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Yuying Liang - One of the best experts on this subject based on the ideXlab platform.
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Pichinde Virus Infection of Outbred Hartley Guinea Pigs as a Surrogate Animal Model for Human Lassa Fever: Histopathological and Immunohistochemical Analyses.
Pathogenetics, 2020Co-Authors: Wun-ju Shieh, Shuiyun Lan, Sherif R. Zaki, Yuying LiangAbstract:Lassa Virus (LASV) is a mammarenaVirus (arenaVirus) that causes zoonotic infection in humans that can lead to fatal hemorrhagic Lassa fever (LF) disease. Currently, there are no FDA-approved vaccines or therapeutics against LASV. Development of treatments against LF and other related arenaVirus-induced hemorrhagic fevers (AHFs) requires relevant animal models that can recapitulate clinical and pathological features of AHF diseases in humans. Laboratory mice are generally resistant to LASV infection, and non-human primates, while being a good animal model for LF, are limited by their high cost. Here, we describe a small, affordable, and convenient animal model that is based on outbred Hartley guinea pigs infected with Pichinde Virus (PICV), a mammarenaVirus that is non-pathogenic in humans, for use as a surrogate model of human LF. We conducted a detailed analysis of tissue histopathology and immunohistochemical analysis of different organs of outbred Hartley guinea pigs infected with different PICV strains that show differential disease phenotypes and pathologies. Comparing to infection with the avirulent PICV strain (P2 or rP2), animals infected with the virulent strain (P18 or rP18) show extensive pathological changes in different organs that sustain high levels of Virus replication. The similarity of tissue pathology and viral antigen distribution between the virulent PICV–guinea pig model and lethal human LASV infection supports a role of this small animal model as a surrogate model of studying human LF in order to understand its pathogenesis and for evaluating potential preventative and therapeutic options against AHFs.
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Virulent infection of outbred Hartley guinea pigs with recombinant Pichinde Virus as a surrogate small animal model for human Lassa fever
Virulence, 2020Co-Authors: Shuiyun Lan, Qinfeng Huang, Wun-ju Shieh, Sherif R. Zaki, Yuying LiangAbstract:ArenaViruses, such as Lassa Virus (LASV), can cause severe and fatal hemorrhagic fevers (e.g., Lassa fever, LF) in humans with no vaccines or therapeutics. Research on arenaVirus-induced hemorrhagic fevers (AHFs) has been hampered by the highly virulent nature of these viral pathogens, which require high biocontainment laboratory, and the lack of an immune-competent small animal model that can recapitulate AHF disease and pathological features. Guinea pig infected with Pichinde Virus (PICV), an arenaVirus that does not cause disease in humans, has been established as a convenient surrogate animal model for AHFs as it can be handled in a conventional laboratory. The PICV strain P18, derived from sequential passaging of the Virus 18 times in strain 13 inbred guinea pigs, causes severe febrile illness in guinea pigs that is reminiscent of lethal LF in humans. As inbred guinea pigs are not readily available and are difficult to maintain, outbred Hartley guinea pigs have been used but they show a high degree of disease heterogeneity upon virulent P18 PICV infection. Here, we describe an improved outbred guinea-pig infection model using recombinant rP18 PICV generated by reverse genetics technique followed by plaque purification, which consistently shows >90% mortality and virulent infection. Comprehensive virological, histopathological, and immunohistochemical analyses of the rP18-Virus infected animals show similar features of human LASV infection. Our data demonstrate that this improved animal model can serve as a safe, affordable, and convenient surrogate small animal model for studying human LF pathogenesis and for evaluating efficacy of preventative or therapeutic approaches.
