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Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation.

ABSTRACT: The emerging novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) was recently isolated from patients with severe pneumonia and renal failure and was associated with an unexplained high crude fatality rate of 56%. We performed a cell line susceptibility study with 28 cell lines. HCoV-EMC was found to infect the human respiratory tract (polarized airway epithelium cell line Calu-3, embryonic fibroblast cell line HFL, and lung adenocarcinoma cell line A549), kidney (embryonic kidney cell line HEK), intestinal tract (colorectal adenocarcinoma cell line Caco-2), liver cells (hepatocellular carcinoma cell line Huh-7), and histiocytes (malignant histiocytoma cell line His-1), as evident by detection of high or increasing viral load in culture supernatants, detection of viral nucleoprotein expression by immunostaining, and/or detection of cytopathic effects. Although an infected human neuronal cell line (NT2) and infected monocyte and T lymphocyte cell lines (THP-1, U937, and H9) had increased viral loads, their relatively lower viral production corroborated with absent nucleoprotein expression and cytopathic effects. This range of human tissue tropism is broader than that for all other HCoVs, including SARS coronavirus, HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63, which may explain the high mortality associated with this disease. A recent cell line susceptibility study showed that HCoV-EMC can infect primate, porcine, and bat cells and therefore may jump interspecies barriers. We found that HCoV-EMC can also infect civet lung fibroblast and rabbit kidney cell lines. These findings have important implications for the diagnosis, pathogenesis, and transmission of HCoV-EMC.

SUBMITTER: Chan JF

PROVIDER: S-EPMC7107374 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation.

Chan Jasper Fuk-Woo JF Chan Kwok-Hung KH Choi Garnet Kwan-Yue GK To Kelvin Kai-Wang KK Tse Herman H Cai Jian-Piao JP Yeung Man Lung ML Cheng Vincent Chi-Chung VC Chen Honglin H Che Xiao-Yan XY Lau Susanna Kar-Pui SK Woo Patrick Chiu-Yat PC Yuen Kwok-Yung KY

The Journal of infectious diseases 20130326 11

The emerging novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) was recently isolated from patients with severe pneumonia and renal failure and was associated with an unexplained high crude fatality rate of 56%. We performed a cell line susceptibility study with 28 cell lines. HCoV-EMC was found to infect the human respiratory tract (polarized airway epithelium cell line Calu-3, embryonic fibroblast cell line HFL, and lung adenocarcinoma cell line A549), kidney (embryonic kidney cell line HEK), ...[more]

PMID: 23532101

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Project description:The recent emergence of a novel human coronavirus (HCoV-EMC) in the Middle East raised considerable concerns, as it is associated with severe acute pneumonia, renal failure, and fatal outcome and thus resembles the clinical presentation of severe acute respiratory syndrome (SARS) observed in 2002 and 2003. Like SARS-CoV, HCoV-EMC is of zoonotic origin and closely related to bat coronaviruses. The human airway epithelium (HAE) represents the entry point and primary target tissue for respiratory viruses and is highly relevant for assessing the zoonotic potential of emerging respiratory viruses, such as HCoV-EMC. Here, we show that pseudostratified HAE cultures derived from different donors are highly permissive to HCoV-EMC infection, and by using reverse transcription (RT)-PCR and RNAseq data, we experimentally determined the identity of seven HCoV-EMC subgenomic mRNAs. Although the HAE cells were readily responsive to type I and type III interferon (IFN), we observed neither a pronounced inflammatory cytokine nor any detectable IFN responses following HCoV-EMC, SARS-CoV, or HCoV-229E infection, suggesting that innate immune evasion mechanisms and putative IFN antagonists of HCoV-EMC are operational in the new host. Importantly, however, we demonstrate that both type I and type III IFN can efficiently reduce HCoV-EMC replication in HAE cultures, providing a possible treatment option in cases of suspected HCoV-EMC infection. IMPORTANCE A novel human coronavirus, HCoV-EMC, has recently been described to be associated with severe respiratory tract infection and fatalities, similar to severe acute respiratory syndrome (SARS) observed during the 2002-2003 epidemic. Closely related coronaviruses replicate in bats, suggesting that, like SARS-CoV, HCoV-EMC is of zoonotic origin. Since the animal reservoir and circumstances of zoonotic transmission are yet elusive, it is critically important to assess potential species barriers of HCoV-EMC infection. An important first barrier against invading respiratory pathogens is the epithelium, representing the entry point and primary target tissue of respiratory viruses. We show that human bronchial epithelia are highly susceptible to HCoV-EMC infection. Furthermore, HCoV-EMC, like other coronaviruses, evades innate immune recognition, reflected by the lack of interferon and minimal inflammatory cytokine expression following infection. Importantly, type I and type III interferon treatment can efficiently reduce HCoV-EMC replication in the human airway epithelium, providing a possible avenue for treatment of emerging virus infections.

Project description:Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis. IMPORTANCE The 2012 outbreak of MERS-CoV raises the specter of another global epidemic, similar to the 2003 SARS-CoV epidemic. MERS-CoV is related to BtCoV HKU5 in target regions that are essential for drug and vaccine testing. Because no small animal model exists to evaluate MERS-CoV pathogenesis or to test vaccines, we constructed a recombinant BtCoV HKU5 that expressed a region of the SARS-CoV spike (S) glycoprotein, thereby allowing the recombinant virus to grow in cell culture and in mice. We show that this recombinant virus targets airway epithelial cells and causes disease in aged mice. We use this platform to (i) identify a broad-spectrum antiviral that can potentially inhibit viruses closely related to MERS-CoV, (ii) demonstrate the absence of increased eosinophilic immune pathology for MERS-CoV N protein-based vaccines, and (iii) mouse adapt this virus to identify viral genetic determinants of cross-species transmission and virulence. This study holds significance as a strategy to control newly emerging viruses.

Dataset Information

Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation.

Publications

Differential cell line susceptibility to the emerging novel human betacoronavirus 2c EMC/2012: implications for disease pathogenesis and clinical manifestation.

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