TMPRSS2 and glycan receptors synergistically facilitate coronavirus entry.

The entry of coronaviruses is initiated by spike recognition of host cellular receptors, involving proteinaceous and/or glycan receptors. Recently, TMPRSS2 was identified as the proteinaceous receptor for HCoV-HKU1 alongside sialoglycan as a glycan receptor. However, the underlying mechanisms for viral entry remain unknown. Here, we investigated the HCoV-HKU1C spike in the inactive, glycan-activated, and functionally anchored states, revealing that sialoglycan binding induces a conformational change of the NTD and promotes the neighboring RBD of the spike to open for TMPRSS2 recognition, exhibiting a synergistic mechanism for the entry of HCoV-HKU1. The RBD of HCoV-HKU1 features an insertion subdomain that recognizes TMPRSS2 through three previously undiscovered interfaces. Furthermore, structural investigation of HCoV-HKU1A in combination with mutagenesis and binding assays confirms a conserved receptor recognition pattern adopted by HCoV-HKU1. These studies advance our understanding of the complex viral-host interactions during entry, laying the groundwork for developing new therapeutics against coronavirus-associated diseases.

An overview on the seven pathogenic human coronaviruses.

To date, seven human coronaviruses (HCoVs) have been detected: HCoV-NL63, HCoV-229E, HCoV-HKU1, HCoV-OC43, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2. Four of these viruses, including HCoV-NL63, -229E, -HKU1 and -OC43, usually cause mild-to-moderate respiratory diseases with a seasonal pattern. Since 2000, three new HCoVs have emerged with a significant mortality rate. Although SARS-CoV and MERS-CoV caused an epidemic in some countries, SARS-CoV-2 escalated into a pandemic. All HCoVs can cause severe complications in the elderly and immunocompromised individuals. The bat origin of HCoVs, the presence of intermediate hosts and the nature of their viral replication suggest that other new coronaviruses may emerge in the future. Despite the fact that all HCoVs share similarities in viral replication, they differ in their accessory proteins, incubation period and pathogenicity. This study aims to review these differences between the seven HCoVs.

Designing AbhiSCoVac - A single potential vaccine for all 'corona culprits': Immunoinformatics and immune simulation approaches.

The coronaviridae family has generated highly virulent viruses, including the ones responsible for three major pandemics in last two decades with SARS in 2002, MERS outbreak in 2012 and the current nCOVID19 crisis that has turned the world breadthless. Future outbreaks are also a plausible threat to mankind. As computational biologists, we are committed to address the need for a universal vaccine that can deter all these pathogenic viruses in a single shot. Notably, the spike proteins present in all these viruses function as credible PAMPs that are majorly sensed by human TLR4 receptors. Our study aims to recognize the amino acid sequence(s) of the viral spike proteins that are precisely responsible for interaction with human TLR4 and to screen the immunogenic epitopes present in them to develop a multi-epitope multi-target chimeric vaccine against the coronaviruses. Molecular design of the constructed vaccine peptide is qualified in silico; additionally, molecular docking and molecular dynamics simulation studies collectively reveal strong and stable interactions of the vaccine construct with TLRs and MHC receptors. In silico cloning is performed for proficient expression in bacterial systems. In silico immune simulation of the vaccine indicates highly immunogenic nature of the vaccine construct without any allergic response. The present biocomputational study hereby innovates a vaccine candidate - AbhiSCoVac hypothesized as a potent remedy to combat all the virulent forms of coronaviruses.

Detection of Serum Cross-Reactive Antibodies and Memory Response to SARS-CoV-2 in Prepandemic and Post-COVID-19 Convalescent Samples.

