Predicting the animal hosts of coronaviruses from compositional biases of spike protein and whole genome sequences through machine learning.

The COVID-19 pandemic has demonstrated the serious potential for novel zoonotic coronaviruses to emerge and cause major outbreaks. The immediate animal origin of the causative virus, SARS-CoV-2, remains unknown, a notoriously challenging task for emerging disease investigations. Coevolution with hosts leads to specific evolutionary signatures within viral genomes that can inform likely animal origins. We obtained a set of 650 spike protein and 511 whole genome nucleotide sequences from 222 and 185 viruses belonging to the family Coronaviridae, respectively. We then trained random forest models independently on genome composition biases of spike protein and whole genome sequences, including dinucleotide and codon usage biases in order to predict animal host (of nine possible categories, including human). In hold-one-out cross-validation, predictive accuracy on unseen coronaviruses consistently reached ~73%, indicating evolutionary signal in spike proteins to be just as informative as whole genome sequences. However, different composition biases were informative in each case. Applying optimised random forest models to classify human sequences of MERS-CoV and SARS-CoV revealed evolutionary signatures consistent with their recognised intermediate hosts (camelids, carnivores), while human sequences of SARS-CoV-2 were predicted as having bat hosts (suborder Yinpterochiroptera), supporting bats as the suspected origins of the current pandemic. In addition to phylogeny, variation in genome composition can act as an informative approach to predict emerging virus traits as soon as sequences are available. More widely, this work demonstrates the potential in combining genetic resources with machine learning algorithms to address long-standing challenges in emerging infectious diseases.

Meta-analysis of diagnostic performance of serological tests for SARS-CoV-2 antibodies up to 25 April 2020 and public health implications.

We reviewed the diagnostic accuracy of SARS-CoV-2 serological tests. Random-effects models yielded a summary sensitivity of 82% for IgM, and 85% for IgG and total antibodies. For specificity, the pooled estimate were 98% for IgM and 99% for IgG and total antibodies. In populations with ≤ 5% of seroconverted individuals, unless the assays have perfect (i.e. 100%) specificity, the positive predictive value would be ≤ 88%. Serological tests should be used for prevalence surveys only in hard-hit areas.

Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus.

BACKGROUND: A novel porcine deltacoronavirus (PDCoV), also known as porcine coronavirus HKU15, was reported in China in 2012 and identified in the U.S. in early 2014. Since then, PDCoV has been identified in a number of U.S. states and linked with clinical disease including acute diarrhea and vomiting in the absence of other identifiable pathogens. Since PDCoV was just recently linked with clinical disease, few specific antibody-based reagents were available to assist in diagnosis of PDCoV and limited serological capabilities were available to detect an antibody response to this virus. Therefore, the overall objective of this project was to develop and validate selected diagnostic reagents and assays for PDCoV antigen and antibody detection. RESULTS: The nucleoprotein of PDCoV was expressed as a recombinant protein and purified for use as an antigen to immunize mice for polyclonal, hyperimmune sera and monoclonal antibody (mAb) production. The resulting mAbs were evaluated for use in fluorescent antibody staining methods to detect PDCoV infected cells following virus isolation attempts and for immunohistochemistry staining of intestinal tissues of infected pigs. The same antigen was used to develop serological tests to detect the antibody response to PDCoV in pigs following infection. Serum samples from swine herds with recent documentation of PDCoV infection and samples from expected naïve herds were used for initial assay optimization. The tests were optimized in a checkerboard fashion to reduce signal to noise ratios using samples of known status. Statistical analysis was performed to establish assay cutoff values and assess diagnostic sensitivities and specificities. At least 629 known negative serum samples and 311 known positive samples were evaluated for each assay. The enzyme linked immunosorbent assay (ELISA) showed diagnostic sensitivity (DSe) of 96.1% and diagnostic specificity (DSp) of 96.2%. The fluorescent microsphere immunoassay (FMIA) showed a DSe of 95.8% and DSp of 98.1%. Both ELISA and FMIA detected seroconversion of challenged pigs between 8-14 days post-infection (DPI). An indirect fluorescent antibody (IFA) test was also developed using cell culture adapted PDCoV for comparative purposes. CONCLUSION: These new, specific reagents and serological assays will allow for improved diagnosis of PDCoV. Since many aspects of PDCoV infection and transmission are still not fully understood, the reagents and assays developed in this project should provide valuable tools to help understand this disease and to aid in the control and surveillance of porcine deltacoronavirus outbreaks.

Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus.

In February 2014, porcine deltacoronavirus (PDCoV) was identified in the United States. We developed a PDCoV real-time reverse transcription PCR that identified PDCoV in 30% of samples tested. Four additional PDCoV genomes from the United States were sequenced; these had ≈99%-100% nt similarity to the other US PDCoV strains.

Update: severe respiratory illness associated with a novel coronavirus--worldwide, 2012-2013.

CDC continues to work closely with the World Health Organization (WHO) and other partners to better understand the public health risk posed by a novel coronavirus that was first reported to cause human infection in September 2012. Genetic sequence analyses have shown that this new virus is different from any other known human coronaviruses, including the one that caused severe acute respiratory syndrome (SARS). As of March 7, 2013, a total of 14 confirmed cases of novel coronavirus infection have been reported to WHO, with eight deaths. Illness onsets have occurred from April 2012 through February 2013. To date, no cases have been reported in the United States.

The emerging novel Middle East respiratory syndrome coronavirus: the "knowns" and "unknowns".

A novel lineage C betacoronavirus, originally named human coronavirus EMC/2012 (HCoV-EMC) and recently renamed Middle East respiratory syndrome coronavirus (MERS-CoV), that is phylogenetically closely related to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5, which we discovered in 2007 from bats in Hong Kong, has recently emerged in the Middle East to cause a severe acute respiratory syndrome (SARS)-like infection in humans. The first laboratory-confirmed case, which involved a 60-year-old man from Bisha, the Kingdom of Saudi Arabia (KSA), who died of rapidly progressive community-acquired pneumonia and acute renal failure, was announced by the World Health Organization (WHO) on September 23, 2012. Since then, a total of 70 cases, including 39 fatalities, have been reported in the Middle East and Europe. Recent clusters involving epidemiologically-linked household contacts and hospital contacts in the Middle East, Europe, and Africa strongly suggested possible human-to-human transmission. Clinical and laboratory research data generated in the past few months have provided new insights into the possible animal reservoirs, transmissibility, and virulence of MERS-CoV, and the optimal laboratory diagnostic options and potential antiviral targets for MERS-CoV-associated infection.

Human rhinovirus and coronavirus detection among allogeneic hematopoietic stem cell transplantation recipients.

Little is known about clinical and virologic manifestations of rhinovirus (HRV) and coronavirus (HCoV) infections after hematopoietic cell transplantation (HCT). We performed surveillance for 1 year and describe the natural history of these infections during the first 100 days after allogeneic HCT, when symptom surveys and upper respiratory samples were collected weekly. Samples were tested using RT-PCR for HRVs and HCoVs (OC43, 229E, HKU1, and NL63). Among 215 patients, 64 (30%) patients had 67 infections. Day 100 cumulative incidence estimate was 22.3% for HRV and 11.1% for HCoV. Median duration of viral shedding was 3 weeks; prolonged shedding of at least 3 months occurred in 6 of 45 patients with HRV and 3 of 22 with HCoV. Six patients with HRV and 9 with HCoV were asymptomatic. HRV infection was associated with rhinorrhea, congestion, postnasal drip, sputum, and cough; HCoV infection was not associated with respiratory symptoms or hepatic dysfunction. Lower respiratory infection developed in 2 patients with HRV before day 100, and 1 each with HRV and HCoV after day 100. HRV and HCoV infections are common in the first 100 days after HCT, viral shedding lasts more than 3 weeks in half, and lower respiratory infection is rare.

Data standardization implementation and applications within and among diagnostic laboratories: integrating and monitoring enteric coronaviruses.

