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.

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.

C oronavirus disease (COVID-19) is a global pandemic, affecting 213 countries with >2.7 million confirmed cases and 190,000 fatalities as of April 25, 2020 (1). Its causative agent was identified as severe acute respiratory syndrome coronavirus (SARS-CoV) 2 (SARS-CoV-2), which belongs to the same coronavirus species as SARS-CoV and SARS-related CoVs (SARSr-CoVs) in horseshoe bats (genus Rhinolophus) (2,3). Given the history among some early case-patients of visiting the Huanan seafood market in Wuhan, China, and its genetic close relatedness to SARSr-CoVs in bats and pangolins (2,4), SARS-CoV-2 was suspected to have emerged from wild animals, particularly bats, similar to SARS-CoV. SARS-CoV was a recombinant virus that originated from Chinese horseshoe bats (Rhinolophus sinicus) before it infected palm civets and then humans (5). Studying cellular tropism may provide clues to the host range and possible origin of zoonotic viruses. For example, SARS-CoV could replicate efficiently in kidney cells of its primary origin, R. sinicus, but not in other tested bat cells (6). To elucidate the possible origin of SARS-CoV-2, we tested susceptibilities of bat cell lines developed from different species commonly found in southern China to infection by SARS-CoV-2 in comparison with SARS-CoV. The selected bat species harbored a diverse set of coronaviruses, including SARSr-CoVs and Middle East respiratory syndrome-related coronaviruses (MERSr-CoVs), which pose potential health threats to humans (7). We also performed structural modeling of the virus/host receptor-binding interface. The Study SARS-CoV strain HKU-39849 was isolated in Hong Kong during the SARS epidemic as previously described (8). SARS-CoV-2 strain HK20 was isolated from a patient with COVID-19 in Hong Kong in early February 2020 (3). Thirteen primary or immortalized bat cell lines from 6 different bat species were subjected to infection with SARS-CoV and SARS-CoV-2 at multiplicity of infection of 0.1 as described previously (6,9,10), except with the addition of 2 µg/mL trypsin. The bat species included Miniopterus pusillus, Pipistrellus abramus (harboring Pipistrellus-BatCoV-HKU5), R. sinicus (harboring SARSr-BatCoVs, Rhinolophus-BatCoV-HKU2, Rhinolophus sinicus-BatCoV-HKU32), Tylonycteris pachypus (harboring Tylonycteris-BatCoV-HKU4), Rousettus leschenaultii (harboring many viruses, including Rousettus-BatCoV-HKU9 and Rousettus-BatCoV-HKU10), and Myotis ricketii (harboring Myotis-BatCoV-HKU6). Vero cells from African green monkey kidney were used as positive control (Figure 1; Appendix, https://wwwnc.cdc.gov/EID/ article/26/12/20-2308-App1.pdf). We determined viral replication efficiency by quantitative reverse transcription PCR (qRT-PCR) on cell culture supernatants (Table 1) (6). Cells were considered susceptible to viral infection if qRT-PCR on day 5 postinfection showed >1 log 10 increase in viral titer with statistical significance (p<0.05 by Student t-test).