Proinflammatory IgG Fc structures in patients with severe COVID-19.

Severe acute respiratory syndrome coronavirus 2 infections can cause coronavirus disease 2019 (COVID-19), which manifests with a range of severities from mild illness to life-threatening pneumonia and multi-organ failure. Severe COVID-19 is characterized by an inflammatory signature, including high levels of inflammatory cytokines, alveolar inflammatory infiltrates and vascular microthrombi. Here we show that patients with severe COVID-19 produced a unique serologic signature, including an increased likelihood of IgG1 with afucosylated Fc glycans. This Fc modification on severe acute respiratory syndrome coronavirus 2 IgGs enhanced interactions with the activating Fcγ receptor FcγRIIIa; when incorporated into immune complexes, Fc afucosylation enhanced production of inflammatory cytokines by monocytes, including interleukin-6 and tumor necrosis factor. These results show that disease severity in COVID-19 correlates with the presence of proinflammatory IgG Fc structures, including afucosylated IgG1.

Role of the B Allele of Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity.

UNLABELLED: Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses. IMPORTANCE: Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and health care sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades: the A and B alleles. The B-allele genes have previously been suggested to be restricted to avian virus species. We introduced a selection of avian virus A- and B-allele segment 8s into human H1N1 and H3N2 virus backgrounds and found that these reassortant viruses were fully competent in mammalian host systems. We also analyzed the currently available public data on the segment 8 gene distribution and found surprisingly little evidence for specific avian host restriction of the B-clade segment. We conclude that B-allele segment 8 genes are, in fact, capable of supporting infection in mammals and that they should be considered during the assessment of the pandemic risk of zoonotic influenza A viruses.

1918 Influenza receptor binding domain variants bind and replicate in primary human airway cells regardless of receptor specificity.

The 1918 influenza pandemic caused ~50 million deaths. Many questions remain regarding the origin, pathogenicity, and mechanisms of human adaptation of this virus. Avian-adapted influenza A viruses preferentially bind α2,3-linked sialic acids (Sia) while human-adapted viruses preferentially bind α2,6-linked Sia. A change in Sia preference from α2,3 to α2,6 is thought to be a requirement for human adaptation of avian influenza viruses. Autopsy data from 1918 cases, however, suggest that factors other than Sia preference played a role in viral binding and entry to human airway cells. Here, we evaluated binding and entry of five 1918 influenza receptor binding domain variants in a primary human airway cell model along with control avian and human influenza viruses. We observed that all five variants bound and entered cells efficiently and that Sia preference did not predict entry of influenza A virus to primary human airway cells evaluated in this model.

Isolating Viral and Host RNA Sequences from Archival Material and Production of cDNA Libraries for High-Throughput DNA Sequencing.

The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus was determined at high coverage in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. This basic methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of infectious diseases.

A forgotten epidemic that changed medicine: measles in the US Army, 1917-18.

A US army-wide measles outbreak in 1917-18 resulted in more than 95,000 cases and more than 3000 deaths. An outbreak investigation implicated measles and streptococcal co-infections in most deaths, and also characterised a parallel epidemic of primary streptococcal pneumonia in soldiers without measles. For the first time, the natural history and pathogenesis of these diseases was able to be well characterised by a broad-interdisciplinary research effort with hundreds of military and civilian physicians and scientists representing disciplines such as internal medicine, pathology, microbiology, radiology, surgery, preventive medicine, and rehabilitation medicine. A clear conceptualisation of bronchopneumonia resulting from viral-bacterial interactions between pathogens was developed, and prevention and treatment approaches were developed and optimised in real time. These approaches were used in the 1918 influenza pandemic, which began as the measles epidemic waned. The outbreak findings remain relevant to the understanding and medical management of severe pneumonia.

Validation of normal human bronchial epithelial cells as a model for influenza A infections in human distal trachea.

Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic.

Pneumocystis encodes a functional endo-ß-1,3-glucanase that is expressed exclusively in cysts.

ß-1,3-glucan is a major cell wall component of Pneumocystis cysts. We have characterized endo-ß-1,3-glucanase (Eng) from 3 species of Pneumocystis. The gene eng is a single-copy gene that encodes a protein containing 786 amino acids in P. carinii and P. murina, and 788 amino acids in P. jirovecii, including a signal peptide for the former 2 but not the latter. Recombinant Eng expressed in Escherichia coli was able to solubilize the major surface glycoprotein of Pneumocystis, thus potentially facilitating switching of the expressed major surface glycoprotein (Msg) variant. Confocal immunofluorescence analysis of P. murina-infected mouse lung sections localized Eng exclusively to the cyst form of Pneumocystis. No Eng was detected after mice were treated with caspofungin, a ß-1,3-glucan synthase inhibitor that is known to reduce the number of cysts. Thus, Eng is a cyst-specific protein that may play a role in Msg variant expression in Pneumocystis.