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Establishment of Bisegmented and Trisegmented Reverse Genetics Systems to Generate Recombinant Pichindé Viruses.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Rekha Dhanwani, Shuiyun Lan, Junjie Shao, Qinfeng Huang, Yanqing Zhou, Yuying LiangAbstract:Pichinde Virus (PICV), isolated from rice rats in Colombia, South America, is an enveloped arenaVirus with a bisegmented RNA genome. The large (L) genomic segment encodes the Z matrix protein and the L RNA-dependent RNA polymerase, whereas the small (S) genomic segment encodes the nucleoprotein (NP) and the glycoprotein (GPC). This article describes the successful development of reverse genetics systems to generate recombinant PICV with either a bisegmented or trisegmented genome. We have successfully demonstrated that these systems can generate high-titered and genetically stable replication-competent Viruses from plasmid transfection into appropriate cell lines. These systems demonstrate the power and versatility of reverse genetic technology to generate recombinant arenaViruses for use in pathogenesis studies and as new viral vaccine vectors.
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Assays to Assess Arenaviral Glycoprotein Function
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Junjie Shao, Xiaoying Liu, Yuying LiangAbstract:ArenaViruses, such as Lassa Virus (LASV) and Pichinde Virus (PICV), are enveloped Viruses with a bi-segmented ambisense RNA genome. The large (L) genomic segment encodes the Z matrix protein and the L RNA-dependent RNA polymerase, whereas the small (S) genomic segment encodes the nucleoprotein (NP) and the glycoprotein precursor complex (GPC). GPC is processed by signal peptidase in the endoplasmic reticulum into the stable signal peptide (SSP) and GP1/GP2, which is further cleaved by the Golgi-resident subtilisin kexin isozyme-1 (SKI-1)/site-1 protease (S1P) into the cellular receptor-recognition subunit GP1 and the transmembrane subunit GP2, which helps promote the membrane fusion reaction to allow Virus entry into the cell. This article describes assays to assess PICV GPC expression, proteolytic processing, fusion function, and GPC-mediated Virus-like particle (VLP) entry into cells under tissue-culture conditions.
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Recombinant tri-segmented Pichinde Virus as a novel live viral vaccine platform
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Rekha Dhanwani, Yuying LiangAbstract:Pichinde Virus (PICV) is a nonpathogenic arenaVirus with a bi-segmented RNA genome (L and S segments) that encodes four viral genes. We have developed a reverse genetics system to generate recombinant tri-segmented PICV (rP18tri) that packages three RNA segments (L, S1, and S2) and can encode up to two foreign genes. Using influenza Virus HA and NP as model antigens, we show that the rP18tri vector can induce strong humoral and cell-mediated immunity, which further increases upon a booster dose. We propose that this novel rP18tri vector can be developed into a useful vaccine platform for other antigens, particularly when strong cellular immunity and prime-boost vaccination strategy are desired.
Judith F. Aronson - One of the best experts on this subject based on the ideXlab platform.
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proteomic analysis of Pichinde Virus infection identifies differential expression of prothymosin alpha
BioMed Research International, 2010Co-Authors: Gavin C. Bowick, Judith F. Aronson, David G. Gorenstein, Bruce A. Luxon, Kizhake V Soman, Heling Wang, Lee Lomas, Norbert K. HerzogAbstract:The arenaViruses include a number of important pathogens including Lassa Virus and Junin Virus. Presently, the only treatment is supportive care and the antiviral Ribavirin. In the event of an epidemic, patient triage may be required to more effectively manage resources; the development of prognostic biomarker signatures, correlating with disease severity, would allow rational triage. Using a pair of arenaViruses, which cause mild or severe disease, we analyzed extracts from infected cells using SELDI mass spectrometry to characterize potential biomarker profiles. EDGE analysis was used to analyze longitudinal expression differences. Extracts from infected guinea pigs revealed protein peaks which could discriminate between mild or severe infection, and between times post-infection. Tandem mass-spectrometry identified several peaks, including the transcriptional regulator prothymosin-α. Further investigation revealed differences in secretion of this peptide. These data show proof of concept that proteomic profiling of host markers could be used as prognostic markers of infectious disease.