Every day, thousands of samples from diverse populations of animals are submitted to veterinary diagnostic laboratories (VDLs) for testing. Each VDL has its own laboratory information management system (LIMS), with processes and procedures to capture submission information, perform laboratory tests, define the boundaries of test results (i.e., positive or negative), and report results, in addition to internal business and accounting applications. Enormous quantities of data are accumulated and stored within VDL LIMSs. There is a need for platforms that allow VDLs to exchange and share portions of laboratory data using standardized, reliable, and sustainable information technology processes. Here we report concepts and applications for standardization and aggregation of data from swine submissions to multiple VDLs to detect and monitor porcine enteric coronaviruses by RT-PCR. Oral fluids, feces, and fecal swabs were the specimens submitted most frequently for enteric coronavirus testing. Statistical algorithms were used successfully to scan and monitor the overall and state-specific percentage of positive submissions. Major findings revealed a consistently recurrent seasonal pattern, with the highest percentage of positive submissions detected during December-February for porcine epidemic diarrhea virus, porcine deltacoronavirus, and transmissible gastroenteritis virus (TGEV). After 2014, very few submissions tested positive for TGEV. Monitoring VDL data proactively has the potential to signal and alert stakeholders early of significant changes from expected detection. We demonstrate the importance of, and applications for, data organized and aggregated by using LOINC and SNOMED CTs, as well as the use of customized messaging to allow inter-VDL exchange of information.

[Diagnostics of selected respiratory virus infections]. / Diagnostyka wybranych zakazen wirusowych ukladu oddechowego.

Viral infections are the most common infectious diseases of the respiratory tract characterized by the considerable mortality (especially among children and elderly people) and considered as the significant economic burden. It has been demonstrated that implementation of rapid diagnostic methods enabled more appropriate treatment of respiratory viral infections, reduced mean hospitalization time and cost, as well as resulted in the significantly decreased mortality. Modern diagnostic methods effectively identify respiratory virus, its antigen or nucleic acids in biological samples by means of the immunological and molecular techniques. This article presents critical overview of those methods with particular emphasis on their clinical usefulness and clinical reliability.

Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses.

With the outbreak of the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, coronaviruses have become a global research hotspot in the field of virology. Coronaviruses mainly cause respiratory and digestive tract diseases, several coronaviruses are responsible for porcine diarrhea, such as porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and emerging swine acute diarrhea syndrome coronavirus (SADS-CoV). Those viruses have caused huge economic losses and are considered as potential public health threats. Porcine torovirus (PToV) and coronaviruses, sharing similar genomic structure and replication strategy, belong to the same order Nidovirales. Here, we developed a multiplex TaqMan-probe-based real-time PCR for the simultaneous detection of PEDV, PDCoV, PToV, and SADS-CoV for the first time. Specific primers and TaqMan fluorescent probes were designed targeting the ORF1a region of PDEV, PToV, and SADS-CoV and the ORF1b region of PDCoV. The method showed high sensitivity and specificity, with a detection limit of 1 × 102 copies/µL for each pathogen. A total of 101 clinical swine samples with signs of diarrhea were analyzed using this method, and the result showed good consistency with conventional reverse transcription PCR (RT-PCR). This method improves the efficiency for surveillance of these emerging and reemerging swine enteric viruses and can help reduce economic losses to the pig industry, which also benefits animal and public health.

Recent discovery and development of inhibitors targeting coronaviruses.

Human coronaviruses (CoVs) are enveloped viruses with a positive-sense single-stranded RNA genome. Currently, six human CoVs have been reported including human coronavirus 229E (HCoV-229E), OC43 (HCoV-OC43), NL63 (HCoV-NL63), HKU1 (HCoV-HKU1), severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), and MiddleEast respiratory syndrome (MERS) coronavirus (MERS-CoV). They cause moderate to severe respiratory and intestinal infections in humans. In this review, we focus on recent advances in the research and development of small-molecule anti-human coronavirus therapies targeting different stages of the CoV life cycle.

