High-throughput #screening identifies mixed lineage kinase 3 as a key #host regulatory #factor in #Zika virus #infection (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

High-throughput screening identifies mixed lineage kinase 3 as a key host regulatory factor in Zika virus infection

Hua Xu, Min Cheng, Xiaojing Chi, Xiuying Liu, Jia Zhou, Tianli Lin, Wei Yang

DOI: 10.1128/JVI.00758-19

 

ABSTRACT

The Zika virus (ZIKV) life cycle involves multiple steps and requires interactions with host factors. However, the inability to systematically identify host regulatory factors for ZIKV has hampered antiviral development and our understanding of pathogenicity. Here, using a bioactive compound library with 2659 small molecules, we applied a high-throughput and imaging-based screen to identify host factors that modulate ZIKV infection. The screen yielded hundreds of hits that markedly inhibited or potentiated ZIKV infection in SNB-19 glioblastoma cells. Among the hits, URMC-099, a mixed lineage kinase 3 (MLK3) inhibitor, significantly facilitated ZIKV replication in both SNB-19 cells and the neonatal mouse brain. Using gene silencing and overexpression, we further confirmed that MLK3 was a host restriction factor against ZIKV. Mechanistically, MLK3 negatively regulated ZIKV replication through inducing the inflammatory cytokines IL-6, IL-8, TNF-α and MCP-1 but did not modulate host interferon related pathways. Importantly, ZIKV activated the MLK3/MKK7/JNK pathway in both SNB-19 cells and neonatal mouse brain. Together, these findings reveal a critical role for MLK3 in regulating ZIKV infection and facilitate the development of anti-ZIKV therapeutics by providing a number of screening hits.

 

IMPORTANCE

Zika fever, an infectious disease caused by the Zika virus (ZIKV), normally results in mild symptoms. Severe infection can cause Guillain–Barré syndrome in adults and birth defects, including microcephaly, in newborns. Although ZIKV was first identified in Uganda in 1947 in rhesus monkeys, a widespread epidemic of ZIKV infection in South and Central America in 2015-2016 raised major concerns. To date, there is no vaccine or specific medicine for ZIKV. The significance of our research is the systematic discovery of small molecule candidates that modulate ZIKV infection, which will allow the development of antiviral therapeutics. In addition, we identified MLK3, a key mediator of host signaling pathways that can be activated during ZIKV infection and limits virus replication by inducing multiple inflammatory cytokines. These findings broaden our understanding of ZIKV pathogenesis.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Zika Virus; Viral pathogenesis.

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#Zika Virus Non-Structural Protein 1 Disrupts Glycosaminoglycans and Causes #Permeability in Developing #Human #Placentas (J Infect Dis., abstract)

[Source: Journal of Infectious Diseases, full page: (LINK). Abstract, edited.]

Zika Virus Non-Structural Protein 1 Disrupts Glycosaminoglycans and Causes Permeability in Developing Human Placentas

Henry Puerta-Guardo, Takako Tabata, Matthew Petitt, Milena Dimitrova, Dustin R Glasner, Lenore Pereira, Eva Harris

The Journal of Infectious Diseases, jiz331, https://doi.org/10.1093/infdis/jiz331

Published: 27 June 2019

 

Abstract

Background

During pregnancy, the Zika flavivirus (ZIKV) infects human placentas, inducing defects in the developing fetus. The flavivirus nonstructural protein 1 (NS1) alters glycosaminoglycans on the endothelium, causing hyperpermeability in vitro and vascular leakage in vivo in a tissue-dependent manner. The contribution of ZIKV NS1 to placental dysfunction during ZIKV infection remains unknown.

Methods

We examined the effect of ZIKV NS1 on expression and release of heparan sulfate (HS), hyaluronic acid (HA), and sialic acid (Sia) on human trophoblast cell lines and anchoring villous explants from first-trimester placentas infected with ZIKV ex vivo. We measured changes in permeability in trophoblasts and stromal cores using a dextran-based fluorescence assay and changes in HA receptor expression using immunofluorescent microscopy.

Results

ZIKV NS1 in the presence and absence of ZIKV increased the permeability of anchoring villous explants. ZIKV NS1 induced shedding of HA and HS and altered expression of CD44 and LYVE-1 HA receptors on stromal fibroblasts and Hofbauer macrophages in villous cores. Hyaluronidase was also stimulated in NS1-treated trophoblasts.

Conclusions

These findings suggest that ZIKV NS1 contributes to placental dysfunction via modulation of glycosaminoglycans on trophoblasts and chorionic villi, resulting in increased permeability of human placentas.

ZIKV NS1, chorionic villi, glycosaminoglycans, permeability, hyaluronic acid, heparan sulfate, hyaluronidase, CD44, LYVE-1, Hofbauer cells

Issue Section: Major Article

This content is only available as a PDF.

© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Keywords: Flavivirus; Zika Virus; Pregnancy; Viral pathogenesis.

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TIM-1 serves as a #receptor for #Ebola virus in vivo, enhancing #viremia and #pathogenesis (PLoS Negl Trop Dis., abstract)

[Source: PLoS Neglected Tropical Diseases, full page: (LINK). Abstract, edited.]

OPEN ACCESS /  PEER-REVIEWED / RESEARCH ARTICLE

TIM-1 serves as a receptor for Ebola virus in vivo, enhancing viremia and pathogenesis

Bethany Brunton, Kai Rogers, Elisabeth K. Phillips, Rachel B. Brouillette, Ruayda Bouls, Noah S. Butler, Wendy Maury

Published: June 26, 2019 / DOI: https://doi.org/10.1371/journal.pntd.0006983 / This is an uncorrected proof.

 

Abstract

Background

T cell immunoglobulin mucin domain-1 (TIM-1) is a phosphatidylserine (PS) receptor, mediating filovirus entry into cells through interactions with PS on virions. TIM-1 expression has been implicated in Ebola virus (EBOV) pathogenesis; however, it remains unclear whether this is due to TIM-1 serving as a filovirus receptor in vivo or, as others have suggested, TIM-1 induces a cytokine storm elicited by T cell/virion interactions. Here, we use a BSL2 model virus that expresses EBOV glycoprotein to demonstrate the importance of TIM-1 as a virus receptor late during in vivo infection.

Methodology/Principal findings

Infectious, GFP-expressing recombinant vesicular stomatitis virus encoding either full length EBOV glycoprotein (EBOV GP/rVSV) or mucin domain deleted EBOV glycoprotein (EBOV GPΔO/rVSV) was used to assess the role of TIM-1 during in vivo infection. GFP-expressing rVSV encoding its native glycoprotein G (G/rVSV) served as a control. TIM-1-sufficient or TIM-1-deficient BALB/c interferon α/β receptor-/- mice were challenged with these viruses. While G/rVSV caused profound morbidity and mortality in both mouse strains, TIM-1-deficient mice had significantly better survival than TIM-1-expressing mice following EBOV GP/rVSV or EBOV GPΔO/rVSV challenge. EBOV GP/rVSV or EBOV GPΔO/rVSV in spleen of infected animals was high and unaffected by expression of TIM-1. However, infectious virus in serum, liver, kidney and adrenal gland was reduced late in infection in the TIM-1-deficient mice, suggesting that virus entry via this receptor contributes to virus load. Consistent with higher virus loads, proinflammatory chemokines trended higher in organs from infected TIM-1-sufficient mice compared to the TIM-1-deficient mice, but proinflammatory cytokines were more modestly affected. To assess the role of T cells in EBOV GP/rVSV pathogenesis, T cells were depleted in TIM-1-sufficient and -deficient mice and the mice were challenged with virus. Depletion of T cells did not alter the pathogenic consequences of virus infection.

Conclusions

Our studies provide evidence that at late times during EBOV GP/rVSV infection, TIM-1 increased virus load and associated mortality, consistent with an important role of this receptor in virus entry. This work suggests that inhibitors which block TIM-1/virus interaction may serve as effective antivirals, reducing virus load at late times during EBOV infection.

 

Author summary

T cell immunoglobulin mucin domain-1 (TIM-1) is one of a number of phosphatidylserine (PS) receptors that mediate clearance of apoptotic bodies by binding PS on the surface of dead or dying cells. Enveloped viruses mimic apoptotic bodies by exposing PS on the outer leaflet of the viral membrane. While TIM-1 has been shown to serve as an adherence factor/receptor for filoviruses in tissue culture, limited studies have investigated the role of TIM-1 as a receptor in vivo. Here, we sought to determine if TIM-1 was critical for Ebola virus glycoprotein-mediated infection using a BSL2 model virus. We demonstrate that loss of TIM-1 expression results in decreased virus load late during infection and significantly reduced virus-elicited mortality. These findings provide evidence that TIM-1 serves as an important receptor for Ebola virus in vivo. Blocking TIM-1/EBOV interactions may be effective antiviral strategy to reduce viral load and pathogenicity at late times of EBOV infection.

