A platform technology for generating subunit vaccines against diverse viral pathogens.

The COVID-19 pandemic response has shown how vaccine platform technologies can be used to rapidly and effectively counteract a novel emerging infectious disease. The speed of development for mRNA and vector-based vaccines outpaced those of subunit vaccines, however, subunit vaccines can offer advantages in terms of safety and stability. Here we describe a subunit vaccine platform technology, the molecular clamp, in application to four viruses from divergent taxonomic families: Middle Eastern respiratory syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), Lassa virus (LASV) and Nipah virus (NiV). The clamp streamlines subunit antigen production by both stabilising the immunologically important prefusion epitopes of trimeric viral fusion proteins while enabling purification without target-specific reagents by acting as an affinity tag. Conformations for each viral antigen were confirmed by monoclonal antibody binding, size exclusion chromatography and electron microscopy. Notably, all four antigens tested remained stable over four weeks of incubation at 40°C. Of the four vaccines tested, a neutralising immune response was stimulated by clamp stabilised MERS-CoV spike, EBOV glycoprotein and NiV fusion protein. Only the clamp stabilised LASV glycoprotein precursor failed to elicit virus neutralising antibodies. MERS-CoV and EBOV vaccine candidates were both tested in animal models and found to provide protection against viral challenge.

A Sensitive and Specific Competitive Enzyme-Linked Immunosorbent Assay for Serodiagnosis of COVID-19 in Animals.

In addition to human cases, cases of COVID-19 in captive animals and pets are increasingly reported. This raises the concern for two-way COVID-19 transmission between humans and animals. Here, we developed a SARS-CoV-2 nucleocapsid protein-based competitive enzyme-linked immunosorbent assay (cELISA) for serodiagnosis of COVID-19 which can theoretically be used in virtually all kinds of animals. We used 187 serum samples from patients with/without COVID-19, laboratory animals immunized with inactive SARS-CoV-2 virions, COVID-19-negative animals, and animals seropositive to other betacoronaviruses. A cut-off percent inhibition value of 22.345% was determined and the analytical sensitivity and specificity were found to be 1:64-1:256 and 93.9%, respectively. Evaluation on its diagnostic performance using 155 serum samples from COVID-19-negative animals and COVID-19 human patients showed a diagnostic sensitivity and specificity of 80.8% and 100%, respectively. The cELISA can be incorporated into routine blood testing of farmed/captive animals for COVID-19 surveillance.

Molecular Evolution of Human Coronavirus 229E in Hong Kong and a Fatal COVID-19 Case Involving Coinfection with a Novel Human Coronavirus 229E Genogroup.

Compared to other human coronaviruses, the genetic diversity and evolution of human coronavirus 229E (HCoV-229E) are relatively understudied. We report a fatal case of COVID-19 pneumonia coinfected with HCoV-229E in Hong Kong. Genome sequencing of SARS-CoV-2 and HCoV-229E from a nasopharyngeal sample of the patient showed that the SARS-CoV-2 strain HK13 was most closely related to SARS-CoV-2 type strain Wuhan-Hu-1 (99.99% nucleotide identity), compatible with his recent history of travel to Wuhan. The HCoV-229E strain HK20-42 was most closely related to HCoV-229E strain SC0865 from the United States (99.86% nucleotide identity). To investigate if it may represent a newly emerged HCoV-229E genotype in Hong Kong, we retrieved 41 archived respiratory samples that tested positive for HCoV-229E from 2004 to 2019. Pneumonia and exacerbations of chronic airway diseases were common among infected patients. Complete RdRp, S, and N gene sequencing of the 41 HCoV-229E strains revealed that our contemporary HCoV-229E strains have undergone significant genetic drift with clustering of strains in chronological order. Two novel genogroups were identified, in addition to previously described genogroups 1 to 4, with recent circulating strains including strain HK20-42 belonging to novel genogroup 6. Positive selection was detected in the spike protein and receptor-binding domain, which may be important for viral evolution at the receptor-binding interphase. Molecular dating analysis showed that HCoV-229E shared the most recent common ancestor with bat and camel/alpaca 229E-related viruses at ∼1884, while camel/alpaca viruses had a relatively recent common ancestor at ∼1999. Further studies are required to ascertain the evolutionary origin and path of HCoV-229E.IMPORTANCE Since its first appearance in the 1960s, the genetic diversity and evolution of human coronavirus 229E (HCoV-229E) have been relatively understudied. In this study, we report a fatal case of COVID-19 coinfected with HCoV-229E in Hong Kong. Genome sequencing revealed that our SARS-CoV-2 strain is highly identical to the SARS-CoV-2 strain from Wuhan, compatible with the patient's recent travel history, whereas our HCoV-229E strain in this study is highly identical to a recent strain in the United States. We also retrieved 41 archived HCoV-229E strains from 2004 to 2019 in Hong Kong for sequence analysis. Pneumonia and exacerbations of chronic airway diseases were common diagnoses among the 41 patients. The results showed that HCoV-229E was evolving in chronological order. Two novel genogroups were identified in addition to the four preexisting HCoV-229E genogroups, with recent circulating strains belonging to novel genogroup 6. Molecular clock analysis dated bat-to-human and bat-to-camelid transmission to as early as 1884.

