Ancestral origin, antigenic resemblance and epidemiological insights of novel coronavirus (SARS-CoV-2): Global burden and Bangladesh perspective.

SARS-CoV-2, a new coronavirus strain responsible for COVID-19, has emerged in Wuhan City, China, and continuing its global pandemic nature. The availability of the complete gene sequences of the virus helps to know about the origin and molecular characteristics of this virus. In the present study, we performed bioinformatic analysis of the available gene sequence data of SARS-CoV-2 for the understanding of evolution and molecular characteristics and immunogenic resemblance of the circulating viruses. Phylogenetic analysis was performed for four types of representative viral proteins (spike, membrane, envelope and nucleoprotein) of SARS-CoV-2, HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, HKU4, HKU5 and BufCoV-HKU26. The findings demonstrated that SARS-CoV-2 exhibited convergent evolutionary relation with previously reported SARS-CoV. It was also depicted that SARS-CoV-2 proteins were highly similar and identical to SARS-CoV proteins, though proteins from other coronaviruses showed a lower level of resemblance. The cross-checked conservancy analysis of SARS-CoV-2 antigenic epitopes showed significant conservancy with antigenic epitopes derived from SARS-CoV. Descriptive epidemiological analysis on several epidemiological indices was performed on available epidemiological outbreak information from several open databases on COVID-19 (SARS-CoV-2). Satellite-derived imaging data have been employed to understand the role of temperature in the environmental persistence of the virus. Findings of the descriptive analysis were used to describe the global impact of newly emerged SARS-CoV-2, and the risk of an epidemic in Bangladesh.

Clinical impact of human coronaviruses 229E and OC43 infection in diverse adult populations.

BACKGROUND: The incidence and clinical impact of coronavirus (CoV) infection in elderly persons and those with underlying cardiopulmonary disease over a long duration is not well described. We determined the incidence and clinical impact of 229E and OC43 CoV in this population during 4 consecutive winters, and compared illnesses to influenza A, respiratory syncytial virus, and human metapneumovirus. METHODS: CoV 229E and OC43 were detected by reverse transcription polymerase chain reaction and serology in 4 adult populations under surveillance for acute respiratory illness during the winters of 1999-2003. Cohorts included healthy young adults, healthy elderly adults, high-risk adults with underlying cardiopulmonary disease, and a hospitalized group. RESULTS: Three hundred ninety-eight CoV infections were identified, with annual infection rates ranging from 2.8% to 26% in prospective cohorts, and prevalence ranging from 3.3% to 11.1% in the hospitalized cohort. The incidence of infections with each strain was similar, although asymptomatic infection and viral coinfection was significantly more common with 229E than OC43 infection. Although the incidence and clinical manifestations were similar for each strain, OC43-infected subjects tended to seek more medical care, as OC43 was twice as common as 229E among the hospitalized cohort. CONCLUSIONS: CoV infections in the elderly are frequent, likely causing substantial medical disease burden.

The first complete genome sequences of clinical isolates of human coronavirus 229E.

Human coronavirus 229E has been identified in the mid-1960s, yet still only one full-genome sequence is available. This full-length sequence has been determined from the cDNA-clone Inf-1 that is based on the lab-adapted strain VR-740. Lab-adaptation might have resulted in genomic changes, due to insufficient pressure to maintain gene integrity of non-essential genes. We present here the first full-length genome sequence of two clinical isolates. Each encoded gene was compared to Inf-1. In general, little sequence changes were noted, most could be attributed to genetic drift, since the clinical isolates originate from 2009 to 2010 and VR740 from 1962. Hot spots of substitutions were situated in the S1 region of the Spike, the nucleocapsid gene, and the non-structural protein 3 gene, whereas several deletions were detected in the 3'UTR. Most notable was the difference in genome organization: instead of an ORF4A and ORF4B, an intact ORF4 was present in clinical isolates.

Differences in neutralizing antigenicity between laboratory and clinical isolates of HCoV-229E isolated in Japan in 2004-2008 depend on the S1 region sequence of the spike protein.