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Pichindé Virus is trafficked through a dynamin 2 endocytic pathway that is dependent on cellular Rab5- and Rab7-mediated endosomes.
Archives of virology, 2008Co-Authors: Eric M. Vela, Lihong Zhang, Tonya M. Colpitts, Robert A. Davey, Judith F. AronsonAbstract:Pichinde Virus (PICV) is a New World arenaVirus that has been shown to enter cells through a clathrin-dependent endocytic pathway. In this study, we determined that PICV is trafficked through the cellular dynamin 2 (dyn2) endocytic pathway. Additionally, the data suggest that PICV entry is pH-dependent and that the Virus travels through Rab5-mediated early and Rab7-mediated late endosomes. In all, this study characterizes the endocytic pathway utilized by the arenaVirus PICV.
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Thioaptamer decoy targeting of AP-1 proteins influences cytokine expression and the outcome of arenaVirus infections
Journal of General Virology, 2007Co-Authors: Susan M. Fennewald, Judith F. Aronson, Erin P. Scott, Lihong Zhang, Xianbin Yang, David G. Gorenstein, Bruce A. Luxon, Robert E. Shope, David W.c. Beasley, Alan D.t. BarrettAbstract:Viral haemorrhagic fever (VHF) is caused by a number of Viruses, including arenaViruses. The pathogenesis is believed to involve dysregulation of cytokine production. The arenaViruses Lassa Virus and Pichinde Virus have a tropism for macrophages and other reticuloendothelial cells and both appear to suppress the normal macrophage response to Virus infection. A decoy thioaptamer, XBY-S2, was developed and was found to bind to AP-1 transcription factor proteins. The P388D1 macrophage-like cell line contains members of the AP-1 family which may act as negative regulators of AP-1-controlled transcription. XBY-S2 was found to bind to Fra-2 and JunB, and enhance the induction of cytokines IL-6, IL-8 and TNF-alpha, while reducing the binding to AP-1 promoter elements. Administration of XBY-S2 to Pichinde Virus-infected guinea pigs resulted in a significant reduction in Pichinde Virus-induced mortality and enhanced the expression of cytokines from primary guinea pig macrophages, which may contribute to its ability to increase survival of Pichinde Virus-infected guinea pigs. These data demonstrate a proof of concept that thioaptamers can be used to modulate the outcome of in vivo viral infections by arenaViruses by the manipulation of transcription factors involved in the regulation of the immune response.
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Differential Signaling Networks Induced by Mild and Lethal Hemorrhagic Fever Virus Infections
Journal of virology, 2006Co-Authors: Gavin C. Bowick, Judith F. Aronson, Susan M. Fennewald, David G. Gorenstein, Bruce A. Luxon, Barry L. Elsom, Norbert K. HerzogAbstract:The family Arenaviridae includes several National Institutes of Allergy and Infections Diseases category A select agents which cause hemorrhagic fever. There are few vaccines available, and treatment is limited to ribavirin, which varies in efficacy. Development of new antiviral compounds has been hindered by a lack of understanding of the molecular basis of pathogenesis. We used two variants of Pichinde Virus, one attenuated and one virulent in the guinea pig model, to delineate the host determinants which lead to either viral clearance or lethal disease. By analyzing protein level changes using pathway analysis, we have identified key intermediates which may be targets for therapeutic intervention.