Isolation of MERS-related coronavirus from lesser bamboo bats that uses DPP4 and infects human-DPP4-transgenic mice.

While a number of human coronaviruses are believed to be originated from ancestral viruses in bats, it remains unclear if bat coronaviruses are ready to cause direct bat-to-human transmission. Here, we report the isolation of a MERS-related coronavirus, Tylonycteris-bat-CoV-HKU4, from lesser bamboo bats. Tylonycteris-bat-CoV-HKU4 replicates efficiently in human colorectal adenocarcinoma and hepatocarcinoma cells with cytopathic effects, and can utilize human-dipeptidyl-peptidase-4 and dromedary camel-dipeptidyl-peptidase-4 as the receptors for cell entry. Flow cytometry, co-immunoprecipitation and surface plasmon resonance assays show that Tylonycteris-bat-CoV-HKU4-receptor-binding-domain can bind human-dipeptidyl-peptidase-4, dromedary camel-dipeptidyl-peptidase-4, and Tylonycteris pachypus-dipeptidyl-peptidase-4. Tylonycteris-bat-CoV-HKU4 can infect human-dipeptidyl-peptidase-4-transgenic mice by intranasal inoculation with self-limiting disease. Positive virus and inflammatory changes were detected in lungs and brains of infected mice, associated with suppression of antiviral cytokines and activation of proinflammatory cytokines and chemokines. The results suggest that MERS-related bat coronaviruses may overcome species barrier by utilizing dipeptidyl-peptidase-4 and potentially emerge in humans by direct bat-to-human transmission.

Differential Tropism of SARS-CoV and SARS-CoV-2 in Bat Cells.

Severe acute respiratory syndrome coronavirus 2 did not replicate efficiently in 13 bat cell lines, whereas severe acute respiratory syndrome coronavirus replicated efficiently in kidney cells of its ancestral host, the Rhinolophus sinicus bat, suggesting different evolutionary origins. Structural modeling showed that RBD/RsACE2 binding may contribute to the differential cellular tropism.

Possible Bat Origin of Severe Acute Respiratory Syndrome Coronavirus 2.

We showed that severe acute respiratory syndrome coronavirus 2 is probably a novel recombinant virus. Its genome is closest to that of severe acute respiratory syndrome-related coronaviruses from horseshoe bats, and its receptor-binding domain is closest to that of pangolin viruses. Its origin and direct ancestral viruses have not been identified.

Middle East Respiratory Syndrome Coronavirus Antibodies in Bactrian and Hybrid Camels from Dubai.