Pediatric Asthma Health Care Utilization, Viral Testing, and Air Pollution Changes During the COVID-19 Pandemic.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic caused dramatic changes in daily routines and health care utilization and delivery patterns in the United States. Understanding the influence of these changes and associated public health interventions on asthma care is important to determine effects on patient outcomes and identify measures that will ensure optimal future health care delivery. OBJECTIVE: We sought to identify changes in pediatric asthma-related health care utilization, respiratory viral testing, and air pollution during the COVID-19 pandemic. METHODS: For the time period January 17 to May 17, 2015 to 2020, asthma-related encounters and weekly summaries of respiratory viral testing data were extracted from Children's Hospital of Philadelphia electronic health records, and pollution data for 4 criteria air pollutants were extracted from AirNow. Changes in encounter characteristics, viral testing patterns, and air pollution before and after Mar 17, 2020, the date public health interventions to limit viral transmission were enacted in Philadelphia, were assessed and compared with data from 2015 to 2019 as a historical reference. RESULTS: After March 17, 2020, in-person asthma encounters decreased by 87% (outpatient) and 84% (emergency + inpatient). Video telemedicine, which was not previously available, became the most highly used asthma encounter modality (61% of all visits), and telephone encounters increased by 19%. Concurrently, asthma-related systemic steroid prescriptions and frequency of rhinovirus test positivity decreased, although air pollution levels did not substantially change, compared with historical trends. CONCLUSIONS: The COVID-19 pandemic in Philadelphia was accompanied by changes in pediatric asthma health care delivery patterns, including reduced admissions and systemic steroid prescriptions. Reduced rhinovirus infections may have contributed to these patterns.

Human respiratory coronavirus HKU1 versus other coronavirus infections in Italian hospitalised patients.

BACKGROUND: Human respiratory coronavirus (hCoV) HKU1 infections were reported for the first time in 2005 in Hong Kong. OBJECTIVE: To investigate epidemiological, clinical, and diagnostic features of HKU1 infections. STUDY DESIGN: Longitudinal, prospective study from November 2005 through May 2006 in a hospitalised patient population. RESULTS: Overall, 48/426 (11.3%) patients were found to be infected by hCoV acute respiratory tract infections (ARTI). Of these, 10 (19.2%) were caused by HKU1 (6 single infections and 4 coinfections) during the period January-May 2006. Diagnosis was made by using RT-PCR for all four hCoVs, and in parallel, in-house developed group-specific monoclonal antibodies (MAbs) for HKU1 and 229E. HKU1-specific MAb was able to retrospectively identify 8 of 10 HKU1 strains detected by RT-PCR. Phylogenetic analysis showed that four HKU1 strains were genotype A and six genotype B. In HKU1-infected patients, the predominant clinical symptom was rhinorrhea (nine patients). Within group II hCoV, HKU1-infected patients had a significantly lower rate of lower ARTI compared to OC43-infected patients. CONCLUSION: HKU1 hCoV strains circulated in northern Italy during the winter-spring season 2005-2006. Both HKU1 genotypes were detected. HKU1-specific MAb may contribute to the rapid diagnosis of HKU1 infections currently performed by RT-PCR.

Detection of the new human coronavirus HKU1: a report of 6 cases.