Human coronavirus (HCoV) is a causative agent of the common cold. Although HCoV is highly prevalent in the world, studies of the genomic and antigenic details of circulating HCoV strains have been limited. In this study, we compared four Japanese isolates with the standard HCoV-229E strain obtained from ATCC (ATCC-VR740) by focusing on the spike (S) protein, a major determinant of neutralizing antigen and pathogenicity. The isolates were found to have nucleotide deletions and a number of sequence differences in the S1 region of the S protein. We compared two of the Japanese isolates with the ATCC-VR740 strain by using virus-neutralizing assays consisting of infectious HCoV-229E particles and vesicular stomatitis virus (VSV)-pseudotyped virus carrying the HCoV-229E S protein. The two clinical isolates (Sendai-H/1121/04 and Niigata/01/08) did not react with antiserum to the ATCC-VR740 strain via the neutralizing test. We then constructed a pseudotype VSV-harboured chimeric S protein with the ATCC S1 and Sendai S2 regions or that with Sendai S1 and ATCC S2 regions and compared them by a neutralization test. The results revealed that the difference in the neutralizing antigenicity depends on the S1 region. This different antigenic phenotype was also confirmed by a neutralizing test with clinically isolated human sera. These results suggest that the HCoV-229E viruses prevalent in Japan are quite different from the laboratory strain ATCC-VR740 in terms of the S sequence and neutralization antigenicity, which is attributed to the difference in the S1 region.

Differentiation between human coronaviruses NL63 and 229E using a novel double-antibody sandwich enzyme-linked immunosorbent assay based on specific monoclonal antibodies.

Human coronaviruses (HCoVs) are responsible for respiratory tract infections ranging from common colds to severe acute respiratory syndrome. HCoV-NL63 and HCoV-229E are two of the four HCoVs that circulate worldwide and are close phylogenetic relatives. HCoV infections can lead to hospitalization of children, elderly individuals, and immunocompromised patients. Globally, approximately 5% of all upper and lower respiratory tract infections in hospitalized children are caused by HCoV-229E and HCoV-NL63. The latter virus has recently been associated with the childhood disease croup. Thus, differentiation between the two viruses is relevant for epidemiology studies. The aim of this study was to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) as a potential tool for identification and differentiation between HCoV-NL63 and HCoV-229E. The nucleocapsid (N) proteins of HCoV-NL63 and HCoV-229E were expressed in an Escherichia coli system and used to immunize mice in order to obtain monoclonal antibodies (MAbs) specific for each virus. Three specific MAbs to HCoV-NL63, one MAb specific to HCoV-229E, and four MAbs that recognized both viruses were obtained. After their characterization, three MAbs were selected in order to develop a differential DAS-ELISA. The described assay could detect up to 3 ng/ml of N protein and 50 50% tissue culture infective doses/ml of virus stock. No cross-reactivity with other human coronaviruses or closely related animal coronaviruses was found. The newly developed DAS-ELISA was species specific, and therefore, it could be considered a potential tool for detection and differentiation of HCoV-NL63 and HCoV-229E infections.

Distant relatives of severe acute respiratory syndrome coronavirus and close relatives of human coronavirus 229E in bats, Ghana.

We tested 12 bat species in Ghana for coronavirus (CoV) RNA. The virus prevalence in insectivorous bats (n = 123) was 9.76%. CoV was not detected in 212 fecal samples from Eidolon helvum fruit bats. Leaf-nosed bats pertaining to Hipposideros ruber by morphology had group 1 and group 2 CoVs. Virus concentrations were < or =45,000 copies/100 mg of bat feces. The diversified group 1 CoV shared a common ancestor with the human common cold virus hCoV-229E but not with hCoV-NL63, disputing hypotheses of common human descent. The most recent common ancestor of hCoV-229E and GhanaBt-CoVGrp1 existed in approximately 1686-1800 ad. The GhanaBt-CoVGrp2 shared an old ancestor (approximately 2,400 years) with the severe acute respiratory syndrome-like group of CoV.

Coronavirus infection and hospitalizations for acute respiratory illness in young children.

There is only limited knowledge on the burden of disease due to both new (HCoV-NL63 and HKU-1) and previously discovered coronaviruses (OC43 and 229E) in children. Respiratory specimens and clinical data were prospectively collected in an active, population-based surveillance study over a 2-year period from children aged <5 years hospitalized with acute respiratory symptoms or fever. These samples were retrospectively tested by real-time RT-PCR for HCoV-NL63, HKU1, OC43, and 229E. Human coronaviruses (HCoVs) were identified in 2.2% of study children <2 years of age. Rates of HCoV-associated hospitalization per 10,000 were 10.2 (95% CI 4.3, 17.6), 4.2 (95% CI 1.9, 6.9), and 0 (95% CI 0, 3.7) in children aged <6 months, 6-23 months, and 24-59 months, respectively. Coronaviruses were identified in a modest number of hospitalized children.

