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This multi-country prospective study of infants aged <1 year aims to assess the frequency of influenza virus and respiratory syncytial virus (RSV) infections associated with hospitalizations, to describe clinical features and antibody response to infection, and to examine predictors of very severe disease requiring intensive care.
Although there is evidence of person-to-person transmission of Middle East respiratory syndrome coronavirus (MERS-CoV) in household and healthcare settings, more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission.
Middle East respiratory syndrome (MERS) is a viral respiratory illness first reported in Saudi Arabia in September 2012 caused by the human coronavirus (CoV), MERS-CoV. Using full-genome sequencing and phylogenetic analysis, scientists have identified three clades and multiple lineages of MERS-CoV in humans and the zoonotic host, dromedary camels. In this study, we have characterized eight MERS-CoV isolates collected from patients in Saudi Arabia in 2015. We have performed full-genome sequencing on the viral isolates, and compared them to the corresponding clinical specimens. All isolates were clade B, lineages 4 and 5. Three of the isolates carry deletions located on three independent regions of the genome in the 5'UTR, ORF1a and ORF3. All novel MERS-CoV strains replicated efficiently in Vero and Huh7 cells. Viruses with deletions in the 5'UTR and ORF1a exhibited impaired viral release in Vero cells. These data emphasize the plasticity of the MERS-CoV genome during human infection.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the etiologic agent associated with coronavirus disease, which emerged in late 2019. In response, we developed a diagnostic panel consisting of 3 real-time reverse transcription PCR assays targeting the nucleocapsid gene and evaluated use of these assays for detecting SARS-CoV-2 infection. All assays demonstrated a linear dynamic range of 8 orders of magnitude and an analytical limit of detection of 5 copies/reaction of quantified RNA transcripts and 1 x 10-1.5 50% tissue culture infectious dose/mL of cell-cultured SARS-CoV-2. All assays performed comparably with nasopharyngeal and oropharyngeal secretions, serum, and fecal specimens spiked with cultured virus. We obtained no false-positive amplifications with other human coronaviruses or common respiratory pathogens. Results from all 3 assays were highly correlated during clinical specimen testing. On February 4, 2020, the Food and Drug Administration issued an Emergency Use Authorization to enable emergency use of this panel.
Prioritization of sequence variants for diagnosis and discovery of Mendelian diseases is challenging, especially in large collections of whole genome sequences (WGS). Fast, scalable solutions are needed for discovery research, for clinical applications, and for curation of massive public variant repositories such as dbSNP and gnomAD. In response, we have developed VVP, the VAAST Variant Prioritizer. VVP is ultrafast, scales to even the largest variant repositories and genome collections, and its outputs are designed to simplify clinical interpretation of variants of uncertain significance.
Middle East respiratory syndrome coronavirus (MERS-CoV) shedding and antibody responses are not fully understood, particularly in relation to underlying medical conditions, clinical manifestations, and mortality. We enrolled MERS-CoV-positive patients at a hospital in Saudi Arabia and periodically collected specimens from multiple sites for real-time reverse transcription PCR and serologic testing. We conducted interviews and chart abstractions to collect clinical, epidemiologic, and laboratory information. We found that diabetes mellitus among survivors was associated with prolonged MERS-CoV RNA detection in the respiratory tract. Among case-patients who died, development of robust neutralizing serum antibody responses during the second and third week of illness was not sufficient for patient recovery or virus clearance. Fever and cough among mildly ill patients typically aligned with RNA detection in the upper respiratory tract; RNA levels peaked during the first week of illness. These findings should be considered in the development of infection control policies, vaccines, and antibody therapeutics.
Among 249 healthcare personnel who worked in hospital units with COVID-19 patients for 1 month, 19 (7.6%) tested positive for SARS-CoV-2 antibodies. Only 11 (57.9%) of the 19 personnel with positive serology reported symptoms of a prior illness, suggesting asymptomatic healthcare personnel could be an important source of SARS-CoV-2 transmission.
One in six nursing home residents and staff with positive SARS-CoV-2 tests ≥90 days after initial infection had specimen cycle thresholds (Ct) <30. Individuals with specimen Ct<30 were more likely to report symptoms but were not different from individuals with high Ct value specimens by other clinical and testing data.
The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccine-mediated protection from disease. To ascertain and rank the risk of VOCs and VOIs, we analyze the ability of 14 variants (614G, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Eta, Theta, Iota, Kappa, Lambda, Mu, and Omicron) to escape from mRNA vaccine-induced antibodies. The variants show differential reductions in neutralization and replication by post-vaccination sera. Although the Omicron variant (BA.1, BA.1.1, and BA.2) shows the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retain moderate neutralizing activity against that variant. Therefore, vaccination remains an effective strategy during the COVID-19 pandemic.