BACKGROUND: A notable feature of coronavirus disease 2019 (COVID-19) is that children are less susceptible to severe disease. Children are known to experience more infections with endemic human coronaviruses (HCoVs) compared to adults. Little is known whether HCoV infections lead to cross-reactive anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies. METHODS: We investigated the presence of cross-reactive anti-SARS-CoV-2 IgG antibodies to spike 1 (S1), S1-receptor-binding domain (S1-RBD), and nucleocapsid protein (NP) by enzyme-linked immunosorbent assays, and neutralizing activity by a SARS-CoV-2 pseudotyped virus neutralization assay, in prepandemic sera collected from children (n = 50) and adults (n = 45), and compared with serum samples from convalescent COVID-19 patients (n = 16). RESULTS: A significant proportion of children (up to 40%) had detectable cross-reactive antibodies to SARS-CoV-2 S1, S1-RBD, and NP antigens, and the anti-S1 and anti-S1-RBD antibody levels correlated with anti-HCoV-HKU1 and anti-HCoV-OC43 S1 antibody titers in prepandemic samples (P < .001). There were marked increases of anti-HCoV-HKU1 and - OC43 S1 (but not anti-NL63 and -229E S1-RBD) antibody titers in serum samples from convalescent COVID-19 patients (P < .001), indicating an activation of cross-reactive immunological memory to ß-coronavirus spike. CONCLUSIONS: We demonstrated cross-reactive anti-SARS-CoV-2 antibodies in prepandemic serum samples from children and young adults. Promoting this cross-reactive immunity and memory response derived from common HCoV may be an effective strategy against SARS-COV-2 and future novel coronaviruses.

Seasonal Human Coronavirus Respiratory Tract Infection in Recipients of Allogeneic Hematopoietic Stem Cell Transplantation.

BACKGROUND: Little is known about characteristics of seasonal human coronaviruses (HCoVs) (NL63, 229E, OC43, and HKU1) after allogeneic stem cell transplantation (allo-HSCT). METHODS: This was a collaborative Spanish and European bone marrow transplantation retrospective multicenter study, which included allo-HSCT recipients (adults and children) with upper respiratory tract disease (URTD) and/or lower respiratory tract disease (LRTD) caused by seasonal HCoV diagnosed through multiplex polymerase chain reaction assays from January 2012 to January 2019. RESULTS: We included 402 allo-HSCT recipients who developed 449 HCoV URTD/LRTD episodes. Median age of recipients was 46 years (range, 0.3-73.8 years). HCoV episodes were diagnosed at a median of 222 days after transplantation. The most common HCoV subtype was OC43 (n = 170 [38%]). LRTD involvement occurred in 121 episodes (27%). HCoV infection frequently required hospitalization (18%), oxygen administration (13%), and intensive care unit (ICU) admission (3%). Three-month overall mortality after HCoV detection was 7% in the whole cohort and 16% in those with LRTD. We identified 3 conditions associated with higher mortality in recipients with LRTD: absolute lymphocyte count <0.1 × 109/mL, corticosteroid use, and ICU admission (hazard ratios: 10.8, 4.68, and 8.22, respectively; P < .01). CONCLUSIONS: Seasonal HCoV after allo-HSCT may involve LRTD in many instances, leading to a significant morbidity.

Endemic Human Coronavirus-Specific Nasal Immunoglobulin A and Serum Immunoglobulin G Dynamics in Lower Respiratory Tract Infections.

Endemic human coronaviruses (HCoV) NL63, 229E, OC43, and HKU1 cause respiratory infection. Following infection, a virus-specific serum antibody rise is usually observed, coinciding with recovery. In some cases, an infection is not accompanied by an immunoglobulin G (IgG) antibody rise in serum in the first month after HCoV infection, even though the infection has cleared in that month and the patient has recovered. We investigated the possible role of nasal immunoglobulin A (IgA). We measured spike (S) and nucleocapsid (N)-specific nasal IgA during and after an HCoV lower respiratory tract infection (LRTI) and compared the IgA responses between subjects with and without a significant IgG rise in serum (IgG responders (n = 31) and IgG non-responders (n = 14)). We found that most IgG responders also exhibited significant nasal IgA rise in the first month after the infection, whereas such an IgA rise was lacking in most IgG non-responders. Interestingly, the serum IgG non-responders presented with a significantly higher nasal IgA when they entered this study than during the acute phase of the LRTI. Our data suggest that nasal IgA could be part of a fast acute response to endemic HCoV infection and may play a role in clearing the infection.

Influenza-like illness symptoms due to endemic human coronavirus reinfections are not influenced by the length of the interval separating reinfections.