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Citation: Brunton B, Rogers K, Phillips EK, Brouillette RB, Bouls R, Butler NS, et al. (2019) TIM-1 serves as a receptor for Ebola virus in vivo, enhancing viremia and pathogenesis. PLoS Negl Trop Dis 13(6): e0006983. https://doi.org/10.1371/journal.pntd.0006983

Editor: Michael R. Holbrook, NIAID Integrated Research Facility, UNITED STATES

Received: November 5, 2018; Accepted: May 17, 2019; Published: June 26, 2019

Copyright: © 2019 Brunton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: Monies received from the National Institutes of Health (NIH) supported this research. WM received financial support from NIH R01 AI077519. KR received financial support from NIH T32 GM067795. RB received financial support from NIH T32GM007337. NSB received financial support from NIH AI127481 and AI125446. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Keywords: Ebola; Viral pathogenesis.

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#Systems analysis of #subjects acutely infected with the #Chikungunya virus (PLoS Pathog., abstract)

[Source: PLoS Pathogens, full page: (LINK). Abstract, edited.]

OPEN ACCESS /  PEER-REVIEWED / RESEARCH ARTICLE

Systems analysis of subjects acutely infected with the Chikungunya virus

Alessandra Soares-Schanoski, Natália Baptista Cruz, Luíza Antunes de Castro-Jorge, Renan Villanova Homem de Carvalho, Cliomar Alves dos Santos, Nancy da Rós, Úrsula Oliveira, Danuza Duarte Costa, Cecília Luíza Simões dos Santos, Marielton dos Passos Cunha, Maria Leonor Sarno Oliveira, Juliana Cardoso Alves, Regina Adalva de Lucena Couto Océa,  [ … ], Helder I. Nakaya

Published: June 18, 2019 / DOI: https://doi.org/10.1371/journal.ppat.1007880 / This is an uncorrected proof.

 

Abstract

The largest ever recorded epidemic of the Chikungunya virus (CHIKV) broke out in 2004 and affected four continents. Acute symptomatic infections are typically associated with the onset of fever and often debilitating polyarthralgia/polyarthritis. In this study, a systems biology approach was adopted to analyze the blood transcriptomes of adults acutely infected with the CHIKV. Gene signatures that were associated with viral RNA levels and the onset of symptoms were identified. Among these genes, the putative role of the Eukaryotic Initiation Factor (eIF) family genes and apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC3A) in the CHIKV replication process were displayed. We further compared these signatures with signatures induced by the Dengue virus infection and rheumatoid arthritis. Finally, we demonstrated that the CHIKV in vitro infection of murine bone marrow-derived macrophages induced IL-1 beta production in a mechanism that is significantly dependent on the inflammasome NLRP3 activation. The observations provided valuable insights into virus-host interactions during the acute phase and can be instrumental in the investigation of new and effective therapeutic interventions.

 

Author summary

The Chikungunya virus (CHIKV) has infected millions of people worldwide and presents a serious public health issue. Acute symptomatic infections caused by contracting this mosquito-transmitted arbovirus are typically associated with an abrupt onset of fever and often debilitating polyarthralgia/ polyarthritis, as well as prolonged periods of disability in some patients. These dramatic effects call for a careful evaluation of the molecular mechanisms involved in this puzzling infection. By analyzing the blood transcriptome of adults acutely infected with CHIKV, we were able to provide a detailed picture of the early molecular events induced by the infection. Additionally, the systems biology approach revealed genes that can be investigated extensively as probable therapeutic targets for the disease.

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Citation: Soares-Schanoski A, Baptista Cruz N, de Castro-Jorge LA, de Carvalho RVH, Santos CAd, Rós Nd, et al. (2019) Systems analysis of subjects acutely infected with the Chikungunya virus. PLoS Pathog 15(6): e1007880. https://doi.org/10.1371/journal.ppat.1007880

Editor: David H. O’Connor, University of Wisconsin, UNITED STATES

Received: February 1, 2019; Accepted: May 30, 2019; Published: June 18, 2019

Copyright: © 2019 Soares-Schanoski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All RNA-seq raw data is available at the NCBI in BioProject: PRJNA507472 and the BioSample Range from SAMN10847030 to SAMN10847088

Funding: H.I.N. is supported by the São Paulo Research Foundation (FAPESP; grants 2017/50137-3, 2012/19278-6, and 2013/08216-2). A.S.S. is supported by Butantan Foundation, CNPq (Grant 443371/2016-4) and Brazilian Health Ministry. R.A. is supported by FINEP Grant 0116005600. D.R.R. has a postdoctoral fellowship from CNPq. J.C.A. has a postdoctoral fellowship from CAPES – Finance Code 001. M.P.C. has a PhD fellowship from FAPESP – 2016/08204-2. I.J.A is supported by São Paulo Research Foundation (FAPESP; grant: CEPID 2013/07467-1). P.L.H is supported by Butantan Foundation, CNPq 306992/2014-0 and Fapesp 2015/25055-8. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Keywords: Chikungunya fever, Viral pathogenesis.

