Neonatal tree shrews were inoculated with sera from HBV-infected patient or tree shrew. Their serum samples and liver biopsies were collected periodically for detection of HBV markers as well as for histopathological and immunohistochemical examinations. Group A consisted of six tree shrews with chronic HBV-infection, and group B consisted of nine tree shrews without chronic HBV infection. RESULTS: Periodical examinations on serum and liver biopsies of the animals in group A showed the progress of HBV infection, and two cases of HCC occurred at their late stage of life. The courses of HBV infection and the hepatic histopathological and immunohistochemical changes in the tree shrews were similar to those in humans. In contrast, neither HCC nor obvious hepatitis histopathological change was found among the tree shrews in group B. CONCLUSIONS: The course of HBV infection and the features of HCC discovered in tree shrews are similar to those of chronically HBV-infected humans. The tree shrew model might be used to investigate the underlying mechanisms favoring susceptibility for chronic HBV infection and disease progression.

microRNA expression in hepatitis B virus infected primary treeshrew hepatocytes and the independence of intracellular miR-122 level for de novo HBV infection in culture.

Infection of Hepatitis B virus (HBV) in hepatocytes has been known to be controlled by multiple cellular factors, while the relationship of the infection and liver microRNAs remains obscure. In this study, a miRNA database, containing 168 unique mature miRNA members from primary hepatocytes of a primate-like animal, northern treeshrew (Tupaia belangeri) that is the only species susceptible for HBV infection other than human and chimpanzee, was established. The relative level of a liver predominant microRNA, miR-122, was markedly increased upon HBV infection of the primary tupaia hepatocyte (PTH). However, introducing neither miR-122 nor its antagonist anti-miR-122 into PTHs, or, HepG2-NTCP that is HepG2 cells with the newly identified receptor sodium taurocholate cotransporting polypeptide (NTCP) did not alter the viral infection on these cells. These data suggest that de novo HBV infection of cultured hepatocytes does not depend on the expression level of intracellular miR-122 of the target cells.

Xanthohumol, a main prenylated chalcone from hops, reduces liver damage and modulates oxidative reaction and apoptosis in hepatitis C virus infected Tupaia belangeri.

Hepatitis C virus (HCV) infection in Tupaia belangeri (Tupaia) represents an important model of HCV infection. Xanthohumol (XN), a major prenylated chalcone from hops, has various biological activities including hepatopreventive and anti-viral activities. In this study, Tupaias infected with HCV RNA positive serum were used to evaluate the effects of XN on liver damage, oxidative reaction, apoptosis and viral protein expression in liver tissues. The Tupaias inoculated with HCV positive serum had elevated serum aminotransferase levels and inflammation, especially hepatic steatosis, and HCV core protein expression in liver tissue. In the animals inoculated with HCV positive serum, XN significantly decreased aminotransferase levels, histological activity index, hepatic steatosis score and transforming growth factor ß1 expression in liver tissue compared with the animals without XN intervention. XN reduced HCV core protein expression in liver tissue compared with those without XN intervention but the difference was not significant. XN significantly decreased malondialdehyde, potentiated superoxide dismutase and glutathione peroxidase, reduced Bax expression, promoted Bcl-xL and inhibited caspase 3 activity in liver tissues compared with the animals without XN intervention. These results indicate that XN may effectively improve hepatic inflammation, steatosis and fibrosis induced by HCV in Tupaias primarily through inhibition of oxidative reaction and regulation of apoptosis and possible suppression of hepatic stellate cell activation. The anti-HCV potential of XN needs further investigation.

Foamy virus in the tree shrew Tupaia belangeri is highly related to simian foamy virus in Macaca mulatta.

Foamy viruses (FVs) are ancient retrovirus that infect most nonhuman primates and several animals, but are rarely reported in tree shrew Tupaia belangeri. In the present study, foamy virus was detected in tree shrew. Phylogenetic analysis indicated that FVtup shared the highest homology with SFVmac (99.3%) in China. The discovery of FVtup indicated that the tree shrew is a new host of foamy virus. FVtup is highly prevalent in Tupaia in China and there is the possibility of cross-species transmission from nonhuman primate to Tupaia.

Sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis B virus infection of Tupaia hepatocytes.

Primary Tupaia hepatocytes (PTHs) are susceptible to woolly monkey hepatitis B virus (WMHBV) infection, but the identity of the cellular receptor(s) mediating WMHBV infection of PTHs remains unclear. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for human hepatitis B virus (HBV) infection of primary human and Tupaia hepatocytes. In this study, a synthetic pre-S1 peptide from WMHBV was found to bind specifically to cells expressing Tupaia NTCP (tsNTCP) and it efficiently blocked WMHBV entry into PTHs; silencing of tsNTCP in PTHs significantly inhibited WMHBV infection. Ectopic expression of tsNTCP rendered HepG2 cells susceptible to WMHBV infection. These data demonstrate that tsNTCP is a functional receptor for WMHBV infection of PTHs. The result also indicates that NTCP's orthologs likely act as a common cellular receptor for all known primate hepadnaviruses.

[Isolation and identification of Tupaia orthoreovirus].