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Alterations in NF-κB and RBP-Jκ by arenaVirus infection of macrophages in vitro and in vivo
Journal of virology, 2002Co-Authors: Susan M. Fennewald, Judith F. Aronson, Lihong Zhang, Norbert K. HerzogAbstract:Pichinde Virus is an arenaVirus that infects guinea pigs and serves as an animal model for human Lassa fever. An attenuated Pichinde Virus variant (P2) and a virulent variant (P18) are being used to delineate pathogenic mechanisms that culminate in shock. In guinea pigs, the infection has been shown to begin in peritoneal macrophages following intraperitoneal inoculation and then spreads to the spleen and other reticuloendothelial organs. We show here that infection of the murine monocytic cell line P388D1 with either Pichinde Virus variant resulted in the induction of inflammatory cytokines and effectors, including interleukin-6 and tumor necrosis factor alpha. Since these genes are regulated in part by the cellular transcription factors NF-κB and RBP-Jκ, we compared the activities of NF-κB and RBP-Jκ in P388D1 cells following infection with Pichinde Virus. The attenuated P2 Virus inhibited NF-κB activation and caused a shift in the size of the RBP-Jκ complex. The virulent P18 Virus showed less inhibition of NF-κB and failed to alter the size of the RBP-Jκ complex. Peritoneal cells from P2-infected guinea pigs showed induction of NF-κB RelA/p50 heterodimer and p50/p50 homodimer and manifested an increase in the size of RBP-Jκ. By contrast, P18 induced large amounts of the NF-κB p50/p50 dimer but failed to induce RelA/p50 or to cause an increase in the RBP-Jκ size. Taken together, these changes suggest that the attenuated viral strain induces an “activation” of macrophages, while the virulent form of the Virus does not.
Jeffrey W. Koehler - One of the best experts on this subject based on the ideXlab platform.
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Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro Virus and Pichinde Virus
Virology Journal, 2016Co-Authors: Christopher P. Stefan, Kitty Chase, Susan Coyne, David A. Kulesh, Timothy D. Minogue, Jeffrey W. KoehlerAbstract:Background Research with high biocontainment pathogens such as Rift Valley fever Virus (RVFV) and Lassa Virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro Virus (PTV) for RVFV infection and Pichinde Virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices. Methods Total nucleic acid from Virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV Virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/μl for PTV and PICV. Results Optimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors. Conclusions PTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies.
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Development of real-time reverse transcriptase qPCR assays for the detection of Punta Toro Virus and Pichinde Virus
Virology journal, 2016Co-Authors: Christopher P. Stefan, Kitty Chase, David A. Kulesh, Timothy D. Minogue, Susan R. Coyne, Jeffrey W. KoehlerAbstract:Research with high biocontainment pathogens such as Rift Valley fever Virus (RVFV) and Lassa Virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro Virus (PTV) for RVFV infection and Pichinde Virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices. Total nucleic acid from Virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV Virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/μl for PTV and PICV. Optimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors. PTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies.
Norbert K. Herzog - One of the best experts on this subject based on the ideXlab platform.
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proteomic analysis of Pichinde Virus infection identifies differential expression of prothymosin alpha
BioMed Research International, 2010Co-Authors: Gavin C. Bowick, Judith F. Aronson, David G. Gorenstein, Bruce A. Luxon, Kizhake V Soman, Heling Wang, Lee Lomas, Norbert K. HerzogAbstract:The arenaViruses include a number of important pathogens including Lassa Virus and Junin Virus. Presently, the only treatment is supportive care and the antiviral Ribavirin. In the event of an epidemic, patient triage may be required to more effectively manage resources; the development of prognostic biomarker signatures, correlating with disease severity, would allow rational triage. Using a pair of arenaViruses, which cause mild or severe disease, we analyzed extracts from infected cells using SELDI mass spectrometry to characterize potential biomarker profiles. EDGE analysis was used to analyze longitudinal expression differences. Extracts from infected guinea pigs revealed protein peaks which could discriminate between mild or severe infection, and between times post-infection. Tandem mass-spectrometry identified several peaks, including the transcriptional regulator prothymosin-α. Further investigation revealed differences in secretion of this peptide. These data show proof of concept that proteomic profiling of host markers could be used as prognostic markers of infectious disease.