We compared paired serum specimens from household contacts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases with detectable SARS-CoV-2 seroconversion with contacts who remained seronegative. No protection from SARS-CoV-2 infection was associated with human coronavirus antibodies; however, an increase in common betacoronavirus antibodies was associated with seroconversion to SARS-CoV-2 in mild to moderately ill cases.
Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Reliable detection and identification of HRSV subgroup A and B infections are essential for accurate disease burden estimates in anticipation of licensure of novel HRSV vaccines and immunotherapies. To ensure continued reliability, molecular assays must remain current with evolving virus strains. We have developed a HRSV subgroup-specific real-time RT-PCR (rRT-PCR) assay for detection and subgroup identification using primers and subgroup-specific probes targeting a conserved region of the nucleoprotein gene combined in a single duplex reaction using all genome sequence data currently available in GenBank. The assay was validated for analytical sensitivity, specificity, reproducibility, and clinical performance with a geographically diverse collection of viral isolates and respiratory specimens in direct comparison with an established pan-HRSV rRT-PCR reference test. The assay was sensitive, reproducibly detecting as few as 5-10 copies/reaction of target RNA. The assay was specific, showing no amplification with a panel of 16 other common respiratory pathogens or predicted by in silico primer/probe analysis. The duplex rRT-PCR assay based on the most current available genome sequence data permits rapid, sensitive and specific detection and subgroup identification of HRSV.
Camels are known carriers for many viral pathogens, including Middle East respiratory syndrome coronavirus (MERS-CoV). It is likely that there are additional, as yet unidentified viruses in camels with the potential to cause disease in humans. In this study, we performed metagenomic sequencing analysis on nasopharyngeal swab samples from 108 MERS-CoV-positive dromedary camels from a live animal market in Abu Dhabi, United Arab Emirates. We obtained a total of 846.72 million high-quality reads from these nasopharyngeal swab samples, of which 2.88 million (0.34%) were related to viral sequences while 512.63 million (60.5%) and 50.87 million (6%) matched bacterial and eukaryotic sequences, respectively. Among the viral reads, sequences related to mammalian viruses from 13 genera in 10 viral families were identified, including Coronaviridae, Nairoviridae, Paramyxoviridae, Parvoviridae, Polyomaviridae, Papillomaviridae, Astroviridae, Picornaviridae, Poxviridae, and Genomoviridae. Some viral sequences belong to known camel or human viruses and others are from potentially novel camel viruses with only limited sequence similarity to virus sequences in GenBank. A total of five potentially novel virus species or strains were identified. Co-infection of at least two recently identified camel coronaviruses was detected in 92.6% of the camels in the study. This study provides a comprehensive survey of viruses in the virome of upper respiratory samples in camels that have extensive contact with the human population.
The etiologic agent of the outbreak of pneumonia in Wuhan China was identified as severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) in January, 2020. The first US patient was diagnosed by the State of Washington and the US Centers for Disease Control and Prevention on January 20, 2020. We isolated virus from nasopharyngeal and oropharyngeal specimens, and characterized the viral sequence, replication properties, and cell culture tropism. We found that the virus replicates to high titer in Vero-CCL81 cells and Vero E6 cells in the absence of trypsin. We also deposited the virus into two virus repositories, making it broadly available to the public health and research communities. We hope that open access to this important reagent will expedite development of medical countermeasures.
Following its emergence in Wuhan, China, in late November or early December 2019, the SARS-CoV-2 virus has rapidly spread throughout the world. On March 11, 2020, the World Health Organization declared Coronavirus Disease 2019 (COVID-19) a pandemic. Genome sequencing of SARS-CoV-2 strains allows for the reconstruction of transmission history connecting these infections. Here, we analyze 346 SARS-CoV-2 genomes from samples collected between 20 February and 15 March 2020 from infected patients in Washington State, USA. We found that the large majority of SARS-CoV-2 infections sampled during this time frame appeared to have derived from a single introduction event into the state in late January or early February 2020 and subsequent local spread, strongly suggesting cryptic spread of COVID-19 during the months of January and February 2020, before active community surveillance was implemented. We estimate a common ancestor of this outbreak clade as occurring between 18 January and 9 February 2020. From genomic data, we estimate an exponential doubling between 2.4 and 5.1 days. These results highlight the need for large-scale community surveillance for SARS-CoV-2 introductions and spread and the power of pathogen genomics to inform epidemiological understanding.
