Background Studies have found different waning rates of neutralising antibodies compared with binding antibodies against SARS-CoV-2. The impact of neutralising antibody waning rate at the individual patient level on the longevity of immunity remains unknown. We aimed to investigate the peak levels and dynamics of neutralising antibody waning and IgG avidity maturation over time, and correlate this with clinical parameters, cytokines, and T-cell responses. Methods We did a longitudinal study of patients who had recovered from COVID-19 up to day 180 post-symptom onset by monitoring changes in neutralising antibody levels using a previously validated surrogate virus neutralisation test. Changes in antibody avidities and other immune markers at different convalescent stages were determined and correlated with clinical features. Using a machine learning algorithm, temporal change in neutralising antibody levels was classified into five groups and used to predict the longevity of neutralising antibody-mediated immunity. Findings We approached 517 patients for participation in the study, of whom 288 consented for outpatient follow-up and collection of serial blood samples. 164 patients were followed up and had adequate blood samples collected for analysis, with a total of 546 serum samples collected, including 128 blood samples taken up to 180 days post-symptom onset. We identified five distinctive patterns of neutralising antibody dynamics as follows: negative, individuals who did not, at our intervals of sampling, develop neutralising antibodies at the 30% inhibition level (19 [12%] of 164 patients); rapid waning, individuals who had varying levels of neutralising antibodies from around 20 days after symptom onset, but seroreverted in less than 180 days (44 [27%] of 164 patients); slow waning, individuals who remained neutralising antibody-positive at 180 days post-symptom onset (52 [29%] of 164 patients); persistent, although with varying peak neutralising antibody levels, these individuals had minimal neutralising antibody decay (52 [32%] of 164 patients); and delayed response, a small group that showed an unexpected increase of neutralising antibodies during late convalescence (at 90 or 180 days after symptom onset; three [2%] of 164 patients). Persistence of neutralising antibodies was associated with disease severity and sustained level of pro-inflammatory cytokines, chemokines, and growth factors. By contrast, T-cell responses were similar among the different neutralising antibody dynamics groups. On the basis of the different decay dynamics, we established a prediction algorithm that revealed a wide range of neutralising antibody longevity, varying from around 40 days to many decades. Interpretation Neutralising antibody response dynamics in patients who have recovered from COVID-19 vary greatly, and prediction of immune longevity can only be accurately determined at the individual level. Our findings emphas...

Among the many questions unanswered for the COVID-19 pandemic are the origin of SARS-CoV-2 and the potential role of intermediate animal host(s) in the early animal-to-human transmission. The discovery of RaTG13 bat coronavirus in China suggested a high probability of a bat origin. Here we report molecular and serological evidence of SARS-CoV-2 related coronaviruses (SC2r-CoVs) actively circulating in bats in Southeast Asia. Whole genome sequences were obtained from five independent bats (Rhinolophus acuminatus) in a Thai cave yielding a single isolate (named RacCS203) which is most related to the RmYN02 isolate found in Rhinolophus malayanus in Yunnan, China. SARS-CoV-2 neutralizing antibodies were also detected in bats of the same colony and in a pangolin at a wildlife checkpoint in Southern Thailand. Antisera raised against the receptor binding domain (RBD) of RmYN02 was able to cross-neutralize SARS-CoV-2 despite the fact that the RBD of RacCS203 or RmYN02 failed to bind ACE2. Although the origin of the virus remains unresolved, our study extended the geographic distribution of genetically diverse SC2r-CoVs from Japan and China to Thailand over a 4800-km range. Cross-border surveillance is urgently needed to find the immediate progenitor virus of SARS-CoV-2.