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SNX11 Identified as an Essential #Host Factor for #SFTS Virus #Infection by #CRISPR Knockout Screening (Virol Sin., abstract)

[Source: Virologica Sinica, full page: (LINK). Abstract, edited.]

SNX11 Identified as an Essential Host Factor for SFTS Virus Infection by CRISPR Knockout Screening

Authors: Tiezhu Liu, Jiajia Li, Yang Liu, Yuanyuan Qu, Aqian Li, Chuan Li, Quanfu Zhang,Wei Wu, Jiandong Li, Yan Liu, Dexin Li, Shiwen Wang, Mifang Liang

Research Article / First Online: 18 June 2019

 

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome (SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. Using a genome-wide CRISPR-based screening strategy, we identified a host cellular protein, sorting nexin 11 (SNX11) which is involved in the intracellular endosomal trafficking pathway, as an essential cell factor for SFTSV infection. An SNX11-KO HeLa cell line was established, and SFTSV replication was significantly reduced. The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum (ER) or Golgi apparatus. pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells, and lysosomal-associated membrane protein 1 (LAMP1) expression was significantly elevated in the SNX11-KO cells. Overall, these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11. Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting, membrane fusion, and other endocytic machinery.

Keywords: CRISPR – screen – Severe fever with thrombocytopenia syndrome virus (SFTSV) – Host factor – Sorting nexin 11 (SNX11)

Electronic supplementary material

The online version of this article ( https://doi.org/10.1007/s12250-019-00141-0) contains supplementary material, which is available to authorized users.

 

Notes

Acknowledgements

This work was supported by the National Key Project for Infectious Disease from the Ministry of Science and Technology (Grant No. 2018ZX10711-001).

Author Contributions

TL performed the experiments and wrote the paper; Jiajia Li, YL, and YQ performed the experiments; AL, QZ, CL, WW, YL, and Jiandong Li contributed reagents/materials/analysis tools. Jiajia Li, TL, ML, YL, Jiandong Li, and DL analyzed and discussed the data. ML and SW designed the project and edited the manuscript. All authors read and approved the final manuscript.

 

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

This article does not contain any studies with human or animal subjects performed by any of the authors.

Keywords: SFTS virus; Bunyavirus; CRISPR; Viral pathogenesis.

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Low #Polymerase #Activity Attributed to PA Drives the Acquisition of the #PB2 E627K #Mutation of #H7N9 #Avian #Influenza Virus in Mammals (mBio, abstract)

[Source: mBio, full page: (LINK). Abstract, edited.]

Low Polymerase Activity Attributed to PA Drives the Acquisition of the PB2 E627K Mutation of H7N9 Avian Influenza Virus in Mammals

Libin Liang, Li Jiang, Junping Li, Qingqing Zhao, Jinguang Wang, Xijun He, Shanyu Huang, Qian Wang, Yuhui Zhao, Guangwen Wang, Nan Sun, Guohua Deng, Jianzhong Shi, Guobin Tian,Xianying Zeng, Yongping Jiang, Liling Liu, Jinxiong Liu, Pucheng Chen, Zhigao Bu, Yoshihiro Kawaoka, Hualan Chen, Chengjun Li

Terence S. Dermody, Editor

DOI: 10.1128/mBio.01162-19

 

ABSTRACT

Avian influenza viruses (AIVs) must acquire mammalian-adaptive mutations before they can efficiently replicate in and transmit among humans. The PB2 E627K mutation is known to play a prominent role in the mammalian adaptation of AIVs. The H7N9 AIVs that emerged in 2013 in China easily acquired the PB2 E627K mutation upon replication in humans. Here, we generate a series of reassortant or mutant H7N9 AIVs and test them in mice. We show that the low polymerase activity attributed to the viral PA protein is the intrinsic driving force behind the emergence of PB2 E627K during H7N9 AIV replication in mice. Four residues in the N-terminal region of PA are critical in mediating the PB2 E627K acquisition. Notably, due to the identity of viral PA protein, the polymerase activity and growth of H7N9 AIV are highly sensitive to changes in expression levels of human ANP32A protein. Furthermore, the impaired viral polymerase activity of H7N9 AIV caused by the depletion of ANP32A led to reduced virus replication in Anp32a−/− mice, abolishing the acquisition of the PB2 E627K mutation and instead driving the virus to acquire the alternative PB2 D701N mutation. Taken together, our findings show that the emergence of the PB2 E627K mutation of H7N9 AIV is driven by the intrinsic low polymerase activity conferred by the viral PA protein, which also involves the engagement of mammalian ANP32A.