[Characterization of human coronavirus 229E infection among patients with respiratory symptom in Beijing, Oct-Dec, 2007].

OBJECTIVE: To know the etiology, prevalence, clinical symptoms associated with the infection of the HCoV-229E in the respiratory specimens sampled from adult patients in Beijing. METHODS: 158 nasopharyngeal swab specimens were collected from adult patients with fever in Beijing between October and December, 2007. We performed the screening of HCoV-229E by real-time RT-PCR and sequencing of HCoV-229E gene fragments derived from conventional PCR. At meantime, we also screened the HCoV-229E positive samples for the co-infection with HCoV-NL63, HCoV-HKU1 and HMPV by real-time RT-PCR. Finally, demographic and clinical data associated with HCoV-229E infection were examined retrospectively. RESULTS: We detected 103 (62.5%) of 158 specimens were positive for HCoV-229E by real-time RT-PCR. When tested for other respiratory viruses, 26 HCoV-229E positive patients were found to be co-infected with other viruses. Of which HCoV-NL63 was observed in 3 specimens (11.5%), HCoV-HKU1 in 3 (11.5%) and HMPV in 20 (76.9%). The main clinical manifestations were noted as: headache (in 70.9%), sore throat (69%), chills (68%), cough (33%), sputum (21.3%), rhinorrhea (21.4%), nasal obstruction (16.5%), and a few of patients were visible as vomiting (6.8%), dyspnea (3.9%), diarrhea (in 1.9%). The rate of HCoV-229E infection in adult patients was found no relative with age and gender. CONCLUSION: Our data showed that HCoV-229E is a common and important pathogen in adult patients with acute respiratory symptoms but usually resulted in milder influenza-like illnesses. There might have a local outbreak of HCoV-229E infection in Beijing, Oct-Dec, 2007.

[Evaluation of Seeplex RV detection kit for detecting rhinovirus, human metapneumovirus, and coronavirus].

BACKGROUND: Direct antigen test (DAT) and culture are primary tests to diagnose infections by respiratory viruses, but are mainly available for the traditional viral pathogens such as respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), and adenovirus in clinical laboratories. The objective of this study was to evaluate a multiplex reverse transcriptase-PCR method using Seeplex RV Detection kit (Seegene, Korea) for the detection of rhinovirus, coronavirus, and human metapneumovirus (hMPV). METHODS: From January to May 2007, nasopharyngeal aspirates (NPAs) from pediatric patients negative for culture and DAT of traditional viral pathogens were tested with Seeplex. All the amplicons were directly sequenced and homology of the sequences was searched in the National Center for Biotechnology Information (NCBI) database. Patients' medical records were reviewed for clinical and demographic features. RESULTS: Forty-seven (26.4%) of 178 NPAs were positive: 18 rhinovirus, 15 hMPV, 4 RSV A, 3 coronavirus OC43, 3 influenza virus A, 2 adenovirus, 1 coronavirus NL63, and 1 RSV B. Based on maximum identity, each of the sequences indicating rhinovirus, hMPV, and coronavirus OC43 matched to the corresponding viruses with homology of 94-98%, 96-99%, and 98-100%, respectively. Seeplex positive patients were 0-11 yr old with a male:female ratio of 1.5:1. Clinical diagnoses included 9 pneumonia, 6 bronchiolitis, 2 cold, 1 asthma exacerbation for rhinovirus; 10 pneumonia, 4 bronchiolitis, and 1 clinical sepsis for hPMV; and 1 pneumonia, 2 croup, and 1 cold for coronavirus. CONCLUSIONS: Multiplex reverse transcriptase-PCR method using Seeplex RV Detection kit is a reliable test to detect rhinovirus, hMPV, and coronavirus. It may improve the diagnostic sensitivity for RSV, influenza virus, PIV, and adenovirus.

Human (non-severe acute respiratory syndrome) coronavirus infections in hospitalised children in France.