After 3 years of its introduction to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared as endemic. Little is known about the severity of the disease manifestation that future infections may cause, especially when reinfections occur after humoral immunity from a previous infection or vaccination has waned. Such knowledge could inform policymakers regarding the frequency of vaccination. Reinfections by endemic human coronaviruses (HCoVs) can serve as a model system for SARS-CoV-2 endemicity. We monitored 44 immunocompetent male adults with blood sampling every 6 months (for 17 years), for the frequency of HCoV (re-)infections, using rises in N-antibodies of HCoV-NL63, HCoV-29E, HCoV-OC43, and HCoV-HKU1 as markers of infection. Disease associations during (re-)infections were examined by comparison of self-reporting records of influenza-like illness (ILI) symptoms, every 6 months, by all participants. During 8,549 follow-up months, we found 364 infections by any HCoV with a median of eight infections per person. Symptoms more frequently reported during HCoV infection were cough, sore throat, and myalgia. Two hundred fifty-one of the 364 infections were species-specific HCoV-reinfections, with a median interval of 3.58 (interquartile range 1.92-5.67) years. The length of the interval between reinfections-being either short or long-had no influence on the frequency of reporting ILI symptoms. All HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1 (re-)infections are associated with the reporting of ILIs. Importantly, in immunocompetent males, these symptoms are not influenced by the length of the interval between reinfections. IMPORTANCE: Little is known about the disease following human coronavirus (HCoV) reinfection occurring years after the previous infection, once humoral immunity has waned. We monitored endemic HCoV reinfection in immunocompetent male adults for up to 17 years. We found no influence of reinfection interval length in the disease manifestation, suggesting that immunocompetent male adults are adequately protected against future HCoV infections.

Phylogenetic characteristics of Non-SARS human coronavirus in southern Taiwan, 2012-2013.

This study characterizes the phylogenetic relatedness of non-SARS human coronaviruses (HCoVs) in southern Taiwan by sequencing the nucleocapsid (N), spike (S), and RNA-dependent RNA polymerase (RdRp) genes directly from ten HCoV PCR-positive respiratory samples collected during 2012-2013. In the N, S1, and RdRp phylogeny, HCoV-OC43 in one and three samples was clustered with genotypes F and G, respectively, and HCoV-OC43 in sample YC101/TWN/2013 represented a recombination event between genotypes F and G. Amino acid substitutions in the S1 protein of HCoV-OC43 were also identified. In the N phylogeny, HCoV-HKU1 in one and two samples clustered with genotypes A and B, respectively, and HCoV-229E in two samples was clustered with genogroup 6. The genotypes and genogroup detected here were in line with the prevalent phylogenetic lineages reported outside of Taiwan during the contemporary period. In summary, three species of non-SARS HCoVs with different genotypes cocirculated in the community, with genetic evolution observed in HCoV-OC43.

Surveillance of endemic coronaviruses during the COVID-19 pandemic in Iran, 2021-2022.

Background: Human coronaviruses (HCoVs) 229E, OC43, HKU1, and NL63 are common viruses that continuously circulate in the human population. Previous studies showed the circulation of HCoVs during the cold months in Iran. We studied the circulation of HCoVs during coronavirus disease 2019 (COVID-19) pandemic to find the impact of pandemic on the circulation of these viruses. Methods: As a cross-sectional survey conducted during 2021 to 2022, of all throat swabs sent to Iran National Influenza Center from patients with severe acute respiratory infection, 590 samples were selected to test for HCoVs using one-step real-time RT-PCR. Results: Overall, 28 out of 590 (4.7%) tested samples were found to be positive for at least one HCoVs. HCoV-OC43 was the most common (14/590 or 2.4%), followed by HCoV-HKU1 (12/590 or 2%) and HCoV-229E (4/590 or 0.6%), while HCoV-NL63 was not detected. HCoVs were detected in patients of all ages and throughout the study period with peaks in the cold months of the year. Conclusions: Our multicenter survey provides insight into the low circulation of HCoVs during the COVID-19 pandemic in Iran in 2021/2022. Hygiene habits and social distancing measures might have important role in decreasing of HCoVs transmission. We believe that surveillance studies are needed to track the pattern of HCoVs distributions and detect changes in the epidemiology of such viruses to set out strategies in order to timely control the future outbreaks of HCoVs throughout the nation.

Clarifying real receptor binding site between coronavirus HCoV-HKU1 and 9-O-Ac-Sia based on molecular docking.

HCoV-HKU1 is a [Formula: see text]-coronavirus with low pathogenicity, which usually leads to respiratory diseases. At present, a controversial issue is that whether the receptor binding site (RBS) of HCoV-HKU1 is located in the N-terminal domain (NTD) or the C-terminal domain (CTD) in the HCoV-HKU1 S protein. To address this issue, we used molecular docking technology to dock the NTD and CTD with 9-oxoacetylated sialic acid (9-O-Ac-Sia), respectively, with the results showing that the RBS of HCoV-HKU1 is located in the NTD (amino acid residues 80-95, 25-32). Our findings clarified the structural basis and molecular mechanism of the HCoV-HKU1 infection, providing important information for the development of therapeutic antibody drugs and the design of vaccines.