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Differential Signaling Networks Induced by Mild and Lethal Hemorrhagic Fever Virus Infections
Journal of virology, 2006Co-Authors: Gavin C. Bowick, Judith F. Aronson, Susan M. Fennewald, David G. Gorenstein, Bruce A. Luxon, Barry L. Elsom, Norbert K. HerzogAbstract:The family Arenaviridae includes several National Institutes of Allergy and Infections Diseases category A select agents which cause hemorrhagic fever. There are few vaccines available, and treatment is limited to ribavirin, which varies in efficacy. Development of new antiviral compounds has been hindered by a lack of understanding of the molecular basis of pathogenesis. We used two variants of Pichinde Virus, one attenuated and one virulent in the guinea pig model, to delineate the host determinants which lead to either viral clearance or lethal disease. By analyzing protein level changes using pathway analysis, we have identified key intermediates which may be targets for therapeutic intervention.
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Alterations in NF-κB and RBP-Jκ by arenaVirus infection of macrophages in vitro and in vivo
Journal of virology, 2002Co-Authors: Susan M. Fennewald, Judith F. Aronson, Lihong Zhang, Norbert K. HerzogAbstract:Pichinde Virus is an arenaVirus that infects guinea pigs and serves as an animal model for human Lassa fever. An attenuated Pichinde Virus variant (P2) and a virulent variant (P18) are being used to delineate pathogenic mechanisms that culminate in shock. In guinea pigs, the infection has been shown to begin in peritoneal macrophages following intraperitoneal inoculation and then spreads to the spleen and other reticuloendothelial organs. We show here that infection of the murine monocytic cell line P388D1 with either Pichinde Virus variant resulted in the induction of inflammatory cytokines and effectors, including interleukin-6 and tumor necrosis factor alpha. Since these genes are regulated in part by the cellular transcription factors NF-κB and RBP-Jκ, we compared the activities of NF-κB and RBP-Jκ in P388D1 cells following infection with Pichinde Virus. The attenuated P2 Virus inhibited NF-κB activation and caused a shift in the size of the RBP-Jκ complex. The virulent P18 Virus showed less inhibition of NF-κB and failed to alter the size of the RBP-Jκ complex. Peritoneal cells from P2-infected guinea pigs showed induction of NF-κB RelA/p50 heterodimer and p50/p50 homodimer and manifested an increase in the size of RBP-Jκ. By contrast, P18 induced large amounts of the NF-κB p50/p50 dimer but failed to induce RelA/p50 or to cause an increase in the RBP-Jκ size. Taken together, these changes suggest that the attenuated viral strain induces an “activation” of macrophages, while the virulent form of the Virus does not.
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Tumor necrosis factor and the pathogenesis of Pichinde Virus infection in guinea pigs.
The American journal of tropical medicine and hygiene, 1995Co-Authors: Judith F. Aronson, Norbert K. Herzog, Thomas R. JerrellsAbstract:Pichinde Virus (PIC) is a reticuloendothelial arenaVirus of the New World tropics. A guinea pig passage–adapted strain of this Virus (adPIC) is uniformly lethal for inbred guinea pigs, while the related, prototype strain (PIC3739) has attenuated virulence. The abilities of adPIC and PIC3739 to induce tumor necrosis factor (TNF) in vivo and in cultured macrophages were compared. Infection with adPIC, but not PIC3739, was associated with detectable serum TNF that peaked in week 2 of infection. Tumor necrosis factor was found in the spleens of adPIC- and PIC3739-infected animals in week 1 of infection ; TNFα mRNA levels in spleens and livers of adPIC infected animals increased and remained high throughout infection, whereas PIC3739-infected organs showed down regulation of TNFα mRNA late in infection. Peritoneal macrophages explanted from adPIC-infected animals showed enhanced lipopolysaccharide-inducible TNF production. Altered regulation of TNF production may play a role in the pathogenesis of guinea pig arenaVirus disease.
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Pathological and virological features of arenaVirus disease in guinea pigs. Comparison of two Pichinde Virus strains.