BACKGROUND: Human coronavirus HKU1 (HCoV-HKU1), a new group 2 coronavirus, was first characterized in 2005 from 2 adults with pneumonia in Hong Kong, China. To the best of our knowledge, there is no other report to date about the detection of this new virus. We report a molecular method allowing for the detection of HCoV-HKU1 and also report the clinical presentation of 6 infected patients. METHODS: We screened 141 specimens (135 nasal samples and 6 stool samples) received in February and March 2005 in our laboratory and obtained from 135 hospitalized patients (61.5% of whom were <5 years old and 34.1% of whom were >20 years old) for HCoV-HKU1. RESULTS: HCoV-HKU1 was detected in 6 (4.4%) of the 135 nasal specimens and in 2 (33.3%) of the 6 stool samples; the positive samples were obtained from 6 patients (5 children and 1 adult). The clinical presentation of these 6 patients was as follows: 3 were admitted to the hospital for acute enteric disease resulting in severe dehydration associated with upper respiratory symptoms; 1 had fever, otitis, and febrile seizure; 1 had a sample obtained to investigate failure to thrive; and 1 had a sample obtained for exploration of X-linked agammaglobulinemia and hyperleucocytosis. CONCLUSION: HCoV-HKU1 can be detected in respiratory and stool samples from children and adults in a part of the world other than Hong Kong. Our results suggest that HCoV-HKU1 could be associated with respiratory and enteric diseases, and its detection can be related to a persistent asymptomatic infection in patients with poor underlying conditions.

Use of an internal control in a quantitative RT-PCR assay for quantitation of porcine epidemic diarrhea virus shedding in pigs.

Porcine epidemic diarrhea virus (PEDV), a member of the family Coronaviridae, has caused a devastating enteric disease in the Korean swine industry. Previously, the differences between virulent field PEDV strains and a Vero cell culture adapted PEDV DR13 strain were determined using restriction fragment length polymorphism analysis (RFLP), and PEDV shedding patterns in pigs were reported. In an extension to these studies, an internal control was constructed and quantitative analysis of virus shedding after oral inoculation was established. A parent field PEDV and a cell culture adapted PEDV DR13 were inoculated orally to colostrum-deprived 1-day-old piglets, commercial 2-week-old pigs, and sows (1-5 ml dose, 10(5.8)-10(6.0) TCID(50)/0.1 ml). PEDV shedding was monitored every day and virus levels were measured using a quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method. In fecal samples from experimentally-inoculated pigs, the level of virus excreted peaked at 2 days after oral inoculation and gradually decreased thereafter. In addition, PEDV from field specimens was quantified using the same RT-PCR assay to determine shedding viral load. This suggests that measurement of PEDV shedding viral load in pigs, by quantitative RT-PCR, may be a useful tool for estimating the transmission potential of PEDV in the swine population.

Diagnostic evaluation of assays for detection of antibodies against porcine epidemic diarrhea virus (PEDV) in pigs exposed to different PEDV strains.

Porcine epidemic diarrhea virus (PEDV) has caused economic losses in the Americas, Asia and Europe in recent years. Reliable serological assays are essential for epidemiological studies and vaccine evaluation. The objective of this study was to compare the ability of five enzyme-linked immunosorbent assays (ELISAs) to detect antibodies against different PEDV strains in pig serum. A total of 732 serum samples from North American or European pigs were tested. Samples included experimental samples from pigs infected with classical (G1a PEDV) or variant genogroup 1 PEDV (G1b PEDV), pandemic genogroup 2 PEDV (G2b PEDV) or non-infected controls. Field samples from herds with confirmed or unknown PEDV exposure were also used. Three indirect ELISAs based on G2b antigens (ELISAs 1, 2 and 3), a competitive ELISA based on the G2b antigen (ELISA 4) and a competitive ELISA based on the G1a antigen (ELISA 5) were compared. Overall, the tests had a moderate agreement (κ=0.61). G1a PEDV infected pigs were earliest detected by ELISA 3, G1b PEDV infected pigs were earliest detected by ELISAs 4 and 5 and the performance of all tests was similar for the G2b PEDV group. ELISA 1 showed the overall lowest detection on experimentally and field derived samples. Diagnostic sensitivity and specificity with a 95% probability interval were estimated to be 68.2% (62.1-74.4%) and 97.5% (95.2-99.0%) for ELISA 1, 73.7% (71.5-79.6%) and 98.4% (96.6-99.5%) for ELISA 2, 86.2% (81.1-90.6%) and 91.6% (87.7-94.8%) for ELISA 3, 78.3% (72.8-83.5%) and 99.7% (98.2-100%) for ELISA 4, and 93.5% (90.3-96.0%) and 91.2% (83.8-97.9%) for ELISA 5. Differences in detection among assays seem to be more related to intrinsic factors of an assay than to the PEDV antigen used.

