A sensitive and simple RT-LAMP assay for sarbecovirus screening in bats.

IMPORTANCE: We report the application of a colorimetric and fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to facilitate mass screening for sarbecoviruses in bats. The assay was evaluated using a total of 838 oral and alimentary samples from bats and demonstrated comparable sensitivity and specificity to quantitative reverse transcription PCR (qRT-PCR), with a simple setup. The addition of SYTO9, a fluorescent nucleic acid stain, also allows for quantitative analysis. The scalability and simplicity of the assay are believed to contribute to improving preparedness for detecting emerging coronaviruses by applying it to field studies and 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.

A Rapid, Simple, Inexpensive, and Mobile Colorimetric Assay COVID-19-LAMP for Mass On-Site Screening of COVID-19.

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional 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.

Immune responses against enterovirus A71 infection: Implications for vaccine success.

Enterovirus A71 (EV-A71) from the Picornaviridae family is an important emerging pathogen causing hand, foot, and mouth disease (HFMD) outbreaks worldwide. EV-A71 also caused fatal neurological complications in young children especially in Asia. On the basis of seroepidemiological studies from many Asian countries, EV-A71 infection is very common. Children of very young age are particularly vulnerable. Large-scale epidemics that occur every 3 to 4 years are associated with accumulation of an immunologically naive younger population. Capsid proteins especially VP1 with the presence of major B- and T-cell epitopes are the most antigenic proteins. The nonstructural proteins mainly contribute to T-cell epitopes that induce cross-reactive immune responses against other enteroviruses. Dominant epitopes and their neutralization magnitudes differ in mice, rabbits, and humans. Neutralizing antibody is sufficient for immune protection, but poorer cellular immunity may lead to severe neurological complications and deaths. Some chemokines/cytokines are consistently found in severely ill patients, for example, IL-6, IL-10, IL-17A, MCP-1, IL-8, MIG, IP-10, IFN-γ, and G-CSF. An increase in white cell counts is a risk factor for severe HFMD. Recent clinical trials on EV-A71 inactivated vaccine showed >90% efficacy and a robust neutralization response that was protective, indicating neutralizing antibody correlates for protection. No protection against other enteroviruses was observed. A comprehensive understanding of the immune responses to EV-A71 infection will benefit the development of diagnostic tools, potential therapeutics, and subunit vaccine candidates. Future development of a multivalent enterovirus vaccine will require knowledge of correlates of protection, understanding of cross-protection and memory T-cell responses among enteroviruses.

Molecular epidemiology of coxsackievirus A6 circulating in Hong Kong reveals common neurological manifestations and emergence of novel recombinant groups.

BACKGROUND: Coxsackievirus A6 (CV-A6) represents the predominant enterovirus serotype in Hong Kong, but its epidemiology in our population was unknown. OBJECTIVES: To examine the clinical and molecular epidemiology of CV-A6 and detect emerging recombinant strains in Hong Kong. STUDY DESIGN: Nasopharyngeal aspirates (NPAs) from patients with febrile or respiratory illness were subject to RT-PCR for CV-A6 and sequencing of 5'-NCR and VP1. CV-A6-positive samples were further subject to 2C and 3D gene sequencing. Complete genome sequencing was performed on potential recombinant strains. RESULTS: Thirty-six (0.35%) NPAs were positive for CV-A6 by 5'-NCR RT-PCR and sequencing, 28 of which confirmed by partial VP1 gene sequencing. Among the 28 patients (mainly young children) with CV-A6 infection, hand-foot-and-mouth disease (HFMD) (43%), herpangina (18%) and tonsillitis (11%) were the most common diagnoses. Seven (25%) patients had neurological manifestations, including febrile seizures, encephalitis and meningitis. VP1 gene analysis showed that 24 CV-A6 strains circulating in Hong Kong belonged to genotype D5, while 4 strains belonged to D4. Further 2C and 3D gene analysis revealed eight potential recombinant strains. Genome sequencing of five selected strains confirmed four recombinant strains: HK459455/2013 belonging to recombination group RJ arisen from CV-A6/CV-A4, HK458288/2015 and HK446377/2015 representing novel group RL arisen from CV-A6/CV-A4, and HK462069/2015 representing novel group RM arisen from CV-A6/EV-A71. Recombination breakpoints located at 3D were identified in the latter three recombinant strains, with HK462069/2015 (from a child with encephalitis) having acquired 3D region from EV-A71. CONCLUSIONS: We identified novel recombinant CV-A6 strains in Hong Kong, with 3D being a common recombination site.

Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand, Foot and Mouth Disease Patients.

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142-156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41-55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates.