Introduction
We describe post‐mortem pulmonary histopathologic findings of COVID‐19 pneumonia in patients with a spectrum of disease course, from rapid demise to prolonged hospitalisation.
Methods ...and results
Histopathologic findings in post‐mortem lung tissue from eight patients who died from COVID‐19 pneumonia were reviewed. Immunohistochemistry (IHC) and next‐generation sequencing (NGS) were performed to detect virus. Diffuse alveolar damage (DAD) was seen in all cases with a spectrum of acute phase and/or organising phase. IHC with monoclonal antibodies against SARS‐CoV‐2 viral nucleoprotein and spike protein detected virus in areas of acute but not organising DAD, with intracellular viral antigen and RNA expression seen predominantly in patients with duration of illness less than 10 days. Major vascular findings included thrombi in medium‐ and large‐calibre vessels, platelet microthrombi detected by CD61 IHC and fibrin microthrombi.
Conclusions
Presence of SARS‐CoV‐2 viral RNA by NGS early in the disease course and expression of viral antigen by IHC exclusively in the acute, but not in the organising phase of DAD, suggests that the virus may play a major role in initiating the acute lung injury of DAD, but when DAD progresses to the organising phase the virus may have been cleared from the lung by the patient's immune response. These findings suggest the possibility of a major change during the disease course of COVID‐19 pneumonia that may have therapeutic implications. Frequent thrombi and microthrombi may also present potential targets for therapeutic intervention.
Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterized by respiratory distress, multiorgan dysfunction, and, in some cases, ...death. The pathological mechanisms underlying COVID-19 respiratory distress and the interplay with aggravating risk factors have not been fully defined. Lung autopsy samples from 18 patients with fatal COVID-19, with symptom onset-to-death times ranging from 3 to 47 days, and antemortem plasma samples from 6 of these cases were evaluated using deep sequencing of SARS-CoV-2 RNA, multiplex plasma protein measurements, and pulmonary gene expression and imaging analyses. Prominent histopathological features in this case series included progressive diffuse alveolar damage with excessive thrombosis and late-onset pulmonary tissue and vascular remodeling. Acute damage at the alveolar-capillary barrier was characterized by the loss of surfactant protein expression with injury to alveolar epithelial cells, endothelial cells, respiratory epithelial basal cells, and defective tissue repair processes. Other key findings included impaired clot fibrinolysis with increased concentrations of plasma and lung plasminogen activator inhibitor-1 and modulation of cellular senescence markers, including p21 and sirtuin-1, in both lung epithelial and endothelial cells. Together, these findings further define the molecular pathological features underlying the pulmonary response to SARS-CoV-2 infection and provide important insights into signaling pathways that may be amenable to therapeutic intervention.
The 1918 influenza pandemic was the most devastating respiratory pandemic in modern human history, with 50-100 million deaths worldwide. Here, we characterized the complete genomes of influenza A ...virus (IAV) from two fatal cases during the fall wave of 1918 influenza A (H1N1) pandemic in the United States, one from Walter Reed Army Hospital in Washington, DC, and the other from Camp Jackson, SC. The two complete IAV genomes were obtained by combining Illumina deep sequencing data from both total RNA and influenza viral genome-enriched libraries along with Sanger sequencing data from PCR across the sequencing gaps. This study confirms the previously reported 1918 IAV genomes and increases the total number of available complete or near-complete influenza viral genomes of the 1918 pandemic from four to six. Sequence comparisons among them confirm that the genomes of the 1918 pandemic virus were highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases. Interestingly, in the Washington, DC, case, evidence is presented of the first reported
-influenza virus co-infection.
This study applied modern molecular biotechnology and high-throughput sequencing to formalin-fixed, paraffin-embedded autopsy lung samples from two fatal cases during the fall wave of the 1918 influenza A (H1N1) pandemic in the United States. Complete influenza genomes were obtained from both cases, which increases the total number of available complete or near-complete influenza genomes of the 1918 pandemic virus from four to six. Sequence analysis confirms that the 1918 pandemic virus was highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases, including the first reported evidence of
-influenza co-infection. Overall, this study offers a detailed view at the molecular level of the very limited samples from the most devastating influenza pandemic in modern human history.
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.
In March 2009, a novel swine-origin influenza A/H1N1 virus was identified. After global spread, the World Health Organization in June declared the first influenza pandemic in 41 years.
To describe ...the clinicopathologic characteristics of 34 people who died following confirmed A/H1N1 infection with emphasis on the pulmonary pathology findings.
We reviewed medical records, autopsy reports, microbiologic studies, and microscopic slides of 34 people who died between May 15 and July 9, 2009, and were investigated either by the New York City Office of Chief Medical Examiner (32 deaths) or through the consultation service of a coauthor (2 deaths).