Pathogenic viruses can harm acutely the life and health of laboratory tree shrews acutely; however, few papers exist regarding natural pathogenic virus infection in this species. Six fecal samples obtained from dead tree shrews were collected. The fecal supernatant infected Vero cell line resulted in cytopathic effects (CPE) after 72 h. The CPE included granulating, shrinking, rounding, seining and falling off. Electron microscopy showed the isolation was spherical, double-layered capsid, and about 75 nm in diameter. The purified isolation genome was 10 segments in a typical 3:3:4 arrangements, as shown by polyacrylamide gel electrophoresis (PAGE). The isolation was confirmed by RT-PCR assays targeting the conserved region of the L1 gene, sequence analysis and reconstruction of a phylogenetic tree. The isolation was a Tupaia Orthoreovirus (TRV), belonging to Mammalian Orthoreovirus (MRV). The obtained strain had the closest phylogenetic relationship to the MRV strain T3/Bat/Germany/342/08. As a zoonotic virus, the novel TRV strain was first isolated from wild tree shrews, which is significant for promoting tree shrew standardization and providing scientific data for preventing zoonotic tree shrew-to-human transmission.

Development of measles virus-based shielded oncolytic vectors: suitability of other paramyxovirus glycoproteins.

Antibody-mediated neutralization may interfere with the efficacy of measles virus (MV) oncolysis. To circumvent vector neutralization, we sought to exchange the envelope glycoproteins, hemagglutinin (H) and fusion (F), with those from the non-crossreactive Tupaia paramyxovirus (TPMV). To sustain efficient particle assembly, we generated hybrid glycoproteins with the MV cytoplasmic tails and the TPMV ectodomains. Hybrid F proteins that partially retained fusion function, and hybrid H proteins that retained fusion support activity, were generated. However, when used in combination, the hybrid proteins did not support membrane fusion. An alternative strategy was developed based on a hybrid F protein and a truncated H protein that supported cell-cell fusion. A hybrid virus expressing these two proteins was rescued, and was able to spread by cell fusion; however, it was only capable of producing minimal amounts of particles. Lack of specific interactions between the matrix and the H protein, in combination with suboptimal F-protein processing and inefficient glycoprotein transport in the rescue cells, accounted for inefficient particle production. Ultimately, this interferes with applications for oncolytic virotherapy. Alternative strategies for the generation of shielded MV are discussed.

Metabolomic analysis of key regulatory metabolites in hepatitis C virus-infected tree shrews.

Metabolomics is a powerful new technology that allows the assessment of global low-molecular-weight metabolites in a biological system and which shows great potential in biomarker discovery. Analysis of the key metabolites in body fluids has become an important part of improving the diagnosis, prognosis, and therapy of diseases. Hepatitis C virus (HCV) is a major leading cause of liver disease worldwide and a serious burden on public health. However, the lack of a small-animal model has hampered the analysis of HCV pathogenesis. We hypothesize that an animal model (Tupaia belangeri chinensis) of HCV would produce a unique characterization of metabolic phenotypes. Ultra-performance liquid-chromatography/electrospray ionization-SYNAPT-high-definition mass spectrometry (UPLC/ESI-SYNAPT-HDMS) coupled with pattern recognition methods and system analysis was carried out to obtain comprehensive metabolomics profiling and pathways of large biological data sets. Taurine, hypotaurine, ether lipid, glycerophospholipid, arachidonic acid, tryptophan, and primary bile acid metabolism pathways were acutely perturbed, and 38 differential metabolites were identified. More important, five metabolite markers were selected via the "significance analysis for microarrays" method as the most discriminant and interesting biomarkers that were effective for the diagnosis of HCV. Network construction has led to the integration of metabolites associated with the multiple perturbation pathways. Integrated network analysis of the key metabolites yields highly related signaling pathways associated with the differentially expressed proteins, which suggests that the creation of new treatment paradigms targeting and activating these networks in their entirety, rather than single proteins, might be necessary for controlling and treating HCV efficiently.

Targeted lentiviral vectors pseudotyped with the Tupaia paramyxovirus glycoproteins.

Lentiviral vectors are vectors of choice for many gene therapy applications. Recently, efficient targeting of lentiviral vectors pseudotyped with the Measles virus (MV) glycoproteins has been reported. However, MV antibodies in patients might limit the clinical use of these vectors. We demonstrate here that lentiviral vectors can also be pseudotyped with the glycoproteins of Tupaia paramyxovirus (TPMV), the hemagglutinin (H) and fusion (F) protein. As this animal paramyxovirus has no known close relatives in humans, we do not expect TPMV antibodies in patients. Because TPMV normally does not infect human cells, 'detargeting' from natural receptors is unnecessary. Similar to the MV system, TPMV glycoproteins can mediate targeted cell entry by displaying different single-chain antibodies (scAb) directed against surface molecules on target cells on the viral hemagglutinin. We generated a panel of H and F proteins with truncated cytoplasmic tails and determined the variants that efficiently pseudotyped lentiviral vectors. The B-cell marker CD20 was used as a model antigen, and CD20-targeted TPMV vectors selectively transduced CD20-positive cells, including quiescent primary human B-cells. Lentiviral vectors pseudotyped with targeted TPMV envelope proteins might be a valuable vector choice when systemic application of targeted lentiviral vectors in humans is required.