The American journal of pathology, 1994Co-Authors: Judith F. Aronson, Norbert K. Herzog, Thomas R. JerrellsAbstract:A guinea pig passage-adapted strain of the arena-Virus Pichinde (adPIC) is highly virulent in inbred guinea pigs, whereas the related strain PIC3739 is attenuated. Both Viruses were macrophage tropic and infected peritoneal, splenic, liver, and alveolar macrophages during experimental Pichinde Virus infection. Infection with the virulent strain was associated with unlimited viral replication in the face of exaggerated delayed-type hypersensitivity response, manifested by the macrophage disappearance reaction. Histopathological lesions unique to adPIC-infected guinea pigs included intestinal villus blunting with mucosal infiltration by pyknotic debris-laden macrophages and apoptosis of crypt epithelial cells. Splenic red pulp necrosis was also significantly associated with adPIC infection but not PIC3739 infection. These findings may provide clues to the pathogenesis of a group of poorly understood human viral hemorrhagic fevers.
Shuiyun Lan - One of the best experts on this subject based on the ideXlab platform.
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Pichinde Virus Infection of Outbred Hartley Guinea Pigs as a Surrogate Animal Model for Human Lassa Fever: Histopathological and Immunohistochemical Analyses.
Pathogenetics, 2020Co-Authors: Wun-ju Shieh, Shuiyun Lan, Sherif R. Zaki, Yuying LiangAbstract:Lassa Virus (LASV) is a mammarenaVirus (arenaVirus) that causes zoonotic infection in humans that can lead to fatal hemorrhagic Lassa fever (LF) disease. Currently, there are no FDA-approved vaccines or therapeutics against LASV. Development of treatments against LF and other related arenaVirus-induced hemorrhagic fevers (AHFs) requires relevant animal models that can recapitulate clinical and pathological features of AHF diseases in humans. Laboratory mice are generally resistant to LASV infection, and non-human primates, while being a good animal model for LF, are limited by their high cost. Here, we describe a small, affordable, and convenient animal model that is based on outbred Hartley guinea pigs infected with Pichinde Virus (PICV), a mammarenaVirus that is non-pathogenic in humans, for use as a surrogate model of human LF. We conducted a detailed analysis of tissue histopathology and immunohistochemical analysis of different organs of outbred Hartley guinea pigs infected with different PICV strains that show differential disease phenotypes and pathologies. Comparing to infection with the avirulent PICV strain (P2 or rP2), animals infected with the virulent strain (P18 or rP18) show extensive pathological changes in different organs that sustain high levels of Virus replication. The similarity of tissue pathology and viral antigen distribution between the virulent PICV–guinea pig model and lethal human LASV infection supports a role of this small animal model as a surrogate model of studying human LF in order to understand its pathogenesis and for evaluating potential preventative and therapeutic options against AHFs.
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Virulent infection of outbred Hartley guinea pigs with recombinant Pichinde Virus as a surrogate small animal model for human Lassa fever
Virulence, 2020Co-Authors: Shuiyun Lan, Qinfeng Huang, Wun-ju Shieh, Sherif R. Zaki, Yuying LiangAbstract:ArenaViruses, such as Lassa Virus (LASV), can cause severe and fatal hemorrhagic fevers (e.g., Lassa fever, LF) in humans with no vaccines or therapeutics. Research on arenaVirus-induced hemorrhagic fevers (AHFs) has been hampered by the highly virulent nature of these viral pathogens, which require high biocontainment laboratory, and the lack of an immune-competent small animal model that can recapitulate AHF disease and pathological features. Guinea pig infected with Pichinde Virus (PICV), an arenaVirus that does not cause disease in humans, has been established as a convenient surrogate animal model for AHFs as it can be handled in a conventional laboratory. The PICV strain P18, derived from sequential passaging of the Virus 18 times in strain 13 inbred guinea pigs, causes severe febrile illness in guinea pigs that is reminiscent of lethal LF in humans. As inbred guinea pigs are not readily available and are difficult to maintain, outbred Hartley guinea pigs have been used but they show a high degree of disease heterogeneity upon virulent P18 PICV infection. Here, we describe an improved outbred guinea-pig infection model using recombinant rP18 PICV generated by reverse genetics technique followed by plaque purification, which consistently shows >90% mortality and virulent infection. Comprehensive virological, histopathological, and immunohistochemical analyses of the rP18-Virus infected animals show similar features of human LASV infection. Our data demonstrate that this improved animal model can serve as a safe, affordable, and convenient surrogate small animal model for studying human LF pathogenesis and for evaluating efficacy of preventative or therapeutic approaches.