Most of the 34 decedents (62%) were between 25 and 49 years old (median, 41.5 years). Tracheitis, bronchiolitis, and diffuse alveolar damage were noted in most cases. Influenza viral antigen was observed most commonly in the epithelium of the tracheobronchial tree but also in alveolar epithelial cells and macrophages. Most cases were reverse transcription-polymerase chain reaction positive for influenza. Histologic and microbiologic autopsy evidence of bacterial pneumonia was detected in 55% of cases. Underlying medical conditions including cardiorespiratory diseases and immunosuppression were present in 91% of cases. Obesity (body mass index, >30) was noted in 72% of adult and adolescent cases.
The pulmonary pathologic findings in fatal disease caused by the novel pandemic influenza virus are similar to findings identified in past pandemics. Superimposed bacterial infections of the respiratory tract were common. Preexisting obesity, cardiorespiratory diseases, and other comorbidities also were prominent findings among the decedents.
The 1918 to 1919 "Spanish" influenza pandemic virus killed up to 50 million people. We report here clinical, pathological, bacteriological, and virological findings in 68 fatal American ...influenza/pneumonia military patients dying between May and October of 1918, a period that includes ∼4 mo before the 1918 pandemic was recognized, and 2 mo (September–October 1918) during which it appeared and peaked. The lung tissues of 37 of these cases were positive for influenza viral antigens or viral RNA, including four from the prepandemic period (May–August). The prepandemic and pandemic peak cases were indistinguishable clinically and pathologically. All 68 cases had histological evidence of bacterial pneumonia, and 94% showed abundant bacteria on Gram stain. Sequence analysis of the viral hemagglutinin receptor-binding domain performed on RNA from 13 cases suggested a trend from a more "avian-like" viral receptor specificity with G222 in prepandemic cases to a more "human-like" specificity associated with D222 in pandemic peak cases. Viral antigen distribution in the respiratory tree, however, was not apparently different between prepandemic and pandemic peak cases, or between infections with viruses bearing different receptor-binding polymorphisms. The 1918 pandemic virus was circulating for at least 4 mo in the United States before it was recognized epidemiologically in September 1918. The causes of the unusually high mortality in the 1918 pandemic were not explained by the pathological and virological parameters examined. These findings have important implications for understanding the origins and evolution of pandemic influenza viruses.
The fifth wave of A(H7N9) virus infection in China from 2016 to 2017 caused great concern due to the large number of individuals infected, the isolation of drug-resistant viruses, and the emergence ...of highly pathogenic strains. Antibodies against neuraminidase (NA) provide added benefit to hemagglutinin-specific immunity and may be important contributors to the effectiveness of A(H7N9) vaccines. We generated a panel of mouse monoclonal antibodies (MAbs) to identify antigenic domains on NA of the novel A(H7N9) virus and compared their functional properties. The loop formed in the region of residue 250 (250 loop) and the domain formed by the loops containing residues 370, 400, and 430 were identified as major antigenic regions. MAbs 1E8, 2F6, 10F4, and 11B2, which recognize these two antigenic domains, were characterized in depth. These four MAbs differ in their abilities to inhibit cleavage of small and large substrates (methyl-umbelliferyl-acetyl neuraminic acid MU-NANA and fetuin, respectively) in NA inhibition assays. 1E8 and 11B2 did not inhibit NA cleavage of either MU-NANA or fetuin, and 2F6 inhibited cleavage of fetuin alone, whereas 10F4 inhibited cleavage of both substrates. All four MAbs reduced the
spread of viruses carrying either the wild-type N9 or N9 with antiviral-resistant mutations but to different degrees. These MAbs have different
levels of effectiveness: 10F4 was the most effective in protecting mice against challenge with A(H7N9) virus, 2F6 was less effective, and 11B2 failed to protect BALB/c mice at the doses tested. Our study confirms that NA-specific antibodies can protect against A(H7N9) infection and suggests that
properties can be used to rank antibodies with therapeutic potential.
The novel A(H7N9) viruses that emerged in China in 2013 continue to infect humans, with a high fatality rate. The most recent outbreak resulted in a larger number of human cases than previous epidemic waves. Due to the absence of a licensed vaccine and the emergence of drug-resistant viruses, there is a need to develop alternative approaches to prevent or treat A(H7N9) infection. We have made a panel of mouse monoclonal antibodies (MAbs) specific for neuraminidase (NA) of A(H7N9) viruses; some of these MAbs are effective in inhibiting viruses that are resistant to antivirals used to treat A(H7N9) patients. Binding avidity, inhibition of NA activity, and plaque formation correlated with the effectiveness of these MAbs to protect mice against lethal A(H7N9) virus challenge. This study identifies
measures that can be used to predict the
efficacy of NA-specific antibodies, providing a way to select MAbs for further therapeutic development.
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 IC50) and reduction in sensitivity to zanamivir (3–7-fold increase in IC50 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.
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