So far, dromedary camels are the only known animal reservoir for Middle East respiratory syndrome (MERS) coronavirus (MERS-CoV). Previous published serological studies showed that sera of Bactrian camels were all negative for MERS-CoV antibodies. However, a recent study revealed that direct inoculation of Bactrian camels intranasally with MERS-CoV can lead to infection with abundant virus shedding and seroconversion. In this study, we examined the presence of MERS-CoV antibodies in Bactrian and hybrid camels in Dubai, the United Arab Emirates (where dromedaries are also present), and Bactrian camels in Xinjiang, China (where dromedaries are absent). For the 29 serum samples from Bactrian camels in Dubai tested by the MERS-CoV spike (S) protein-based enzyme-linked immunosorbent assay (S-ELISA) and neutralization antibody test, 14 (48%) and 12 (41%), respectively, were positive for MERS-CoV antibodies. All the 12 serum samples that were positive with the neutralization antibody test were also positive for the S-ELISA. For the 11 sera from hybrid camels in Dubai tested with the S-ELISA and neutralization antibody test, 6 (55%) and 9 (82%), respectively, were positive for MERS-CoV antibodies. All the 6 serum samples that were positive for the S-ELISA were also positive with the neutralization antibody test. There was a strong correlation between the antibody levels detected by S-ELISA and neutralizing antibody titers, with a Spearman coefficient of 0.6262 (P < 0.0001; 95% confidence interval, 0.5062 to 0.7225). All 92 Bactrian camel serum samples from Xinjiang were negative for MERS-CoV antibodies tested using both S-ELISA and the neutralization antibody test. Bactrian and hybrid camels are potential sources of MERS-CoV infection.IMPORTANCE Since its first appearance in 2012, Middle East respiratory syndrome (MERS) has affected >25 countries, with >2,400 cases and an extremely high fatality rate of >30%. The total number of mortalities due to MERS is already greater than that due to severe acute respiratory syndrome. MERS coronavirus (MERS-CoV) has been confirmed to be the etiological agent. So far, dromedaries are the only known animal reservoir for MERS-CoV. Previously published serological studies showed that sera of Bactrian camels were all negative for MERS-CoV antibodies. In this study, we observed that 41% of the Bactrian camel sera and 55% of the hybrid camel sera from Dubai (where dromedaries are also present), but none of the sera from Bactrian camels in Xinjiang (where dromedaries are absent), were positive for MERS-CoV antibodies. Based on these results, we conclude that in addition to dromedaries, Bactrian and hybrid camels are also potential sources of MERS-CoV infection.

Identification of a Novel Betacoronavirus (Merbecovirus) in Amur Hedgehogs from China.

While dromedaries are the immediate animal source of Middle East Respiratory Syndrome (MERS) epidemic, viruses related to MERS coronavirus (MERS-CoV) have also been found in bats as well as hedgehogs. To elucidate the evolution of MERS-CoV-related viruses and their interspecies transmission pathway, samples were collected from different mammals in China. A novel coronavirus related to MERS-CoV, Erinaceus amurensis hedgehog coronavirus HKU31 (Ea-HedCoV HKU31), was identified from two Amur hedgehogs. Genome analysis supported that Ea-HedCoV HKU31 represents a novel species under Merbecovirus, being most closely related to Erinaceus CoV from European hedgehogs in Germany, with 79.6% genome sequence identity. Compared to other members of Merbecovirus, Ea-HedCoV HKU31 possessed unique non-structural proteins and putative cleavage sites at ORF1ab. Phylogenetic analysis showed that Ea-HedCoV HKU31 and BetaCoV Erinaceus/VMC/DEU/2012 were closely related to NeoCoV and BatCoV PREDICT from African bats in the spike region, suggesting that the latter bat viruses have arisen from recombination between CoVs from hedgehogs and bats. The predicted HKU31 receptor-binding domain (RBD) possessed only one out of 12 critical amino acid residues for binding to human dipeptidyl peptidase 4 (hDPP4), the MERS-CoV receptor. The structural modeling of the HKU31-RBD-hDPP4 binding interphase compared to that of MERS-CoV and Tylonycteris bat CoV HKU4 (Ty-BatCoV HKU4) suggested that HKU31-RBD is unlikely to bind to hDPP4. Our findings support that hedgehogs are an important reservoir of Merbecovirus, with evidence of recombination with viruses from bats. Further investigations in bats, hedgehogs and related animals are warranted to understand the evolution of MERS-CoV-related viruses.

Novel Bat Alphacoronaviruses in Southern China Support Chinese Horseshoe Bats as an Important Reservoir for Potential Novel Coronaviruses.

While bats are increasingly recognized as a source of coronavirus epidemics, the diversity and emergence potential of bat coronaviruses remains to be fully understood. Among 1779 bat samples collected in China, diverse coronaviruses were detected in 32 samples from five different bat species by RT-PCR. Two novel alphacoronaviruses, Rhinolophus sinicus bat coronavirus HKU32 (Rs-BatCoV HKU32) and Tylonycteris robustula bat coronavirus HKU33 (Tr-BatCoV HKU33), were discovered from Chinese horseshoe bats in Hong Kong and greater bamboo bats in Guizhou Province, respectively. Genome analyses showed that Rs-BatCoV HKU32 is closely related to BatCoV HKU10 and related viruses from diverse bat families, whereas Tr-BatCoV HKU33 is closely related to BtNv-AlphaCoV and similar viruses exclusively from bats of Vespertilionidae family. The close relatedness of Rs-BatCoV HKU32 to BatCoV HKU10 which was also detected in Pomona roundleaf bats from the same country park suggests that these viruses may have the tendency of infecting genetically distant bat populations of close geographical proximity with subsequent genetic divergence. Moreover, the presence of SARSr-CoV ORF7a-like protein in Rs-BatCoV HKU32 suggests a common evolutionary origin of this accessory protein with SARS-CoV, also from Chinese horseshoe bats, an apparent reservoir for coronavirus epidemics. The emergence potential of Rs-BatCoV HKU32 should be explored.