Contemporary avian influenza A virus subtype H1, H6, H7, H10, and H15 hemagglutinin genes encode a mammalian virulence factor similar to the 1918 pandemic virus H1 hemagglutinin.

UNLABELLED: Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. IMPORTANCE: Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections with these subtypes may be important in controlling future outbreaks.

The natural history of influenza infection in the severely immunocompromised vs nonimmunocompromised hosts.

INTRODUCTION: Medical advances have led to an increase in the world's population of immunosuppressed individuals. The most severely immunocompromised patients are those who have been diagnosed with a hematologic malignancy, solid organ tumor, or who have other conditions that require immunosuppressive therapies and/or solid organ or stem cell transplants. MATERIALS AND METHODS: Medically attended patients with a positive clinical diagnosis of influenza were recruited prospectively and clinically evaluated. Nasal washes and serum were collected. Evaluation of viral shedding, nasal and serum cytokines, clinical illness, and clinical outcomes were performed to compare severely immunocompromised individuals to nonimmunocompromised individuals with influenza infection. RESULTS: Immunocompromised patients with influenza had more severe disease/complications, longer viral shedding, and more antiviral resistance while demonstrating less clinical symptoms and signs on clinical assessment. CONCLUSIONS: Immunocompromised patients are at risk for more severe or complicated influenza induced disease, which may be difficult to prevent with existing vaccines and antiviral treatments. Specific issues to consider when managing a severely immunocompromised host include the development of asymptomatic shedding, multi-drug resistance during prolonged antiviral therapy, and the potential high risk of pulmonary involvement. CLINICAL TRIALS REGISTRATION: ClinicalTrials.gov identifier NCT00533182.

Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells.

We studied the changes in expression of microRNAs (miRNAs or miRs) and mRNA in normal human bronchial epithelial cells as they differentiate from an undifferentiated monolayer to a differentiated pseudostratified epithelium after 28 days of air-liquid interface (ALI) culture. After 28 days in ALI, the epithelial cells differentially expressed basal, ciliated, and goblet cell markers. Using Affymetrix microarrays, 20 human miRNAs were found to be up-regulated, whereas 35 miRNAs were found to be down-regulated in differentiated cells compared with undifferentiated cells. An analysis of changes in global mRNA expression revealed that 1,201 probe sets demonstrated an 8-fold change (FC) or greater at Day 28 of ALI culture. Of these, 816 were up-regulated and 385 were down-regulated. With differentiation, miR-449a increased (FC, 38.15), and was related to changes in mRNA for cell division cycle 25 homolog A (FC, 0.11). MiR-455 decreased (FC, 0.12) and was related to changes in mRNA for the epithelial cell marker, mucin 1 (FC, 136). Transfection with anti-miR-449 or miR-455-3p resulted in changes in target protein expression (cell division cycle 25 homolog A and mucin 1, respectively), whereas transfection with reporter genes with 3'-untranslated regions of these targets confirmed control of expression through that structure. Therefore, changes in specific miRNAs during human airway epithelial cell differentiation control gene and protein expression important for differentiation.

High-throughput RNA sequencing of a formalin-fixed, paraffin-embedded autopsy lung tissue sample from the 1918 influenza pandemic.

Most biopsy and autopsy tissues are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. The RNA genome of the 1918 pandemic influenza virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Here, the full genome of the 1918 virus at 3000× coverage was determined in one high-throughput sequencing run of a library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments. Bacterial sequences associated with secondary bacterial pneumonias were also detected. Host transcripts were well represented in the library. Compared to a 2009 pandemic influenza virus FFPE post-mortem library, the 1918 sample showed significant enrichment for host defence and cell death response genes, concordant with prior animal studies. This methodological approach should assist in the analysis of FFPE tissue samples isolated over the past century from a variety of diseases.

Identification of a novel splice variant form of the influenza A virus M2 ion channel with an antigenically distinct ectodomain.

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle.

Human H-ficolin inhibits replication of seasonal and pandemic influenza A viruses.

