Comparison of innate immune responses to pathogenic and putative non-pathogenic hantaviruses in vitro☆
Highlights
► Pathogenic hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. However, not all hantaviruses are human pathogens. ► We have examined the pathogenesis of different hantavirus groups by comparing the innate immune responses induced in the host cell following infection by pathogenic (Sin Nombre, Hantaan, and Seoul virus) and putative non-pathogenic (Prospect Hill, Tula, and Thottapalayam virus) hantaviruses. ► The degree of innate immune inhibition may play an important role to distinguish pathogenic and non-pathogenic hantaviruses.
Introduction
Hantavirus is a genus of rodent-borne, lipid-enveloped, negative-sense, single-stranded RNA viruses belonging to the family Bunyaviridae. Viruses of this family are characterized by a trisegmented genome, designated according to size as the large (L), medium (M), and small (S) segments, respectively, which encode four proteins: the RNA-dependent RNA polymerase, two envelope glycoproteins (Gn and Gc), and the nucleocapsid protein (N) (Elliott et al., 1991). Hantaviruses are broadly classified into Old World and New World categories based on geographic distribution. The transmission of hantaviruses to humans occurs via inhalation of contaminated aerosolized excreta from rodents (Nuzum et al., 1988). Pathogenic hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in Eurasia (Old World hantavirus) and hantavirus cardiopulmonary syndrome (HCPS) in the Americas (New World hantavirus). However, not all hantaviruses are human pathogens; for example, there is no known association between the Prospect Hill, Tula, and Thottapalayam virus, and human disease (Carey et al., 1971, Lee et al., 1982b, Lee et al., 1985, Plyusnin et al., 1994, Song et al., 2007, Vapalahti et al., 1996).
Virus infection elicits a cascade of rapid innate immune responses in the host that are aimed at blocking viral replication through the activation of an early defense mechanism. Activation of the innate immune responses generally leads to the induction of type-I Interferon (IFN-α/β) as one of the first lines of defense against viral infection (Bonjardim, 2005, Conzelmann, 2005). However, IFN-α/β is not only synthesized in response to viral infection, but also induced at low levels in the absence of viral infection (Taniguchi and Takaoka, 2001). The regulation of IFN responses may differ among virus species, cell lines, signaling pathways, and integrin receptors.
The expression of IFN-β requires the activation of transcription factors, such as IFN regulatory factor (IRF)-3 and nuclear factor kappa B (NF-κB), both of which play a major role in the induction of IFN by viruses in the cytoplasm (Bonjardim, 2005, Haller et al., 2006, Hiscott, 2007). Viral infection triggers the phosphorylation and dimerization of IRF-3 and its subsequent translocation to the nucleus where it initiates IFN-β gene expression. Secreted IFN-β binds to the type-I IFN receptors (IFNARs) and initiates activation of the IFN signaling pathway. The expression of IFN-stimulated genes (ISGs), including 2′,5′ oligoadenylate synthetases (2′,5′-OAS), protein kinase R (PKR), and myxovirus resistance protein (Mx), is activated via Janus kinase/signal transducers and activators of the transcription (JAK-STAT) pathway (Borden et al., 2007, Randall and Goodbourn, 2008).
In the study reported here, we have compared innate immune responses to the pathogenic hantaviruses (PaV), including Sin Nombre virus (SNV), Hantaan virus (HTNV), and Seoul virus (SEOV), and the putative non-pathogenic hantaviruses (NPaV), including Prospect Hill virus (PHV), Tula virus (TULV), and Thottapalayam virus (TPMV) (Table 1). Our results provide interesting insights into the differences in human virulence among hantavirus strains and demonstrate that infection with NPaV may induce a strong innate immune response through the activation of such effectors as IFN-β and the MxA gene during the early post-infection period.
Section snippets
Cells and plasmid
Vero E6 (African green monkey epithelial kidney cell, ATCC C1008 CRL-1586) and A549 (Human epithelial lung cell, ATCC CCL-185) cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Lonza) and RPMI-1640 (Lonza, Basel, Switzerland) supplemented with 5% fetal bovine serum (FBS), 10 mM HEPES buffer, 2 mM l-glutamine, and 50 ng/ml gentamycin. The pEGFP-c1-hIRF3 plasmid was kindly provided by Dr. Man-Seong Park (College of Medicine, Hallym University, Korea).
