39 On 31 st December 2019, the World Health Organization was informed of a cluster of cases of 40 pneumonia of unknown etiology in Wuhan, China. Subsequent investigations identified a novel 41 coronavirus, now named as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), 42 from the affected patients. Highly sensitive and specific laboratory diagnostics are important for 43 controlling the rapidly evolving SARS-CoV-2-associated Coronavirus Disease 2019 (COVID-44 19) epidemic. In this study, we developed and compared the performance of three novel real-time 45 RT-PCR assays targeting the RNA-dependent RNA polymerase (RdRp)/helicase (Hel), spike (S), 46 and nucleocapsid (N) genes of SARS-CoV-2 with that of the reported RdRp-P2 assay which is 47 used in >30 European laboratories. Among the three novel assays, the COVID-19-RdRp/Hel 48 assay had the lowest limit of detection in vitro (1.8 TCID 50 /ml with genomic RNA and 11.2 RNA 49 copies/reaction with in vitro RNA transcripts). Among 273 specimens from 15 patients with 50 laboratory-confirmed COVID-19 in Hong Kong, 77 (28.2%) were positive by both the COVID-51 19-RdRp/Hel and RdRp-P2 assays. The COVID-19-RdRp/Hel assay was positive for an 52 additional 42 RdRd-P2-negative specimens [119/273 (43.6%) vs 77/273 (28.2%), P<0.001], 53including 29/120 (24.2%) respiratory tract specimens and 13/153 (8.5%) non-respiratory tract 54 specimens. The mean viral load of these specimens was 3.21×10 4 RNA copies/ml (range, 55 2.21×10 2 to 4.71×10 5 RNA copies/ml). The COVID-19-RdRp/Hel assay did not cross-react with 56 other human-pathogenic coronaviruses and respiratory pathogens in cell culture and clinical 57 specimens, whereas the RdRp-P2 assay cross-reacted with SARS-CoV in cell culture. The highly 58 sensitive and specific COVID-19-RdRp/Hel assay may help to improve the laboratory diagnosis 59 of COVID-19. 60 61 on March 16, 2020 by guest http://jcm.asm.org/ Downloaded from 4

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The pandemic novel coronavirus infection, Coronavirus Disease 2019 , has affected at least 190 countries or territories, with 465,915 confirmed cases and 21,031 deaths. In a containment-based strategy, rapid, sensitive and specific testing is important in epidemiological control and clinical management. Using 96 SARS-CoV-2 and 104 non-SARS-CoV-2 coronavirus genomes and our in-house program, GolayMetaMiner, four specific regions longer than 50 nucleotides in the SARS-CoV-2 genome were identified. Primers were designed to target the longest and previously untargeted nsp2 region and optimized as a probe-free real-time reverse transcription-polymerase chain reaction (RT-PCR) assay. The new COVID-19-nsp2 assay had a limit of detection (LOD) of 1.8 TCID 50 /mL and did not amplify other human-pathogenic coronaviruses and respiratory viruses. Assay reproducibility in terms of cycle threshold (Cp) values was satisfactory, with the total imprecision (% CV) values well below 5%. Evaluation of the new assay using 59 clinical specimens from 14 confirmed cases showed 100% concordance with our previously developed Int.

A B S T R A C TZika virus (ZIKV) is a human-pathogenic flavivirus that has recently emerged as a global public health threat. ZIKV infection may be associated with congenital malformations in infected fetuses and severe neurological and systemic complications in infected adults. There are currently limited treatment options for ZIKV infection. AR-12 (OSU-03012) is a celecoxib derivative cellular kinase inhibitor that has broad-spectrum antiviral activities. In this study, we investigated the antiviral activity and mechanism of AR-12 against ZIKV. We evaluated the in vitro anti-ZIKV activity of AR-12, using cell protection and virus yield reduction assays, in multiple clinically relevant cell lines, and the in vivo treatment effects of AR-12 in a lethal mouse model using type I interferon receptordeficient A129 mice. AR-12 inhibited ZIKV strains belonging to both the African and Asian/American lineages in Huh-7 and/or neuronal cells. AR12's IC 50 against ZIKV was consistently < 2 μM in these cells. ZIKV-infected A129 mice treated with intraperitoneally or orally administered AR-12 had significantly higher survival rate (50.0%-83.3% vs 0%, P < 0.05), less body weight loss, and lower blood and tissue ZIKV RNA loads than untreated control A129 mice. These anti-ZIKV effects were likely the results of down-regulation of the PI3K/Akt pathway by AR-12. Clinical trials using the clinically available and broad-spectrum AR-12 as an empirical treatment should be considered especially for patients residing in or returning from areas endemic of ZIKV and other arboviral infections who present with an acute undifferentiated febrile illness.