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Establishment of Bisegmented and Trisegmented Reverse Genetics Systems to Generate Recombinant Pichindé Viruses.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Rekha Dhanwani, Shuiyun Lan, Junjie Shao, Qinfeng Huang, Yanqing Zhou, Yuying LiangAbstract:Pichinde Virus (PICV), isolated from rice rats in Colombia, South America, is an enveloped arenaVirus with a bisegmented RNA genome. The large (L) genomic segment encodes the Z matrix protein and the L RNA-dependent RNA polymerase, whereas the small (S) genomic segment encodes the nucleoprotein (NP) and the glycoprotein (GPC). This article describes the successful development of reverse genetics systems to generate recombinant PICV with either a bisegmented or trisegmented genome. We have successfully demonstrated that these systems can generate high-titered and genetically stable replication-competent Viruses from plasmid transfection into appropriate cell lines. These systems demonstrate the power and versatility of reverse genetic technology to generate recombinant arenaViruses for use in pathogenesis studies and as new viral vaccine vectors.
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Characterization of virulence-associated determinants in the envelope glycoprotein of Pichinde Virus
Virology, 2012Co-Authors: Naveen Kumar, Jialong Wang, Shuiyun Lan, Shamika Danzy, Lisa Mclay Schelde, Jill Seladi-schulman, Yuying LiangAbstract:We use a small animal model, based on guinea pigs infected with a non-pathogenic Pichinde Virus (PICV), to understand the virulence mechanisms of arenaVirus infections in the hosts. PICV P2 strain causes a mild febrile reaction in guinea pigs, while P18 causes severe disease with clinical and pathological features reminiscent of Lassa hemorrhagic fever in humans. The envelope glycoproteins (GPC) of P2 and P18 Viruses differ at positions 119, 140, and 164, all localized to the receptor-binding G1 subunit. We found that lentiviral pseudotyped virions (VLPs) bearing P18 GPC show more efficient cell entry than those with P2 GPC, and that the E140 residue plays a critical role in this process. Infection of guinea pigs with the recombinant Viruses containing the E140K change demonstrated that E140 of GPC is a necessary virulence determinant of P18 infections, possibly by enhancing the ability of Virus to enter target cells.
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Molecular determinants of Pichinde Virus infection of guinea pigs--a small animal model system for arenaviral hemorrhagic fevers.
Annals of the New York Academy of Sciences, 2009Co-Authors: Yuying Liang, Shuiyun LanAbstract:ArenaViruses are enveloped single-strand RNA Viruses that mostly have natural hosts in rodents. Upon infection of humans, several arenaViruses can cause severe hemorrhagic fever diseases, including Lassa fever that is endemic in West Africa. The virulence mechanism of these deadly arenaViruses can be studied in a safe and economical small animal model - guinea pigs infected by a non-pathogenic arenaVirus Pichinde Virus (PICV), a virulent strain of which can cause similar disease syndromes in guinea pigs as arenaviral hemorrhagic fevers in humans. We have recently developed molecular clones for both the virulent and avirulent strains of PICV. Using the available reverse genetics tools, we are characterizing the molecular determinants of virulent arenaVirus infections in vivo.