Atypical Antibody Dynamics During Human Coronavirus HKU1 Infections.

Human coronavirus HKU1 (HCoV-HKU1) is one of the four endemic coronaviruses. It has been suggested that there is a difference in incidence, with PCR-confirmed HCoV-NL63 and HCoV-OC43 infections occurring more commonly, whereas HCoV-HKU1 is the least seen. Lower incidence of HCoV-HKU1 infection has also been observed in serological studies. The current study aimed to investigate antibody dynamics during PCR-confirmed HCoV-HKU1 infections using serum collected during infection and 1 month later. We expressed a new HCoV-HKU1 antigen consisting of both the linker and carboxy-terminal domain of the viral nucleocapsid protein and implemented it in ELISA. We also applied a spike-based Luminex assay on serum samples from PCR-confirmed infections by the four endemic HCoVs. At least half of HCoV-HKU1-infected subjects consistently showed no antibody rise via either assay, and some subjects even exhibited substantial antibody decline. Investigation of self-reported symptoms revealed that HCoV-HKU1-infected subjects rated their illness milder than subjects infected by other HCoVs. In conclusion, HCoV-HKU1 infections reported in this study displayed atypical antibody dynamics and milder symptoms when compared to the other endemic HCoVs.

Epidemiology of Non-SARS-CoV2 Human Coronaviruses (HCoVs) in People Presenting with Influenza-like Illness (ILI) or Severe Acute Respiratory Infections (SARI) in Senegal from 2012 to 2020.

In addition to emerging coronaviruses (SARS-CoV, MERS, SARS-CoV-2), there are seasonal human coronaviruses (HCoVs): HCoV-OC43, HCoV-229E, HCoV-NL63 and HCoV-HKU1. With a wide distribution around the world, HCoVs are usually associated with mild respiratory disease. In the elderly, young children and immunocompromised patients, more severe or even fatal respiratory infections may be observed. In Africa, data on seasonal HCoV are scarce. This retrospective study investigated the epidemiology and genetic diversity of seasonal HCoVs during nine consecutive years of influenza-like illness surveillance in Senegal. Nasopharyngeal swabs were collected from ILI outpatients or from SARI hospitalized patients. HCoVs were diagnosed by qRT-PCR and the positive samples were selected for molecular characterization. Among 9337 samples tested for HCoV, 406 (4.3%) were positive: 235 (57.9%) OC43, 102 (25.1%) NL63, 58 (14.3%) 229E and 17 (4.2%) HKU1. The four types circulated during the study period and a peak was noted between November and January. Children under five were the most affected. Co-infections were observed between HCoV types (1.2%) or with other viruses (76.1%). Genetically, HCoVs types showed diversity. The results highlighted that the impact of HCoVs must be taken into account in public health; monitoring them is therefore particularly necessary both in the most sensitive populations and in animals.

A Novel Method to Reduce ELISA Serial Dilution Assay Workload Applied to SARS-CoV-2 and Seasonal HCoVs.

Assays using ELISA measurements on serially diluted serum samples have been heavily used to measure serum reactivity to SARS-CoV-2 antigens and are widely used in virology and elsewhere in biology. We test a method using Bayesian hierarchical modelling to reduce the workload of these assays and measure reactivity of SARS-CoV-2 and HCoV antigens to human serum samples collected before and during the COVID-19 pandemic. Inflection titers for SARS-CoV-2 full-length spike protein (S1S2), spike protein receptor-binding domain (RBD), and nucleoprotein (N) inferred from 3 spread-out dilutions correlated with those inferred from 8 consecutive dilutions with an R2 value of 0.97 or higher. We confirm existing findings showing a small proportion of pre-pandemic human serum samples contain cross-reactive antibodies to SARS-CoV-2 S1S2 and N, and that SARS-CoV-2 infection increases serum reactivity to the beta-HCoVs OC43 and HKU1 S1S2. In serial dilution assays, large savings in resources and/or increases in throughput can be achieved by reducing the number of dilutions measured and using Bayesian hierarchical modelling to infer inflection or endpoint titers. We have released software for conducting these types of analysis.