AIM: This study has two objectives: to study the clinical symptoms associated with the detection of the four human coronaviruses (HCoVs), 229E, OC43, NL63 and HKU1 types, in the respiratory specimens sampled from hospitalised children in France between September 2004 and May 2005; and to develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay allowing for the simultaneous detection of the four HCoVs. METHODS: 1002 respiratory specimens were tested for HCoVs. The clinical and epidemiological data were compared on the basis of the type HCoV infection. RESULTS: A hundred coronaviruses, 33 NL63, 2229E, 27 OC43 and 38 HKU1, were detected in 97 (9.8%) of 1002 samples negative in routine tests. The clinical and epidemiological characteristics of the study children were compared in three groups, 24 OC43-, 27 NL63- and 34 HKU1-infected children. HCoVs were identified mainly in children with upper and lower respiratory tract infections (50.5% vs. 29.4%). The significant difference in clinical presentation between the three coronavirus groups was the very low association between lower respiratory tract illness and HKU1 detection. CONCLUSIONS: HCoV detection in hospitalised children without any other respiratory virus detection was associated with upper and a significant rate of lower respiratory tract illness. The four types of HCoVs were detected, and new types NL63 and HKU1 represented a substantial portion of detection. The multiplex RT-PCR enabled a sensitive one-time detection and the characterisation of all of the known HCoV types with the exception severe acute respiratory syndrome-coronavirus.

Evaluation of Seeplex(TM) RV Detection Kit for Detecting Rhinovirus, Human Metapneumovirus, and Coronavirus / 대한진단검사의학회지

BACKGROUND: Direct antigen test (DAT) and culture are primary tests to diagnose infections by respiratory viruses, but are mainly available for the traditional viral pathogens such as respiratory syncytial virus (RSV), influenza virus, parainfluenza virus (PIV), and adenovirus in clinical laboratories. The objective of this study was to evaluate a multiplex reverse transcriptase-PCR method using Seeplex(TM) RV Detection kit (Seegene, Korea) for the detection of rhinovirus, coronavirus, and human metapneumovirus (hMPV). METHODS: From January to May 2007, nasopharyngeal aspirates (NPAs) from pediatric patients negative for culture and DAT of traditional viral pathogens were tested with Seeplex(TM). All the amplicons were directly sequenced and homology of the sequences was searched in the National Center for Biotechnology Information (NCBI) database. Patients' medical records were reviewed for clinical and demographic features. RESULTS: Forty-seven (26.4%) of 178 NPAs were positive: 18 rhinovirus, 15 hMPV, 4 RSV A, 3 coronavirus OC43, 3 influenza virus A, 2 adenovirus, 1 coronavirus NL63, and 1 RSV B. Based on maximum identity, each of the sequences indicating rhinovirus, hMPV, and coronavirus OC43 matched to the corresponding viruses with homology of 94-98%, 96-99%, and 98-100%, respectively. Seeplex(TM) positive patients were 0-11 yr old with a male:female ratio of 1.5:1. Clinical diagnoses included 9 pneumonia, 6 bronchiolitis, 2 cold, 1 asthma exacerbation for rhinovirus; 10 pneumonia, 4 bronchiolitis, and 1 clinical sepsis for hPMV; and 1 pneumonia, 2 croup, and 1 cold for coronavirus. CONCLUSIONS: Multiplex reverse transcriptase-PCR method using Seeplex(TM) RV Detection kit is a reliable test to detect rhinovirus, hMPV, and coronavirus. It may improve the diagnostic sensitivity for RSV, influenza virus, PIV, and adenovirus.

Generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-PCR and nonfluorescent low-density microarray.

A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.

Coronavirus HKU1 and other coronavirus infections in Hong Kong.

We have recently described the discovery of a novel coronavirus, coronavirus HKU1 (CoV-HKU1), associated with community-acquired pneumonia. However, the clinical spectrum of disease and the epidemiology of CoV-HKU1 infections in relation to infections with other respiratory viruses are unknown. In this 12-month prospective study, 4,181 nasopharyngeal aspirates from patients with acute respiratory tract infections were subjected to reverse transcription-PCRs specific for CoV-HKU1 and human coronaviruses NL63 (HCoV-NL63), OC43 (HCoV-OC43), and 229E (HCoV-229E). Coronaviruses were detected in 87 (2.1%) patients, with 13 (0.3%) positive for CoV-HKU1, 17 (0.4%) positive for HCoV-NL63, 53 (1.3%) positive for HCoV-OC43, and 4 (0.1%) positive for HCoV-229E. Of the 13 patients with CoV-HKU1 infections, 11 were children and 8 had underlying diseases. Similar to the case for other coronaviruses, upper respiratory infection was the most common presentation of CoV-HKU1 infections, although pneumonia, acute bronchiolitis, and asthmatic exacerbation also occurred. Despite a shorter duration of fever (mean, 1.7 days) and no difference in maximum temperature in children with CoV-HKU1 infections compared to patients with most other respiratory virus infections, a high incidence of febrile seizures (50%) was noted, which was significantly higher than those for HCoV-OC43 (14%), adenovirus (9%), human parainfluenza virus 1 (0%), and respiratory syncytial virus (8%) infections. CoV-HKU1 and HCoV-OC43 infections peaked in winter, although cases of the former also occurred in spring to early summer. This is in contrast to HCoV-NL63 infections, which mainly occurred in early summer and autumn but were absent in winter. Two genotypes of CoV-HKU1 cocirculated during the study period. Continuous studies over a longer period are warranted to ascertain the seasonal variation and relative importance of the different coronaviruses. Similar studies in other countries are required to better determine the epidemiology and genetic diversity of CoV-HKU1.