The collectins have been shown to have a role in host defense against influenza A virus (IAV) and other significant viral pathogens (e.g., HIV). The ficolins are a related group of innate immune proteins that are present at relatively high concentrations in serum, but also in respiratory secretions; however, there has been little study of the role of ficolins in viral infection. In this study, we demonstrate that purified recombinant human H-ficolin and H-ficolin in human serum and bronchoalveolar lavage fluid bind to IAV and inhibit viral infectivity and hemagglutination activity in vitro. Removal of ficolins from human serum or bronchoalveolar lavage fluid reduces their antiviral activity. Inhibition of IAV did not involve the calcium-dependent lectin activity of H-ficolin. We demonstrate that H-ficolin is sialylated and that removal of sialic acid abrogates IAV inhibition, while addition of the neuraminidase inhibitor oseltamivir potentiates neutralization, hemagglutinin inhibition, and viral aggregation caused by H-ficolin. Pandemic and mouse-adapted strains of IAV are generally not inhibited by the collectins surfactant protein D or mannose binding lectin because of a paucity of glycan attachments on the hemagglutinin of these strains. In contrast, H-ficolin inhibited both the mouse-adapted PR-8 H1N1 strain and a pandemic H1N1 strain from 2009. H-ficolin also fixed complement to a surface coated with IAV. These findings suggest that H-ficolin contributes to host defense against IAV.

Overlapping signals for translational regulation and packaging of influenza A virus segment 2.

Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1-4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1-4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.

The ability of pandemic influenza virus hemagglutinins to induce lower respiratory pathology is associated with decreased surfactant protein D binding.

Pandemic influenza viral infections have been associated with viral pneumonia. Chimeric influenza viruses with the hemagglutinin segment of the 1918, 1957, 1968, or 2009 pandemic influenza viruses in the context of a seasonal H1N1 influenza genome were constructed to analyze the role of hemagglutinin (HA) in pathogenesis and cell tropism in a mouse model. We also explored whether there was an association between the ability of lung surfactant protein D (SP-D) to bind to the HA and the ability of the corresponding chimeric virus to infect bronchiolar and alveolar epithelial cells of the lower respiratory tract. Viruses expressing the hemagglutinin of pandemic viruses were associated with significant pathology in the lower respiratory tract, including acute inflammation, and showed low binding activity for SP-D. In contrast, the virus expressing the HA of a seasonal influenza strain induced only mild disease with little lung pathology in infected mice and exhibited strong in vitro binding to SP-D.

Rapid selection of a transmissible multidrug-resistant influenza A/H3N2 virus in an immunocompromised host.

BACKGROUND: The overall impact of influenza virus infection in immunocompromised patients is largely unknown. Antigenic drift and genetic variations during prolonged influenza infection have been demonstrated. In this report we describe a multidrug-resistant H3N2 influenza virus isolated from an immunocompromised patient after 5 days of therapy. METHODS: Multiple nasal wash samples were collected from an infected patient, and viral isolates were characterized. Sensitivity to antiviral agents was evaluated. Fitness and transmissibility were assessed in ferrets and tissue culture. RESULTS: An in-frame 4-amino acid deletion emerged in the neuraminidase (NA) gene of an H3N2 virus after 5 days of oseltamivir therapy. No other changes in the NA or hemagglutinin genes were noted. Drug sensitivity assays revealed resistance to oseltamivir (>10-fold increase in 50% inhibitory concentration [IC(50)]) and reduction in sensitivity to zanamivir (3-7-fold increase in IC(50) or 50% effective concentration). No change in fitness or transmissibility was observed. CONCLUSIONS: An in-frame NA gene deletion was rapidly selected for in an immunocompromised patient, resulting in decreased sensitivity of the isolate to available NA inhibitors without a change in fitness or transmissibility. This finding has implications for our understanding of the emergence of antiviral resistance and treatment of patients with influenza A infection, especially those who are immunocompromised.

Ewing sarcoma family of tumors in unusual sites: confirmation by rt-PCR.

Ewing sarcoma family tumors originating in the palate or adrenal gland are extremely rare and may cause difficulty in diagnosis. More common tumors primary to these sites need to be excluded before one arrives at the correct diagnosis. We have recently diagnosed 2 such cases. The 1st case was that of a 24-year-old woman who presented with a swelling in the right side of the hard palate. The 2nd case was diagnosed in a 28-year-old woman who presented with a mass in the right adrenal gland. In both cases, the diagnosis of Ewing sarcoma family of tumors was confirmed by immunohistochemical studies and reverse transcriptase-polymerase chain reaction (RT-PCR). The hard palate case is the 1st and the adrenal gland the 3rd case of Ewing sarcoma family of tumors arising in these sites, in which the diagnosis was confirmed by RT-PCR and/or cytogenetics. Accurate diagnosis of Ewing sarcoma family of tumors is crucial for the management of patients, and when found in such rare locations, diagnosis should be supported by immunohistochemical and/or molecular genetic studies.

Pathogenicity of influenza viruses with genes from the 1918 pandemic virus: functional roles of alveolar macrophages and neutrophils in limiting virus replication and mortality in mice.

