Time Course and Cellular Localization of SARS-CoV Nucleoprotein and RNA in Lungs from Fatal Cases of SARS

BACKGROUND: Cellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) in the lungs of patients with SARS is important in confirming the etiological association of the virus with disease as well as in understanding the pathogenesis of the disease. To our knowledge, there have been no comprehensive studies investigating viral infection at the cellular level in humans. METHODS AND FINDINGS: We collected the largest series of fatal cases of SARS with autopsy material to date by merging the pathological material from two regions involved in the 2003 worldwide SARS outbreak in Hong Kong, China, and Toronto, Canada. We developed a monoclonal antibody against the SARS-CoV nucleoprotein and used it together with in situ hybridization (ISH) to analyze the autopsy lung tissues of 32 patients with SARS from Hong Kong and Toronto. We compared the results of these assays with the pulmonary pathologies and the clinical course of illness for each patient. SARS-CoV nucleoprotein and RNA were detected by immunohistochemistry and ISH, respectively, primarily in alveolar pneumocytes and, less frequently, in macrophages. Such localization was detected in four of the seven patients who died within two weeks of illness onset, and in none of the 25 patients who died later than two weeks after symptom onset. CONCLUSIONS: The pulmonary alveolar epithelium is the chief target of SARS-CoV, with macrophages infected subsequently. Viral replication appears to be limited to the first two weeks after symptom onset, with little evidence of continued widespread replication after this period. If antiviral therapy is considered for future treatment, it should be focused on this two-week period of acute clinical disease

In-Flight Transmission of SARS-CoV-2.

Four persons with severe acute respiratory syndrome coronavirus 2 infection had traveled on the same flight from Boston, Massachusetts, USA, to Hong Kong, China. Their virus genetic sequences are identical, unique, and belong to a clade not previously identified in Hong Kong, which strongly suggests that the virus can be transmitted during air travel

Influenza H5N1 and H1N1 Virus Replication and Innate Immune Responses in Bronchial Epithelial Cells Are Influenced by the State of Differentiation

Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE) cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004) and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998), the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of α2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis

Systems-Level Comparison of Host-Responses Elicited by Avian H5N1 and Seasonal H1N1 Influenza Viruses in Primary Human Macrophages

Human disease caused by highly pathogenic avian influenza (HPAI) H5N1 can lead to a rapidly progressive viral pneumonia leading to acute respiratory distress syndrome. There is increasing evidence from clinical, animal models and in vitro data, which suggests a role for virus-induced cytokine dysregulation in contributing to the pathogenesis of human H5N1 disease. The key target cells for the virus in the lung are the alveolar epithelium and alveolar macrophages, and we have shown that, compared to seasonal human influenza viruses, equivalent infecting doses of H5N1 viruses markedly up-regulate pro-inflammatory cytokines in both primary cell types in vitro. Whether this H5N1-induced dysregulation of host responses is driven by qualitative (i.e activation of unique host pathways in response to H5N1) or quantitative differences between seasonal influenza viruses is unclear. Here we used microarrays to analyze and compare the gene expression profiles in primary human macrophages at 1, 3, and 6 h after infection with H5N1 virus or low-pathogenic seasonal influenza A (H1N1) virus. We found that host responses to both viruses are qualitatively similar with the activation of nearly identical biological processes and pathways. However, in comparison to seasonal H1N1 virus, H5N1 infection elicits a quantitatively stronger host inflammatory response including type I interferon (IFN) and tumor necrosis factor (TNF)-α genes. A network-based analysis suggests that the synergy between IFN-β and TNF-α results in an enhanced and sustained IFN and pro-inflammatory cytokine response at the early stage of viral infection that may contribute to the viral pathogenesis and this is of relevance to the design of novel therapeutic strategies for H5N1 induced respiratory disease

A single residue substitution in the receptor-binding domain of H5N1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles

© 2012 Tang 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.Background: Serological studies for influenza infection and vaccine response often involve microneutralization and hemagglutination inhibition assays to evaluate neutralizing antibodies against human and avian influenza viruses, including H5N1. We have previously characterized lentiviral particles pseudotyped with H5-HA (H5pp) and validated an H5pp-based assay as a safe alternative for high-throughput serological studies in BSL-2 facilities. Here we show that H5-HAs from different clades do not always give rise to efficient production of H5pp and the underlying mechanisms are addressed. Methodology/Findings: We have carried out mutational analysis to delineate the molecular determinants responsible for efficient packaging of HA from A/Cambodia/40808/2005 (H5Cam) and A/Anhui/1/2005 (H5Anh) into H5pp. Our results demonstrate that a single A134V mutation in the 130-loop of the receptor binding domain is sufficient to render H5Anh the ability to generate H5Anh-pp efficiently, whereas the reverse V134A mutation greatly hampers production of H5Cam-pp. Although protein expression in total cell lysates is similar for H5Anh and H5Cam, cell surface expression of H5Cam is detected at a significantly higher level than that of H5Anh. We further demonstrate by several independent lines of evidence that the behaviour of H5Anh can be explained by a stronger binding to sialic acid receptors implicating residue 134. Conclusions: We have identified a single A134V mutation as the molecular determinant in H5-HA for efficient incorporation into H5pp envelope and delineated the underlying mechanism. The reduced binding to sialic acid receptors as a result of the A134V mutation not only exerts a critical influence in pseudotyping efficiency of H5-HA, but has also an impact at the whole virus level. Because A134V substitution has been reported as a naturally occurring mutation in human host, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 virusesThis work was supported by grants from the Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID#08070972), the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, and the RESPARI project of the Institut Pasteur International Network

Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection

Seroprevalence of Pandemic H1N1 Antibody among Health Care Workers in Hong Kong Following Receipt of Monovalent 2009 H1N1 Influenza Vaccine

Background: Healthcare workers in many countries are recommended to receive influenza vaccine to protect themselves as well as patients. A monovalent H1N1 vaccine became available in Hong Kong in December 2009 and around 10% of local healthcare workers had received the vaccine by February 2010. Methods: We conducted a cross-sectional study of the prevalence of antibody to pandemic (H1N1) 2009 among HCWs in Hong Kong in February-March 2010 following the first pandemic wave and the pH1N1 vaccination campaign. In this study we focus on the subset of healthcare workers who reported receipt of non-adjuvanted monovalent 2009 H1N1 vaccine (Panenza, Sanofi Pasteur). Sera collected from HCWs were tested for antibody against the pH1N1 virus by hemagglutination inhibition (HI) and viral neutralization (VN) assays. Results: We enrolled 703 HCWs. Among 104 HCWs who reported receipt of pH1N1 vaccine, 54% (95% confidence interval (CI): 44%-63%) had antibody titer ≥1:40 by HI and 42% (95% CI: 33%-52%) had antibody titer ≥1:40 by VN. The proportion of HCWs with antibody titer ≥1:40 by HI and VN significantly decreased with age, and the proportion with antibody titer ≥1:40 by VN was marginally significantly lower among HCWs who reported prior receipt of 2007-08 seasonal influenza vaccine (odds ratio: 0.43; 95% CI: 0.19-1.00). After adjustment for age, the effect of prior seasonal vaccine receipt was not statistically significant. Conclusions: Our findings suggest that monovalent H1N1 vaccine may have had suboptimal immunogenicity in HCWs in Hong Kong. Larger studies are required to confirm whether influenza vaccine maintains high efficacy and effectiveness in HCWs. © 2011 Zhou et al.published_or_final_versio

Influenza A H5N1 Replication Sites in Humans

Tissue tropism and pathogenesis of influenza A virus subtype H5N1 disease in humans is not well defined. In mammalian experimental models, H5N1 influenza is a disseminated disease. However, limited previous data from human autopsies have not shown evidence of virus dissemination beyond the lung. We investigated a patient with fatal H5N1 influenza. Viral RNA was detected by reverse transcription–polymerase chain reaction in lung, intestine, and spleen tissues, but positive-stranded viral RNA indicating virus replication was confined to the lung and intestine. Viral antigen was detected in pneumocytes by immunohistochemical tests. Tumor necrosis factor-α mRNA was seen in lung tissue. In contrast to disseminated infection documented in other mammals and birds, H5N1 viral replication in humans may be restricted to the lung and intestine, and the major site of H5N1 viral replication in the lung is the pneumocyte

The Effects of Temperature and Relative Humidity on the Viability of the SARS Coronavirus

The main route of transmission of SARS CoV infection is presumed to be respiratory droplets. However the virus is also detectable in other body fluids and excreta. The stability of the virus at different temperatures and relative humidity on smooth surfaces were studied. The dried virus on smooth surfaces retained its viability for over 5 days at temperatures of 22–25°C and relative humidity of 40–50%, that is, typical air-conditioned environments. However, virus viability was rapidly lost (>3 log10) at higher temperatures and higher relative humidity (e.g., 38°C, and relative humidity of >95%). The better stability of SARS coronavirus at low temperature and low humidity environment may facilitate its transmission in community in subtropical area (such as Hong Kong) during the spring and in air-conditioned environments. It may also explain why some Asian countries in tropical area (such as Malaysia, Indonesia or Thailand) with high temperature and high relative humidity environment did not have major community outbreaks of SARS

Extended Viral Shedding of MERS-CoV Clade B Virus in Llamas Compared with African Clade C Strain

Middle East respiratory syndrome coronavirus (MERS-CoV) clade B viruses are found in camelids and humans in the Middle East, but clade C viruses are not. We provide experimental evidence for extended shedding of MERS-CoV clade B viruses in llamas, which might explain why they outcompete clade C strains in the Arabian Peninsula.This study was performed as part of the Zoonotic Anticipation and Preparedness Initiative (ZAPI project) (Innovative Medicines initiative [IMI] grant 115760), with assistance and financial support from IMI and the European Commission and contributions from EFPIA partners. J.R. was partially supported by the VetBioNet project (EU Grant Agreement INFRA-2016-1 Nº731014) and the crowdfunding initiative #Yomecorono, available online at https://www.yomecorono.com (accessed on June 16, 2022). IRTA is supported by CERCA Programme/Generalitat de Catalunya.info:eu-repo/semantics/publishedVersio