The novel coronavirus pandemic incited unprecedented demand for assays that detect viral nucleic acids, viral proteins, and corresponding antibodies. The 320 molecular diagnostics in receipt of US Food and Drug Administration emergency use authorization mainly focus on viral detection; however, no currently approved test can be used to infer infectiousness, that is, the presence of replicable virus. As the number of tests conducted increased, persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA positivity by reverse-transcription polymerase chain reaction (RT-PCR) in some individuals led to concerns over quarantine guidelines. To this end, we attempted to design an assay that reduces the frequency of positive test results from individuals who do not shed culturable virus. We describe multiplex quantitative RT-PCR assays that detect genomic RNA (gRNA) and subgenomic RNA (sgRNA) species of SARS-CoV-2, including spike, nucleocapsid, membrane, envelope, and ORF8. Viral RNA abundances calculated from these assays were compared with antigen presence, self-reported symptoms, and culture outcome (virus isolation) using samples from a 14-day longitudinal household transmission study. By characterizing the clinical and molecular dynamics of infection, we show that sgRNA detection has higher predictive value for culture outcome compared to detection of gRNA alone. Our findings suggest that sgRNA presence correlates with active infection and may help identify individuals shedding culturable virus.
Previous COVID-19 vaccine efficacy (VE) studies have estimated neutralizing and binding antibody concentrations that correlate with protection from symptomatic infection; how these estimates compare to those generated in response to SARS-CoV-2 infection is unclear. Here, we assessed quantitative neutralizing and binding antibody concentrations using standardized SARS-CoV-2 assays on 3,067 serum specimens collected during 27 July 2020 to 27 August 2020 from COVID-19-unvaccinated persons with detectable anti-SARS-CoV-2 antibodies. Neutralizing and binding antibody concentrations were severalfold lower in the unvaccinated study population compared to published concentrations at 28 days postvaccination. In this convenience sample, ~88% of neutralizing and ~63 to 86% of binding antibody concentrations met or exceeded concentrations associated with 70% COVID-19 VE against symptomatic infection; ~30% of neutralizing and 1 to 14% of binding antibody concentrations met or exceeded concentrations associated with 90% COVID-19 VE. Our study not only supports observations of infection-induced immunity and current recommendations for vaccination postinfection to maximize protection against COVID-19, but also provides a large data set of pre-COVID-19 vaccination anti-SARS-CoV-2 antibody concentrations that will serve as an important comparator in the current setting of vaccine-induced and hybrid immunity. As new SARS-CoV-2 variants emerge and displace circulating virus strains, we recommend that standardized binding antibody assays that include spike protein-based antigens be utilized to estimate antibody concentrations correlated with protection from COVID-19. These estimates will be helpful in informing public health guidance, such as the need for additional COVID-19 vaccine booster doses to prevent symptomatic infection. IMPORTANCE Although COVID-19 vaccine efficacy (VE) studies have estimated antibody concentrations that correlate with protection from COVID-19, how these estimates compare to those generated in response to SARS-CoV-2 infection is unclear. We assessed quantitative neutralizing and binding antibody concentrations using standardized assays on serum specimens collected from COVID-19-unvaccinated persons with detectable antibodies. We found that most unvaccinated persons with qualitative antibody evidence of prior infection had quantitative antibody concentrations that met or exceeded concentrations associated with 70% VE against COVID-19. However, only a small proportion had antibody concentrations that met or exceeded concentrations associated with 90% VE, suggesting that persons with prior COVID-19 would benefit from vaccination to maximize protective antibody concentrations against COVID-19.
To assess transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a detention facility experiencing a coronavirus disease outbreak and evaluate testing strategies, we conducted a prospective cohort investigation in a facility in Louisiana, USA. We conducted SARS-CoV-2 testing for detained persons in 6 quarantined dormitories at various time points. Of 143 persons, 53 were positive at the initial test, and an additional 58 persons were positive at later time points (cumulative incidence 78%). In 1 dormitory, all 45 detained persons initially were negative; 18 days later, 40 (89%) were positive. Among persons who were SARS-CoV-2 positive, 47% (52/111) were asymptomatic at the time of specimen collection; 14 had replication-competent virus isolated. Serial SARS-CoV-2 testing might help interrupt transmission through medical isolation and quarantine. Testing in correctional and detention facilities will be most effective when initiated early in an outbreak, inclusive of all exposed persons, and paired with infection prevention and control.
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