Kelvin K. W. To

Human infections with the emerging avian influenza A H7N9 virus from wet market poultry: clinical analysis and characterisation of viral genome

Summary Background Human infection with avian influenza A H7N9 virus emerged in eastern China in February, 2013, and has been associated with exposure to poultry. We report the clinical and microbiological features of patients infected with influenza A H7N9 virus and compare genomic features of the human virus with those of the virus in market poultry in Zhejiang, China. Methods Between March 7 and April 8, 2013, we included hospital inpatients if they had new-onset respiratory symptoms, unexplained radiographic infiltrate, and laboratory-confirmed H7N9 virus infection. We recorded histories and results of haematological, biochemical, radiological, and microbiological investigations. We took throat and sputum samples, used RT-PCR to detect M, H7, and N9 genes, and cultured samples in Madin-Darby canine kidney cells. We tested for co-infections and monitored serum concentrations of six cytokines and chemokines. We collected cloacal swabs from 86 birds from epidemiologically linked wet markets and inoculated embryonated chicken eggs with the samples. We identified and subtyped isolates by RT-PCR sequencing. RNA extraction, complementary DNA synthesis, and PCR sequencing were done for one human and one chicken isolate. We characterised and phylogenetically analysed the eight gene segments of the viruses in the patients and the chickens isolates, and constructed phylogenetic trees of H, N, PB2, and NS genes. Findings We identified four patients (mean age 56 years), all of whom had contact with poultry 3–8 days before disease onset. They presented with fever and rapidly progressive pneumonia that did not respond to antibiotics. Patients were leucopenic and lymphopenic, and had impaired liver or renal function, substantially increased serum cytokine or chemokine concentrations, and disseminated intravascular coagulation with disease progression. Two patients died. Sputum specimens were more likely to test positive for the H7N9 virus than were samples from throat swabs. The viral isolate from the patient was closely similar to that from an epidemiologically linked market chicken. All viral gene segments were of avian origin. The H7 of the isolated viruses was closest to that of the H7N3 virus from domestic ducks in Zhejiang, whereas the N9 was closest to that of the wild bird H7N9 virus in South Korea. We noted Gln226Leu and Gly186Val substitutions in human virus H7 (associated with increased affinity for α-2,6-linked sialic acid receptors) and the PB2 Asp701Asn mutation (associated with mammalian adaptation). Ser31Asn mutation, which is associated with adamantane resistance, was noted in viral M2. Interpretation Cross species poultry-to-person transmission of this new reassortant H7N9 virus is associated with severe pneumonia and multiorgan dysfunction in human beings. Monitoring of the viral evolution and further study of disease pathogenesis will improve disease management, epidemic control, and pandemic preparedness. Funding Larry Chi-Kin Yung, National Key Program for Infectious Diseases of China.

Delayed Clearance of Viral Load and Marked Cytokine Activation in Severe Cases of Pandemic H1N1 2009 Influenza Virus Infection

Abstract Background. Infections caused by the pandemic H1N1 2009 influenza virus range from mild upper respiratory tract syndromes to fatal diseases. However, studies comparing virological and immunological profile of different clinical severity are lacking. Methods. We conducted a retrospective cohort study of 74 patients with pandemic H1N1 infection, including 23 patients who either developed acute respiratory distress syndrome (ARDS) or died (ARDS-death group), 14 patients with desaturation requiring oxygen supplementation and who survived without ARDS (survived-without-ARDS group), and 37 patients with mild disease without desaturation (mild-disease group). We compared their pattern of clinical disease, viral load, and immunological profile. Results. Patients with severe disease were older, more likely to be obese or having underlying diseases, and had lower respiratory tract symptoms, especially dyspnea at presentation. The ARDS-death group had a slower decline in nasopharyngeal viral loads, had higher plasma levels of proinflammatory cytokines and chemokines, and were more likely to have bacterial coinfections (30.4%), myocarditis (21.7%), or viremia (13.0%) than patients in the survived-without-ARDS or the mild-disease groups. Reactive hemophagocytosis, thrombotic phenomena, lymphoid atrophy, diffuse alveolar damage, and multiorgan dysfunction similar to fatal avian influenza A H5N1 infection were found at postmortem examinations. Conclusions. The slower control of viral load and immunodysregulation in severe cases mandate the search for more effective antiviral and immunomodulatory regimens to stop the excessive cytokine activation resulting in ARDS and death.

Effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza A H1N1pdm09 virus infection: a meta-analysis of individual participant data

BACKGROUND Neuraminidase inhibitors were widely used during the 2009-10 influenza A H1N1 pandemic, but evidence for their effectiveness in reducing mortality is uncertain. We did a meta-analysis of individual participant data to investigate the association between use of neuraminidase inhibitors and mortality in patients admitted to hospital with pandemic influenza A H1N1pdm09 virus infection. METHODS We assembled data for patients (all ages) admitted to hospital worldwide with laboratory confirmed or clinically diagnosed pandemic influenza A H1N1pdm09 virus infection. We identified potential data contributors from an earlier systematic review of reported studies addressing the same research question. In our systematic review, eligible studies were done between March 1, 2009 (Mexico), or April 1, 2009 (rest of the world), until the WHO declaration of the end of the pandemic (Aug 10, 2010); however, we continued to receive data up to March 14, 2011, from ongoing studies. We did a meta-analysis of individual participant data to assess the association between neuraminidase inhibitor treatment and mortality (primary outcome), adjusting for both treatment propensity and potential confounders, using generalised linear mixed modelling. We assessed the association with time to treatment using time-dependent Cox regression shared frailty modelling. FINDINGS We included data for 29,234 patients from 78 studies of patients admitted to hospital between Jan 2, 2009, and March 14, 2011. Compared with no treatment, neuraminidase inhibitor treatment (irrespective of timing) was associated with a reduction in mortality risk (adjusted odds ratio [OR] 0·81; 95% CI 0·70-0·93; p=0·0024). Compared with later treatment, early treatment (within 2 days of symptom onset) was associated with a reduction in mortality risk (adjusted OR 0·48; 95% CI 0·41-0·56; p<0·0001). Early treatment versus no treatment was also associated with a reduction in mortality (adjusted OR 0·50; 95% CI 0·37-0·67; p<0·0001). These associations with reduced mortality risk were less pronounced and not significant in children. There was an increase in the mortality hazard rate with each days delay in initiation of treatment up to day 5 as compared with treatment initiated within 2 days of symptom onset (adjusted hazard ratio [HR 1·23] [95% CI 1·18-1·28]; p<0·0001 for the increasing HR with each days delay). INTERPRETATION We advocate early instigation of neuraminidase inhibitor treatment in adults admitted to hospital with suspected or proven influenza infection. FUNDING F Hoffmann-La Roche.

Viral load in patients infected with pandemic H1N1 2009 influenza A virus.

Viral shedding profile of infections caused by the pandemic H1N1 2009 influenza A virus has not been reported. The aim of this study was to determine the viral load in different body sites. Viral loads of pandemic H1N1 virus in respiratory specimens, stool, urine, and serum were determined by quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR). Respiratory specimens from patients with seasonal influenza were used as historical controls. Initial pre‐treatment viral load were compared between these two groups. Serial respiratory specimens from patients with pandemic H1N1 virus infection were obtained for analysis of viral dynamics. Twenty‐two pandemic H1N1 cases and 44 seasonal influenza historical controls were included. The mean initial viral load before oseltamivir therapy was 1.84 × 108 copies/ml for pandemic H1N1 virus compared with 3.28 × 108 copies/ml in seasonal influenza historical controls (P = 0.085). Among patients with pandemic H1N1 virus infection, peak viral load occurred on the day of onset of symptoms, and declined gradually afterwards, with no virus being detectable in respiratory specimens by RT‐PCR 8 days and by culture 5 days after the onset of symptoms respectively, except in one patient. Pandemic H1N1 virus was detected in stool and in urine from 4/9 and 1/14 patients, respectively. Viral culture was also positive from the stool sample with the highest viral load. Younger age was associated with prolonged shedding in the respiratory tract and higher viral load in the stool. Data from this quantitative analysis of viral shedding may have implications for formulating infection control measures. J. Med. Virol. 82:1–7, 2010.

Middle East Respiratory Syndrome Coronavirus: Another Zoonotic Betacoronavirus Causing SARS-Like Disease