Comprehensive detection and identification of human coronaviruses, including the SARS-associated coronavirus, with a single RT-PCR assay.

The SARS-associated human coronavirus (SARS-HCoV) is a newly described, emerging virus conclusively established as the etiologic agent of the severe acute respiratory syndrome (SARS). This study presents a single-tube RT-PCR assay that can detect with high analytical sensitivity the SARS-HCoV, as well as several other coronaviruses including other known human respiratory coronaviruses (HCoV-OC43 and HCoV-229E). Species identification is provided by sequencing the amplicon, although a rapid screening test by restriction enzyme analysis has proved to be very useful for the analysis of samples obtained during the SARS outbreak in Toronto, Canada.

Frequent detection of human coronaviruses in clinical specimens from patients with respiratory tract infection by use of a novel real-time reverse-transcriptase polymerase chain reaction.

During the past years, human coronaviruses (HCoVs) have been increasingly identified as pathogens associated with more-severe respiratory tract infection (RTI). Diagnostic tests for HCoVs are not frequently used in the routine setting. It is likely that, as a result, the precise role that HCoVs play in RTIs is greatly underestimated. We describe a rapid, sensitive, and highly specific quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) for the detection of HCoV that can easily be implemented in the routine diagnostic setting. HCoV was detected in 28 (11%) of the 261 clinical specimens obtained from patients presenting with symptoms of RTI ranging from common cold to severe pneumonia. Only 1 (0.4%) of the 243 control specimens obtained from patients without symptoms of RTI showed the presence of HCoV. We conclude that HCoVs can be frequently detected in patients presenting with RTI. Real-time RT-PCR provides a tool for large-scale epidemiological studies to further clarify the role that coronavirus infection plays in RTI in humans.

Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-PCR assays.

There is a need for rapid, sensitive, and accurate diagnosis of lower respiratory tract infections in children, elderly, and immunocompromised patients, who are susceptible to serious complications. The multiplex RT-nested PCR assay has been used widely for simultaneous detection of non-related viruses involved in infectious diseases because of its high specificity and sensitivity. A new multiplex RT-PCR assay is described in this report. This approach includes nested primer sets targeted to conserve regions of human parainfluenza virus haemagglutinin, human coronavirus spike protein, and human enterovirus and rhinovirus polyprotein genes. It permits rapid, sensitive, and simultaneous detection and typing of the four types of parainfluenza viruses (1, 2, 3, 4AB), human coronavirus 229E and OC43, and the generic detection of enteroviruses and rhinoviruses. The testing of 201 clinical specimens with this multiplex assay along with other one formerly described by our group to simultaneously detect and type the influenza viruses, respiratory syncytial viruses, and a generic detection of all serotypes of adenovirus, covers the detection of most viruses causing respiratory infectious disease in humans. The results obtained were compared with conventional viral culture, immunofluorescence assay, and a third multiplex RT-PCR assay for all human parainfluenza viruses types described previously. In conclusion, both multiplex RT-PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population.

Direct diagnosis of human respiratory coronaviruses 229E and OC43 by the polymerase chain reaction.

An RT-PCR-hybridization was developed that amplified genetic material from the M protein gene of HCoV-229E and HCoV-OC43. The analytic sensitivity of these original primers were compared with primers defined in the N gene and described previously. The results show that 0.05 TCID50 of HCoV-229E and 0.01 TCID50 of HCoV-OC43 can be detected by this molecular method using the original method. Detection of HCoV-229E and HCoV-OC43 in clinical specimens is possible using this method: 348 respiratory specimens (202 sputum and 146 nasal aspirates) were tested with this RT-PCR-hybridization and 12 human coronavirus are detected (3%). The method could provide a useful tool for demonstrating the role of human coronavirus in infections of the respiratory tract.