Roles of Sialyl Glycans in HCoV-OC43, HCoV-HKU1, MERS-CoV and SARS-CoV-2 Infections.

Ongoing seasonal HCoV-OC43 and HCoV-HKU1 (common cold), an ongoing zoonotic infection of highly lethal MERS-CoV in humans (MERS disease), and an ongoing pandemic SARS-CoV-2 (COVID-19) with high mutability giving some variants causing severe illness and death have been reported to attach to sialyl receptors via their spike (S) glycoproteins and via additional short spikes, hemagglutinin-esterase (HE) glycoproteins, for HCoV-OC43 and HCoV-HKU1. There is lack of zoonotic viruses that are origins of HCoV-HKU1 and the first recorded pandemic CoV (SARS-CoV-2) for studies. In this chapter, we review current knowledge of the roles of sialyl glycans in infections with these viruses in distinct infection stages. Determination of the similarities and differences in roles of sialyl glycans in infections with these viruses could lead to a better understanding of the pathogenesis and transmission that is essential for combating infections with CoVs that recognize sialyl glycans.

QSAR based virtual screening derived identification of a novel hit as a SARS CoV-229E 3CLpro Inhibitor: GA-MLR QSAR modeling supported by molecular Docking, molecular dynamics simulation and MMGBSA calculation approaches.

Congruous coronavirus drug targets and analogous lead molecules must be identified as quickly as possible to produce antiviral therapeutics against human coronavirus (HCoV SARS 3CLpro) infections. In the present communication, we bear recognized a HIT candidate for HCoV SARS 3CLpro inhibition. Four Parametric GA-MLR primarily based QSAR model (R2:0.84, R2adj:0.82, Q2loo: 0.78) was once promoted using a dataset over 37 structurally diverse molecules along QSAR based virtual screening (QSAR-VS), molecular docking (MD) then molecular dynamic simulation (MDS) analysis and MMGBSA calculations. The QSAR-based virtual screening was utilized to find novel lead molecules from an in-house database of 100 molecules. The QSAR-vS successfully offered a hit molecule with an improved PEC50 value from 5.88 to 6.08. The benzene ring, phenyl ring, amide oxygen and nitrogen, and other important pharmacophoric sites are revealed via MD and MDS studies. Ile164, Pro188, Leu190, Thr25, His41, Asn46, Thr47, Ser49, Asn189, Gln191, Thr47, and Asn141 are among the key amino acid residues in the S1 and S2 pocket. A stable complex of a lead molecule with the HCoV SARS 3CLpro was discovered using MDS. MM-GBSA calculations resulted from MD simulation results well supported with the binding energies calculated from the docking results. The results of this study can be exploited to develop a novel antiviral target, such as an HCoV SARS 3CLpro Inhibitor.

First Detection of Human Coronavirus HKU1 in Greece, in an Immunocompromised Patient With Severe Lower Respiratory Tract Infection.

Human coronavirus HKU1 (HCoV-HKU1) is a RNA virus which gets in the human cells by binding to the receptor of N-acetyl-9-O-acetylneuraminic acid. Human Coronaviruses (HCoVs), including HCoV-HKU1, are globally found. HCoV-HKU1 is responsible for upper and lower respiratory tract infections, usually with mild symptoms. In severe cases, HCoV-HKU1 can cause life-threatening respiratory illness especially in vulnerable hosts such as elderly, children and immunocompromised patients. In Greece, Respiratory Syncytial Virus (RSV) and influenza are the most common viruses causing respiratory tract infections. Traditionally, HCoVs are responsible for less than 3% of respiratory infections in Greek population. HCoVs 229E and OC43 have been shown to circulate in Greece. We report the first case of lung infection in an immunocompromised woman due to HCoV-HKU1, that has never been before detected in Greece. HCoV-HKU1 is related to severe disease even in healthy individuals and must be considered in the differential diagnosis of severe respiratory infections.

High Levels of Common Cold Coronavirus Antibodies in Convalescent Plasma Are Associated With Improved Survival in COVID-19 Patients.