Molecular epidemiology, evolution and phylogeny of SARS coronavirus.

Shortly after its emergence in southern China in 2002/2003, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) was confirmed to be the cause of SARS. Subsequently, SARS-related CoVs (SARSr-CoVs) were found in palm civets from live animal markets in Guangdong and in various horseshoe bat species, which were believed to be the ultimate reservoir of SARSr-CoV. Till November 2018, 339 SARSr-CoV genomes have been sequenced, including 274 from human, 18 from civets and 47 from bats [mostly from Chinese horseshoe bats (Rhinolophus sinicus), n = 30; and greater horseshoe bats (Rhinolophus ferrumequinum), n = 9]. The human SARS-CoVs and civet SARSr-CoVs were collected in 2003/2004, while bat SARSr-CoVs were continuously isolated in the past 13 years even after the cessation of the SARS epidemic. SARSr-CoVs belong to the subgenus Sarbecovirus (previously lineage B) of genus Betacoronavirus and occupy a unique phylogenetic position. Overall, it is observed that the SARSr-CoV genomes from bats in Yunnan province of China possess the highest nucleotide identity to those from civets. It is evident from both multiple alignment and phylogenetic analyses that some genes of a particular SARSr-CoV from bats may possess higher while other genes possess much lower nucleotide identity to the corresponding genes of SARSr-CoV from human/civets, resulting in the shift of phylogenetic position in different phylogenetic trees. Our current model on the origin of SARS is that the human SARS-CoV that caused the epidemic in 2002/2003 was probably a result of multiple recombination events from a number of SARSr-CoV ancestors in different horseshoe bat species.

Replication of MERS and SARS coronaviruses in bat cells offers insights to their ancestral origins.

Previous findings of Middle East Respiratory Syndrome coronavirus (MERS-CoV)-related viruses in bats, and the ability of Tylonycteris-BatCoV HKU4 spike protein to utilize MERS-CoV receptor, human dipeptidyl peptidase 4 hDPP4, suggest a bat ancestral origin of MERS-CoV. We developed 12 primary bat cell lines from seven bat species, including Tylonycteris pachypus, Pipistrellus abramus and Rhinolophus sinicus (hosts of Tylonycteris-BatCoV HKU4, Pipistrellus-BatCoV HKU5, and SARS-related-CoV respectively), and tested their susceptibilities to MERS-CoVs, SARS-CoV, and human coronavirus 229E (HCoV-229E). Five cell lines, including P. abramus and R. sinicus but not T. pachypus cells, were susceptible to human MERS-CoV EMC/2012. However, three tested camel MERS-CoV strains showed different infectivities, with only two strains capable of infecting three and one cell lines respectively. SARS-CoV can only replicate in R. sinicus cells, while HCoV-229E cannot replicate in any bat cells. Bat dipeptidyl peptidase 4 (DPP4) sequences were closely related to those of human and non-human primates but distinct from dromedary DPP4 sequence. Critical residues for binding to MERS-CoV spike protein were mostly conserved in bat DPP4. DPP4 was expressed in the five bat cells susceptible to MERS-CoV, with significantly higher mRNA expression levels than those in non-susceptible cells (P = 0.0174), supporting that DPP4 expression is critical for MERS-CoV infection in bats. However, overexpression of T. pachypus DPP4 failed to confer MERS-CoV susceptibility in T. pachypus cells, suggesting other cellular factors in determining viral replication. The broad cellular tropism of MERS-CoV should prompt further exploration of host diversity of related viruses to identify its ancestral origin.

Receptor Usage of a Novel Bat Lineage C Betacoronavirus Reveals Evolution of Middle East Respiratory Syndrome-Related Coronavirus Spike Proteins for Human Dipeptidyl Peptidase 4 Binding.