The Spanish influenza pandemic of 1918 to 1919 swept the globe and resulted in the deaths of at least 20 million people. The basis of the pulmonary damage and high lethality caused by the 1918 H1N1 influenza virus remains largely unknown. Recombinant influenza viruses bearing the 1918 influenza virus hemagglutinin (HA) and neuraminidase (NA) glycoproteins were rescued in the genetic background of the human A/Texas/36/91 (H1N1) (1918 HA/NA:Tx/91) virus. Pathogenesis experiments revealed that the 1918 HA/NA:Tx/91 virus was lethal for BALB/c mice without the prior adaptation that is usually required for human influenza A H1N1 viruses. The increased mortality of 1918 HA/NA:Tx/91-infected mice was accompanied by (i) increased (>200-fold) viral replication, (ii) greater influx of neutrophils into the lung, (iii) increased numbers of alveolar macrophages (AMs), and (iv) increased protein expression of cytokines and chemokines in lung tissues compared with the levels seen for control Tx/91 virus-infected mice. Because pathological changes in AMs and neutrophil migration correlated with lung inflammation, we assessed the role of these cells in the pathogenesis associated with 1918 HA/NA:Tx/91 virus infection. Neutrophil and/or AM depletion initiated 3 or 5 days after infection did not have a significant effect on the disease outcome following a lethal 1918 HA/NA:Tx/91 virus infection. By contrast, depletion of these cells before a sublethal infection with 1918 HA/NA:Tx/91 virus resulted in uncontrolled virus growth and mortality in mice. In addition, neutrophil and/or AM depletion was associated with decreased expression of cytokines and chemokines. These results indicate that a human influenza H1N1 virus possessing the 1918 HA and NA glycoproteins can induce severe lung inflammation consisting of AMs and neutrophils, which play a role in controlling the replication and spread of 1918 HA/NA:Tx/91 virus after intranasal infection of mice.

Novel origin of the 1918 pandemic influenza virus nucleoprotein gene.

The nucleoprotein (NP) gene of the 1918 pandemic influenza A virus has been amplified and sequenced from archival material. The NP gene is known to be involved in many aspects of viral function and to interact with host proteins, thereby playing a role in host specificity. The 1918 NP amino acid sequence differs at only six amino acids from avian consensus sequences, consistent with reassortment from an avian source shortly before 1918. However, the nucleotide sequence of the 1918 NP gene has more than 170 differences from avian strain consensus sequences, suggesting substantial evolutionary distance from known avian strain sequences. Both the gene and protein sequences of the 1918 NP fall within the mammalian clade upon phylogenetic analysis. The evolutionary distance of the 1918 NP sequences from avian and mammalian strain sequences is examined, using several different parameters. The results suggest that the 1918 strain did not retain the previously circulating human NP. Nor is it likely to have obtained its NP by reassortment with an avian strain similar to those now characterized. The results are consistent with the existence of a currently unknown host for influenza, with an NP similar to current avian strain NPs at the amino acid level but with many synonymous nucleotide differences, suggesting evolutionary isolation from the currently characterized avian influenza virus gene pool.

Lung pathology of severe acute respiratory syndrome (SARS): a study of 8 autopsy cases from Singapore.

Severe acute respiratory syndrome (SARS) is an infectious condition caused by the SARS-associated coronavirus (SARS-CoV), a new member in the family Coronaviridae. To evaluate the lung pathology in this life-threatening respiratory illness, we studied postmortem lung sections from 8 patients who died from SARS during the spring 2003 Singapore outbreak. The predominant pattern of lung injury in all 8 cases was diffuse alveolar damage. The histology varied according to the duration of illness. Cases of 10 or fewer days' duration demonstrated acute-phase diffuse alveolar damage (DAD), airspace edema, and bronchiolar fibrin. Cases of more than 10 days' duration exhibited organizing-phase DAD, type II pneumocyte hyperplasia, squamous metaplasia, multinucleated giant cells, and acute bronchopneumonia. In acute-phase DAD, pancytokeratin staining was positive in hyaline membranes along alveolar walls and highlighted the absence of pneumocytes. Multinucleated cells were shown to be both type II pneumocytes and macrophages by pancytokeratin, thyroid transcription factor-1, and CD68 staining. SARS-CoV RNA was identified by reverse transcriptase-polymerase chain reaction in 7 of 8 cases in fresh autopsy tissue and in 8 of 8 cases in formalin-fixed, paraffin-embedded lung tissue, including the 1 negative case in fresh tissue. Understanding the pathology of DAD in SARS patients may provide the basis for therapeutic strategies. Further studies of the pathogenesis of SARS may reveal new insight into the mechanisms of DAD.