Viruses
The SNV (strain Convict Creek
Differential efficiencies of hantavirus replication in Vero E6 and A549 cells
The replication of the PaV and NPaV hantaviruses in Vero E6 and A549 cells was determined on each post-infection day up to post-infection day 7 (Fig. 1). Vero E6 cells, which are IFN-deficient cells, are unable to produce type-I IFN because of a chromosomal defect (Diaz et al., 1988, Wathelet et al., 1992), whereas A549 cells are IFN-competent cells. In Vero E6 cells, both PaV and NPaV grew with similar kinetics and replication efficiency, yielding titers of 1 × 105 to 1 × 106 PFU/ml within 7 days
Discussion
Hantaviruses are distributed worldwide and occur in both pathogenic (etiologic agents of HFRS and HCPS) and non-pathogenic hantaviruses. The differences between pathogenic and non-pathogenic hantaviruses are currently unclear. Interestingly, infection with pathogenic and non-pathogenic hantaviruses differentially activate the innate immunity in vitro (Alff et al., 2006, Geimonen et al., 2002, Handke et al., 2009, Kraus et al., 2004, Spiropoulou et al., 2007), and this difference in the innate
Acknowledgment
This work was supported in part by the Brain Korea 21 Project of the Ministry of Education and Human Resources Development, Republic of Korea.
References (51)
Interferons (IFNs) are key cytokines in both innate and adaptive antiviral immune responses and viruses counteract IFN action
Microbes and Infection
(2005)- et al.
A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection
Virology
(2008) - et al.
The interferon response circuit: induction and suppression by pathogenic viruses
Virology
(2006) - et al.
Interferon, Mx, and viral countermeasures
Cytokine and Growth Factor Reviews
(2007) - et al.
The Mx GTPase family of interferon-induced antiviral proteins
Microbes and Infection
(2007) Convergence of the NF-kappaB and IRF pathways in the regulation of the innate antiviral response
Cytokine and Growth Factor Reviews
(2007)- et al.
Inhibition of puumala and tula hantaviruses in vero cells by MxA protein
Virology
(1996) - et al.
New hemorrhagic-fever with renal syndrome-related virus in indigenous wild rodents in United-States
Lancet
(1982) - et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(−Delta Delta C) method
Methods
(2001) - et al.
The pathogenic NY-1 hantavirus G1 cytoplasmic tail inhibits RIG-I- and TBK-1-directed interferon responses
Journal of Virology
(2006)
The NY-1 hantavirus Gn cytoplasmic tail coprecipitates TRAF3 and inhibits cellular interferon responses by disrupting TBK1–TRAF3 complex formation
Journal of Virology
Interferons at age 50: past, current and future impact on biomedicine
Nature Reviews Drug Discovery
Thottapalayam virus: a presumptive arbovirus isolated from a shrew in India
Indian Journal of Medical Research
Transcriptional activation of alpha/beta interferon genes: interference by nonsegmented negative-strand RNA viruses
Journal of Virology
Homozygous deletion of the alpha- and beta 1-interferon genes in human leukemia and derived cell lines
Proceedings of the National Academy of Sciences of the United States of America
Bunyaviridae genome structure and gene expression
Current Topics in Microbiology and Immunology
Inhibition of bunyaviruses, phleboviruses, and hantaviruses by human MxA protein
Journal of Virology
Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins
Journal of Virology
Pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses
Proceedings of the National Academy of Sciences of the United States of America
Transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes
Journal of Virology
Mx proteins: mediators of innate resistance to RNA viruses
Revue Scientifique et Technique
Hantaan virus triggers TLR3-dependent innate immune responses
Journal of Immunology
Induction of MxA gene expression by influenza A virus requires type I or type III interferon signaling
Journal of Virology
Virus detection and identification with serological tests
Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon-beta promoter
Journal of Medical Virology
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The findings and conclusions in this report are those of the authors and do not necessarily represent the views of CDC.