Background: COVID-19 Convalescent plasma (CCP) is safe and effective, particularly if given at an early stage of the disease. Our study aimed to identify an association between survival and specific antibodies found in CCP. Patients and Methods: Patients ≥18 years of age who were hospitalized with moderate to severe COVID-19 infection and received CCP at the MD Anderson Cancer Center between 4/30/2020 and 8/20/2020 were included in the study. We quantified the levels of anti-SARS-CoV-2 antibodies, as well as antibodies against antigens of other coronavirus strains, in the CCP units and compared antibody levels with patient outcomes. For each antibody, a Bayesian exponential survival time regression model including prognostic variables was fit, and the posterior probability of a beneficial effect (PBE) of higher antibody level on survival time was computed. Results: CCP was administered to 44 cancer patients. The median age was 60 years (range 37-84) and 19 (43%) were female. Twelve patients (27%) died of COVID-19-related complications. Higher levels of two non-SARS-CoV-2-specific antibodies, anti-HCoV-OC43 spike IgG and anti-HCoV-HKU1 spike IgG, had PBE = 1.00, and 4 SARS-CoV-2-specific antibodies had PBEs between 0.90 and 0.95. Other factors associated with better survival were shorter time to CCP administration, younger age, and female sex. Conclusions: Common cold coronavirus spike IgG antibodies anti-HCoV-OC43 and anti-HCoV-HKU1 may target a common domain for SARS-CoV-2 and other coronaviruses. They provide a promising therapeutic target for monoclonal antibody production.

Human coronavirus spike protein-host receptor recognition.

A variety of coronaviruses (CoVs) have infected humans and caused mild to severe respiratory diseases that could result in mortality. The human CoVs (HCoVs) belong to the genera of α- and ß-CoVs that originate in rodents and bats and are transmitted to humans via zoonotic contacts. The binding of viral spike proteins to the host cell receptors is essential for mediating fusion of viral and host cell membranes to cause infection. The SARS-CoV-2 originated in bats (RaTG13 SARS-CoV) and is transmitted to humans via pangolins. The presence of 'PRRA' sequence motif in SARS-CoV-2 spike proteins from human, dog, cat, mink, tiger and lion suggests a common viral entry mechanism into host cells. In this review, we discuss structural features of HCoV spike proteins and recognition of host protein and carbohydrate receptors.

Human Coronavirus Infections in Israel: Epidemiology, Clinical Symptoms and Summer Seasonality of HCoV-HKU1.

Human coronaviruses (HCoVs) cause mild to severe respiratory diseases. Six types of HCoVs have been discovered, the most recent one termed the Middle East respiratory syndrome coronavirus (MERS-CoV). The aim of this study is to monitor the circulation of HCoV types in the population during 2015⁻2016 in Israel. HCoVs were detected by real-time PCR analysis in 1910 respiratory samples, collected from influenza-like illness (ILI) patients during the winter sentinel influenza survey across Israel. Moreover, 195 HCoV-positive samples from hospitalized patients were detected during one year at Soroka University Medical Center. While no MERS-CoV infections were detected, 10.36% of patients in the survey were infected with HCoV-OC43 (43.43%), HCoV-NL63 (44.95%), and HCoV-229E (11.62%) viruses. The HCoVs were shown to co-circulate with respiratory syncytial virus (RSV) and to appear prior to influenza virus infections. HCoV clinical symptoms were more severe than those of RSV infections but milder than influenza symptoms. Hospitalized patients had similar HCoV types percentages. However, while it was absent from the public winter survey, 22.6% of the patients were HCoV-HKU1 positives, mainly during the spring-summer period.

Co-circulation of four human coronaviruses (HCoVs) in Queensland children with acute respiratory tract illnesses in 2004.

Acute respiratory illnesses (ARIs) with unconfirmed infectious aetiologies peak at different times of the year. Molecular diagnostic assays reduce the number of unconfirmed ARIs compared to serology- or culture-based techniques. Screening of 888 inpatient and outpatient respiratory specimens spanning late autumn through to early spring, 2004, identified the presence of a human coronavirus (HCoV) on 74 occasions (8.3% of all specimens and 26.3% of all respiratory virus detections). Prevalence peaked in August (late winter in the southern hemisphere) when they were detected in 21.9% of specimens tested. HCoV-HKU1 and HCoV-OC43 comprised 82.4% of all HCoVs detected. Positive specimens were used to develop novel reverse transcriptase real-time PCRs (RT-rtPCRs) for HCoV detection. An objective clinical severity score was assigned to each positive HCoV patient. Severity scores were similar to those from a random selection of young children who were positive for respiratory syncytial virus at a different time but from the same specimen population. During the cooler months of 2004, sensitive and specific RT-rtPCRs identified the concurrent circulation of all four HCoVs, a quarter of which co-occurred with another virus and most of which were from children under the age of two years.