Although bats are known to harbor Middle East Respiratory Syndrome coronavirus (MERS-CoV)-related viruses, the role of bats in the evolutionary origin and pathway remains obscure. We identified a novel MERS-CoV-related betacoronavirus, Hp-BatCoV HKU25, from Chinese pipistrelle bats. Although it is closely related to MERS-CoV in most genome regions, its spike protein occupies a phylogenetic position between that of Ty-BatCoV HKU4 and Pi-BatCoV HKU5. Because Ty-BatCoV HKU4 but not Pi-BatCoV HKU5 can use the MERS-CoV receptor human dipeptidyl peptidase 4 (hDPP4) for cell entry, we tested the ability of Hp-BatCoV HKU25 to bind and use hDPP4. The HKU25-receptor binding domain (RBD) can bind to hDPP4 protein and hDPP4-expressing cells, but it does so with lower efficiency than that of MERS-RBD. Pseudovirus assays showed that HKU25-spike can use hDPP4 for entry to hDPP4-expressing cells, although with lower efficiency than that of MERS-spike and HKU4-spike. Our findings support a bat origin of MERS-CoV and suggest that bat CoV spike proteins may have evolved in a stepwise manner for binding to hDPP4.

Pulmonary and extrapulmonary complications of human rhinovirus infection in critically ill patients.

BACKGROUND: Human rhinovirus (HRV) is frequently detected in patients with respiratory tract infection. However, the full clinical spectrum of HRV infection in critically ill patients is not well characterized. OBJECTIVE: To evaluate the clinical and virological characteristics of critically ill patients with HRV infection. STUDY DESIGN: HRV-specific reverse transcription-polymerase chain reaction (RT-PCR) was performed on nasopharyngeal aspirate (NPA) specimens from 294 adult patients who required admission into the intensive care unit (ICU). Clinical characteristics were analyzed. HRV genotyping using the 5'UTR-VP4-VP2 region was performed. RESULTS: HRV was detected in NPA specimens of 22 patients (7.5%) by RT-PCR. Dyspnea was the most common presenting symptom (16/22; 72.7%), but seizure also occurred in 5 (22.7%) patients. Exacerbation of underlying disease occurred in 12 (54.5%) patients. Four (18.2%) patients died, and HRV was considered to play a role as the cause of death in 3 patients. Thirteen (59.1%) patients had pneumonia, and the most common radiological finding was consolidation (6/13; 46.2%). Streptococcus pneumoniae was the most common co-pathogen among patients with pneumonia. Among the 9 patients without pneumonia, 3 patients had exacerbation of underlying lung diseases, 3 patients had acute pulmonary edema, 2 patients with diabetes mellitus had acute complications from poor glycemic control, and 1 patient had status epilepticus. HRV-A was the most common species (64.3%), but there was no clear relationship between HRV species and clinical presentation. CONCLUSION: Both pulmonary and extrapulmonary complications of HRV were common in critically ill patients.

A six-year descriptive epidemiological study of human coronavirus infections in hospitalized patients in Hong Kong.

We conducted a six-year epidemiological study on human coronaviruses (HCoVs) circulating in Hong Kong, using 8275 nasopharyngeal samples from patients with acute respiratory tract infections. HCoVs were detected in 77 (0.93%) of the samples by a pan-HCoV RT-PCR assay. The most frequently detected HCoV species was HCoV-OC43 (0.58%), followed by HCoV-229E (0.15%), HCoV-HKU1 (0.13%) and HCoV-NL63 (0.07%). HCoVs were detected throughout the study period (September 2008-August 2014), with the highest detection rate from September 2010 to August 2011 (22/1500, 1.47%). Different seasonal patterns of each HCoV species in Hong Kong were noted. HCoV-OC43 was predominant in the fall and winter, whereas HCoV-HKU1 showed peak activity in winter, with a few cases occurred in spring and summer. HCoV-229E mainly occurred in winter and spring, while HCoV-NL63 was predominant in summer and autumn. HCoVs most commonly infect the elderly and young children, with median age of 79.5 years (range, 22 days to 95 years). Intriguingly, the detection rate of HCoV-OC43 in the age group of > 80 years (26/2380, 1.09%) was significantly higher than that in the age group of 0-10 years (12/2529, 0.47%) (P < 0.05). These data provides new insight into the epidemiology of coronaviruses.