 

IMPORTANCE

The emergence of the PB2 E627K substitution is critical in the mammalian adaptation and pathogenesis of AIV. H7N9 AIVs that emerged in 2013 possess a prominent ability in gaining the PB2 E627K mutation in humans. Here, we demonstrate that the acquisition of the H7N9 PB2 E627K mutation is driven by the low polymerase activity conferred by the viral PA protein in human cells, and four PA residues are collectively involved in this process. Notably, the H7N9 PA protein leads to significant dependence of viral polymerase function on human ANP32A protein, and Anp32a knockout abolishes PB2 E627K acquisition in mice. These findings reveal that viral PA and host ANP32A are crucial for the emergence of PB2 E627K during adaptation of H7N9 AIVs to humans.

Keywords: Avian Influenza; H7N9; Viral pathogenesis.

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PD1+CCR2+CD8+ T Cells Infiltrate the #CNS during Acute #JEV #Infection (Virol Sin., abstract)

[Source: Virologica Sinica, full page: (LINK). Abstract, edited.]

PD1+CCR2+CD8+ T Cells Infiltrate the Central Nervous System during Acute Japanese Encephalitis Virus Infection

Authors: Fang Zhang, Linlin Qi, Tong Li, Xiaojing Li, Dan Yang, Shengbo Cao, Jing Ye, Bin Wei

Research Article / First Online: 18 June 2019

 

Abstract

Japanese encephalitis (JE) is a viral encephalitis disease caused by Japanese encephalitis virus (JEV) infection. Uncontrolled inflammatory responses in the central nervous system (CNS) are a hallmark of severe JE. Although the CCR2–CCL2 axis is important for monocytes trafficking during JEV infection, little is known about its role in CNS trafficking of CD8+ T cells. Here, we characterized a mouse model of JEV infection, induced via intravenous injection (i.v.) and delineated the chemokines and infiltrating peripheral immune cells in the brains of infected mice. The CNS expression of chemokines, Ccl2, Ccl3, and Ccl5, and their receptors, Ccr2 or Ccr5, was significantly up-regulated after JEV infection and was associated with the degree of JE pathogenesis. Moreover, JEV infection resulted in the migration of a large number of CD8+T cells into the CNS. In the brains of JEV-infected mice, infiltrating CD8+ T cells expressed CCR2 and CCR5 and were found to comprise mainly effector T cells (CD44+CD62L−). JEV infection dramatically enhanced the expression of programmed death 1 (PD-1) on infiltrating CD8+ T cells in the brain, as compared to that on peripheral CD8+ T cells in the spleen. This effect was more pronounced on infiltrating CCR2+CD8+ T cells than on CCR2−CD8+ T cells. In conclusion, we identified a new subset of CD8+ T cells (PD1+CCR2+CD8+ T cells) present in the CNS of mice during acute JEV infection. These CD8+ T cells might play a role in JE pathogenesis.

Keywords: Japanese encephalitis virus (JEV) – CD8+ T cell – CCL2 – CCR2 – PD-1

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Fang Zhang and Linlin Qi have contributed equally to this work.

Electronic supplementary material

The online version of this article ( https://doi.org/10.1007/s12250-019-00134-z) contains supplementary material, which is available to authorized users.

 

Notes

Acknowledgements

This work was supported by grants from the Key Research and Development Program of the Ministry of Science and Technology of China (2016YFD500407), Precision Medicine program of Ministry of Science and Technology of China (2016YFC0905902), and the National Natural Science Foundation of China (81630043, 81571552). We thank the Core Facility and Technical Support in the Wuhan Investigate of Virology. We are grateful to Ding Gao and Juan Min for technical support for flow cytometry and mass cytometry, as well as Xuefang An and Fan Zhang for valuable assistance in the animal studies.

Author Contributions

BW and JY conceptualized and designed the study. LLQ, TL and FZ performed the experiments in this study, and analyzed the data. SBC and JY contributed virus strain and virus infection techniques to this study. DY and XJL participated in part of experimental work. FZ wrote the manuscript. All authors read and approved the final manuscript.

 

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

The study was approved by the Animal Ethics Committee of Wuhan Institute of Virology. All institutional and national guidelines for the care and use of laboratory animals were followed.

Keywords: Japanese encephalitis; Viral pathogenesis; Animal models.

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