SUMMARY The source of the severe acute respiratory syndrome (SARS) epidemic was traced to wildlife market civets and ultimately to bats. Subsequent hunting for novel coronaviruses (CoVs) led to the discovery of two additional human and over 40 animal CoVs, including the prototype lineage C betacoronaviruses, Tylonycteris bat CoV HKU4 and Pipistrellus bat CoV HKU5; these are phylogenetically closely related to the Middle East respiratory syndrome (MERS) CoV, which has affected more than 1,000 patients with over 35% fatality since its emergence in 2012. All primary cases of MERS are epidemiologically linked to the Middle East. Some of these patients had contacted camels which shed virus and/or had positive serology. Most secondary cases are related to health care-associated clusters. The disease is especially severe in elderly men with comorbidities. Clinical severity may be related to MERS-CoVs ability to infect a broad range of cells with DPP4 expression, evade the host innate immune response, and induce cytokine dysregulation. Reverse transcription-PCR on respiratory and/or extrapulmonary specimens rapidly establishes diagnosis. Supportive treatment with extracorporeal membrane oxygenation and dialysis is often required in patients with organ failure. Antivirals with potent in vitro activities include neutralizing monoclonal antibodies, antiviral peptides, interferons, mycophenolic acid, and lopinavir. They should be evaluated in suitable animal models before clinical trials. Developing an effective camel MERS-CoV vaccine and implementing appropriate infection control measures may control the continuing epidemic.

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.

Two Years after Pandemic Influenza A/2009/H1N1: What Have We Learned?

SUMMARY The world had been anticipating another influenza pandemic since the last one in 1968. The pandemic influenza A H1N1 2009 virus (A/2009/H1N1) finally arrived, causing the first pandemic influenza of the new millennium, which has affected over 214 countries and caused over 18,449 deaths. Because of the persistent threat from the A/H5N1 virus since 1997 and the outbreak of the severe acute respiratory syndrome (SARS) coronavirus in 2003, medical and scientific communities have been more prepared in mindset and infrastructure. This preparedness has allowed for rapid and effective research on the epidemiological, clinical, pathological, immunological, virological, and other basic scientific aspects of the disease, with impacts on its control. A PubMed search using the keywords “pandemic influenza virus H1N1 2009” yielded over 2,500 publications, which markedly exceeded the number published on previous pandemics. Only representative works with relevance to clinical microbiology and infectious diseases are reviewed in this article. A significant increase in the understanding of this virus and the disease within such a short amount of time has allowed for the timely development of diagnostic tests, treatments, and preventive measures. These findings could prove useful for future randomized controlled clinical trials and the epidemiological control of future pandemics.

Convalescent Plasma Treatment Reduced Mortality in Patients With Severe Pandemic Influenza A (H1N1) 2009 Virus Infection

Treatment of severe pandemic influenza A(H1N1) 2009 virus infection with convalescent plasma suppressed the viral load and cytokine response, thereby reducing the subsequent risk of complication and death. Further studies by double-blind randomized controlled trial of plasma treatment in these patients are warranted.

Interspecies transmission and emergence of novel viruses: lessons from bats and birds

As exemplified by coronaviruses and influenza viruses, bats and birds are natural reservoirs for providing viral genes during evolution of new virus species and viruses for interspecies transmission. These warm-blooded vertebrates display high species biodiversity, roosting and migratory behavior, and a unique adaptive immune system, which are favorable characteristics for asymptomatic shedding, dissemination, and mixing of different viruses for the generation of novel mutant, recombinant, or reassortant RNA viruses. The increased intrusion of humans into wildlife habitats and overcrowding of different wildlife species in wet markets and farms have also facilitated the interspecies transmission between different animal species.

Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus.

Summary Objectives Middle East respiratory syndrome coronavirus (MERS-CoV) has emerged to cause fatal infections in patients in the Middle East and traveler-associated secondary cases in Europe and Africa. Person-to-person transmission is evident in outbreaks involving household and hospital contacts. Effective antivirals are urgently needed. Methods We used small compound-based forward chemical genetics to screen a chemical library of 1280 known drugs against influenza A virus in Biosafety Level-2 laboratory. We then assessed the anti-MERS-CoV activities of the identified compounds and of interferons, nelfinavir, and lopinavir because of their reported anti-coronavirus activities in terms of cytopathic effect inhibition, viral yield reduction, and plaque reduction assays in Biosafety Level-3 laboratory. Results Ten compounds were identified as primary hits in high-throughput screening. Only mycophenolic acid exhibited low EC50 and high selectivity index. Additionally, ribavirin and interferons also exhibited in-vitro anti-MERS-CoV activity. The serum concentrations achievable at therapeutic doses of mycophenolic acid and interferon-β1b were 60–300 and 3–4 times higher than the concentrations at which in-vitro anti-MERS-CoV activities were demonstrated, whereas that of ribavirin was ∼2 times lower. Combination of mycophenolic acid and interferon-β1b lowered the EC50 of each drug by 1–3 times. Conclusions Interferon-β1b with mycophenolic acid should be considered in treatment trials of MERS.