A sensitive and specific antigen detection assay for Middle East respiratory syndrome coronavirus.

Since its emergence in 2012, more than 900 laboratory-confirmed cases of Middle East respiratory syndrome (MERS) have been reported with a fatality rate of more than 30%. However, no antigen detection assay for commercial use is available for diagnosis. In this study, the full-length nucleocapsid protein (NP) gene of MERS coronavirus (MERS-CoV) was cloned and expressed in Escherichia coli. A MERS-CoV NP capture enzyme-linked immunosorbent assay (ELISA) using two MERS-CoV-NP-specific monoclonal antibodies (MAbs) generated was developed. The ELISA was evaluated using 129 nasopharyngeal aspirates (NPAs) positive for various respiratory viruses and simulated positive NPAs by adding serial dilutions of MERS-CoV. Using a cutoff OD of 0.19, all 129 NPAs positive for respiratory viruses showed very low OD, with a specificity of 100%. For the two simulated MERS-CoV-positive NPAs with serial dilutions of live MERS-CoV, all samples with ≥10 50% tissue culture infective dose (TCID50)/0.1 mL showed positive results. For the 10 additional NPAs with 20 and 200 TCID50/0.1 mL of live MERS-CoV added, all were positive. A highly sensitive and specific MAbs-based antigen capture ELISA has been developed for MERS. This sensitive and specific antigen capture ELISA should be useful for detection of MERS-CoV in human and dromedaries and in field studies.

Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination.

UNLABELLED: Despite the identification of horseshoe bats as the reservoir of severe acute respiratory syndrome (SARS)-related coronaviruses (SARSr-CoVs), the origin of SARS-CoV ORF8, which contains the 29-nucleotide signature deletion among human strains, remains obscure. Although two SARS-related Rhinolophus sinicus bat CoVs (SARSr-Rs-BatCoVs) previously detected in Chinese horseshoe bats (Rhinolophus sinicus) in Yunnan, RsSHC014 and Rs3367, possessed 95% genome identities to human and civet SARSr-CoVs, their ORF8 protein exhibited only 32.2 to 33% amino acid identities to that of human/civet SARSr-CoVs. To elucidate the origin of SARS-CoV ORF8, we sampled 348 bats of various species in Yunnan, among which diverse alphacoronaviruses and betacoronaviruses, including potentially novel CoVs, were identified, with some showing potential interspecies transmission. The genomes of two betacoronaviruses, SARSr-Rf-BatCoV YNLF_31C and YNLF_34C, from greater horseshoe bats (Rhinolophus ferrumequinum), possessed 93% nucleotide identities to human/civet SARSr-CoV genomes. Although these two betacoronaviruses displayed lower similarities than SARSr-Rs-BatCoV RsSHC014 and Rs3367 in S protein to civet SARSr-CoVs, their ORF8 proteins demonstrated exceptionally high (80.4 to 81.3%) amino acid identities to that of human/civet SARSr-CoVs, compared to SARSr-BatCoVs from other horseshoe bats (23.2 to 37.3%). Potential recombination events were identified around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. The expression of ORF8 subgenomic mRNA suggested that the ORF8 protein may be functional in SARSr-Rf-BatCoVs. The high Ka/Ks ratio among human SARS-CoVs compared to that among SARSr-BatCoVs supported that ORF8 is under strong positive selection during animal-to-human transmission. Molecular clock analysis using ORF1ab showed that SARSr-Rf-BatCoV YNLF_31C and YNLF_34C diverged from civet/human SARSr-CoVs in approximately 1990. SARS-CoV ORF8 originated from SARSr-CoVs of greater horseshoe bats through recombination, which may be important for animal-to-human transmission. IMPORTANCE: Although horseshoe bats are the primary reservoir of SARS-related coronaviruses (SARSr-CoVs), it is still unclear how these bat viruses have evolved to cross the species barrier to infect civets and humans. Most human SARS-CoV epidemic strains contain a signature 29-nucleotide deletion in ORF8, compared to civet SARSr-CoVs, suggesting that ORF8 may be important for interspecies transmission. However, the origin of SARS-CoV ORF8 remains obscure. In particular, SARSr-Rs-BatCoVs from Chinese horseshoe bats (Rhinolophus sinicus) exhibited <40% amino acid identities to human/civet SARS-CoV in the ORF8 protein. We detected diverse alphacoronaviruses and betacoronaviruses among various bat species in Yunnan, China, including two SARSr-Rf-BatCoVs from greater horseshoe bats that possessed ORF8 proteins with exceptionally high amino acid identities to that of human/civet SARSr-CoVs. We demonstrated recombination events around ORF8 between SARSr-Rf-BatCoVs and SARSr-Rs-BatCoVs, leading to the generation of civet SARSr-CoVs. Our findings offer insight into the evolutionary origin of SARS-CoV ORF8 protein, which was likely acquired from SARSr-CoVs of greater horseshoe bats through recombination.