Cerebral pyogranuloma associated with systemic coronavirus infection in a ferret.

A 2-year-old male ferret was presented with central nervous system signs. Computed tomography (CT) of the brain revealed a well-defined contrast-enhancing lesion on the rostral forebrain that appeared extraparenchymal. Surgical excision of the mass was performed and the ferret was euthanised during the procedure. Histopathology of the excised mass showed multiple meningeal nodular lesions with infiltrates of epithelioid macrophages, occasionally centred on degenerated neutrophils and surrounded by a broad rim of plasma cells, features consistent with pyogranulomatous meningitis. The histopathological features in this ferret were similar to those in cats with feline infectious peritonitis. Definitive diagnosis was assessed by immunohistochemistry, confirming a ferret systemic coronavirus (FSCV) associated disease. This is the first case of coronavirus granuloma described on CT-scan in the central nervous system of a ferret.

A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against porcine deltacoronavirus.

Recently, porcine deltacoronavirus (PDCoV) has been proven to be associated with enteric disease in piglets. Diagnostic tools for serological surveys of PDCoV remain in the developmental stage when compared with those for other porcine coronaviruses. In our study, an indirect enzyme-linked immunosorbent assay (ELISA) (rPDCoV-N-ELISA) was developed to detect antibodies against PDCoV using a histidine-tagged recombinant nucleocapsid (N) protein as an antigen. The rPDCoV-N-ELISA did not cross-react with antisera against porcine epidemic diarrhea virus, swine transmissible gastroenteritis virus, porcine group A rotavirus, classical swine fever virus, porcine circovirus-2, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus; the receiver operating characteristic (ROC) curve analysis revealed 100% sensitivity and 90.4% specificity of the rPDCoV-N-ELISA based on samples of known status (n=62). Analyses of field samples (n=319) using the rPDCoV-N-ELISA indicated that 11.59% of samples were positive for antibodies against PDCoV. These data demonstrated that the rPDCoV-N-ELISA can be used for epidemiological investigations of PDCoV and that PDCoV had a low serum prevalence in pig population in Heilongjiang province, northeast China.

Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction.

OBJECTIVE: To determine the frequencies of human rhinovirus (HRV), respiratory syncytial virus (RSV), and coronavirus (HCV) infection in children with acute otitis media (AOM). METHODS: Middle ear fluids (MEF) collected by tympanocentesis and nasopharyngeal aspirates (NPA) at the time of the AOM diagnosis were examined by reverse transcriptase polymerase chain reaction for HRV, RSV, and HCV RNA. PATIENTS: Ninety-two children aged 3 months to 7 years during a 1-year period. RESULTS: Virus RNA was detected in a total of 69 children (75%) and in 44 MEF samples (48%) and 57 NPA samples (62%) at the time of AOM diagnosis. HRV RNA was detected in both MEF and NPA in 18 (20%), in MEF alone in 4 (4%), and in NPA alone in 10 (11%). RSV was detected in both MEF and NPA in 12 (13%), in MEF alone in 5 (5%), and in NPA alone in 9 (10%). HCV RNA was detected in both MEF and NPA in 5 (5%), in MEF alone in 2 (2%), and in NPA alone in 9 (10%). Dual viral infections were detected in 5% of children. HRV and RSV were detected simultaneously in 2 MEF samples and in 2 NPA samples; RSV and HCV were detected in 1 NPA sample. Bacterial pathogens were detected in 56 (62%) MEF from 91 children. Viral RNA was detected in 20 (57%) MEF of 35 bacteria-negative and in 25 (45%) of 56 bacteria-positive MEF samples. No important differences in the risk of treatment failure, relapse, or occurrence of late secretory otitis media were noted between children with virus-positive and virus-negative MEF aspirates. CONCLUSION: These findings highlight the importance of common respiratory viruses, particularly HRV and RSV, in predisposing to and causing AOM in young children.