Discovery of a novel coronavirus, China Rattus coronavirus HKU24, from Norway rats supports the murine origin of Betacoronavirus 1 and has implications for the ancestor of Betacoronavirus lineage A.

UNLABELLED: We discovered a novel Betacoronavirus lineage A coronavirus, China Rattus coronavirus (ChRCoV) HKU24, from Norway rats in China. ChRCoV HKU24 occupied a deep branch at the root of members of Betacoronavirus 1, being distinct from murine coronavirus and human coronavirus HKU1. Its unique putative cleavage sites between nonstructural proteins 1 and 2 and in the spike (S) protein and low sequence identities to other lineage A betacoronaviruses (ßCoVs) in conserved replicase domains support ChRCoV HKU24 as a separate species. ChRCoV HKU24 possessed genome features that resemble those of both Betacoronavirus 1 and murine coronavirus, being closer to Betacoronavirus 1 in most predicted proteins but closer to murine coronavirus by G+C content, the presence of a single nonstructural protein (NS4), and an absent transcription regulatory sequence for the envelope (E) protein. Its N-terminal domain (NTD) demonstrated higher sequence identity to the bovine coronavirus (BCoV) NTD than to the mouse hepatitis virus (MHV) NTD, with 3 of 4 critical sugar-binding residues in BCoV and 2 of 14 contact residues at the MHV NTD/murine CEACAM1a interface being conserved. Molecular clock analysis dated the time of the most recent common ancestor of ChRCoV HKU24, Betacoronavirus 1, and rabbit coronavirus HKU14 to about the year 1400. Cross-reactivities between other lineage A and B ßCoVs and ChRCoV HKU24 nucleocapsid but not spike polypeptide were demonstrated. Using the spike polypeptide-based Western blot assay, we showed that only Norway rats and two oriental house rats from Guangzhou, China, were infected by ChRCoV HKU24. Other rats, including Norway rats from Hong Kong, possessed antibodies only against N protein and not against the spike polypeptide, suggesting infection by ßCoVs different from ChRCoV HKU24. ChRCoV HKU24 may represent the murine origin of Betacoronavirus 1, and rodents are likely an important reservoir for ancestors of lineage A ßCoVs. IMPORTANCE: While bats and birds are hosts for ancestors of most coronaviruses (CoVs), lineage A ßCoVs have never been found in these animals and the origin of Betacoronavirus lineage A remains obscure. We discovered a novel lineage A ßCoV, China Rattus coronavirus HKU24 (ChRCoV HKU24), from Norway rats in China with a high seroprevalence. The unique genome features and phylogenetic analysis supported the suggestion that ChRCoV HKU24 represents a novel CoV species, occupying a deep branch at the root of members of Betacoronavirus 1 and being distinct from murine coronavirus. Nevertheless, ChRCoV HKU24 possessed genome characteristics that resemble those of both Betacoronavirus 1 and murine coronavirus. Our data suggest that ChRCoV HKU24 represents the murine origin of Betacoronavirus 1, with interspecies transmission from rodents to other mammals having occurred centuries ago, before the emergence of human coronavirus (HCoV) OC43 in the late 1800s. Rodents are likely an important reservoir for ancestors of lineage A ßCoVs.

First report of severe parainfluenza virus 4B and rhinovirus C coinfection in a liver transplant recipient treated with immunoglobulin.

We describe the first reported case of severe pneumonia due to coinfection by parainfluenza virus type 4B and rhinovirus C in a liver transplant recipient. The patient responded promptly to intravenous immunoglobulin and timely infection control measures prevented spreading of the infections. This report highlights respiratory viral coinfections as a possible cause of severe morbidity in transplant recipients and the importance of efficient molecular diagnostic technologies with major impact on clinical practice in a transplant center. It also describes a potential therapeutic strategy for such patients.