To date, limited genetic changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described. Here, we report a 382-nucleotide (nt) deletion in SARS-CoV-2 that truncates open reading frame 7b (ORF7b) and ORF8, removing the ORF8 transcription regulatory sequence (TRS) and eliminating ORF8 transcription. The earliest 382-nt deletion variant was detected in Singapore on 29 January 2020, with the deletion viruses circulating in the country and accounting for 23.6% (45/191) of SARS-CoV-2 samples screened in this study. SARS-CoV-2 with the same deletion has since been detected in Taiwan, and other ORF7b/8 deletions of various lengths, ranging from 62 nt to 345 nt, have been observed in other geographic locations, including Australia, Bangladesh, and Spain. Mutations or deletions in ORF8 of SARS-CoV have been associated with reduced replicative fitness and virus attenuation. In contrast, the SARS-CoV-2 382-nt deletion viruses showed significantly higher replicative fitness in vitro than the wild type, while no difference was observed in patient viral load, indicating that the deletion variant viruses retained their replicative fitness. A robust antibody response to ORF8 has been observed in SARS-CoV-2 infection, suggesting that the emergence of ORF8 deletions may be due to immune-driven selection and that further deletion variants may emerge during the sustained transmission of SARS-CoV-2 in humans. IMPORTANCE During the SARS epidemic in 2003/2004, a number of deletions were observed in ORF8 of SARS-CoV, and eventually deletion variants became predominant, leading to the hypothesis that ORF8 was an evolutionary hot spot for adaptation of SARS-CoV to humans. However, due to the successful control of the SARS epidemic, the importance of these deletions for the epidemiological fitness of SARS-CoV in humans could not be established. The emergence of multiple SARS-CoV-2 strains with ORF8 deletions, combined with evidence of a robust immune response to ORF8, suggests that the lack of ORF8 may assist with host immune evasion. In addition to providing a key insight into the evolutionary behavior of SARS-CoV-2 as the virus adapts to its new human hosts, the emergence of ORF8 deletion variants may also impact vaccination strategies.

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Summary Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern pose a challenge to the effectiveness of current vaccines. A vaccine that could prevent infection caused by known and future variants of concern as well as infection with pre-emergent sarbecoviruses (i.e., those with potential to cause disease in humans in the future) would be ideal. Here we provide data showing that potent cross-clade pan-sarbecovirus neutralizing antibodies are induced in survivors of severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) infection who have been immunized with the BNT162b2 messenger RNA (mRNA) vaccine. The antibodies are high-level and broad-spectrum, capable of neutralizing not only known variants of concern but also sarbecoviruses that have been identified in bats and pangolins and that have the potential to cause human infection. These findings show the feasibility of a pan-sarbecovirus vaccine strategy. (Funded by the Singapore National Research Foundation and National Medical Research Council.)

In response to the coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, multiple diagnostic tests are required for acute disease diagnosis, contact tracing, monitoring asymptomatic infection rates and assessing herd immunity. While PCR remains the frontline test of choice in the acute diagnostic setting, serological tests are urgently needed. Unlike PCR tests which are highly specific, cross-reactivity is a major challenge for COVID-19 antibody tests considering there are six other coronaviruses known to infect humans. SARS-CoV is genetically related to SARS-CoV-2 sharing approximately 80% sequence identity and both belong to the species SARS related coronavirus in the genus Betacoronavirus of family Coronaviridae. We developed and compared the performance of four different serological tests to comprehensively assess the cross-reactivity between COVID-19 and SARS patient sera. There is significant cross-reactivity when N protein of either virus is used. The S1 or RBD regions from the spike (S) protein offers better specificity. Amongst the different platforms, capture ELISA performed best. We found that SARS survivors all have significant levels of antibodies remaining in their blood 17 years after infection. Anti-N antibodies waned more than anti-RBD antibodies, and the latter is known to play a more important role in providing protective immunity.

bioRxiv preprint To date, the SARS-CoV-2 genome has been considered genetically more stable than 20 SARS-CoV or MERS-CoV. Here we report a 382-nt deletion covering almost the entire 21 open reading frame 8 (ORF8) of SARS-CoV-2 obtained from eight hospitalized patients 22 in Singapore. The deletion also removes the ORF8 transcription-regulatory sequence 23 (TRS), which in turn enhances the downstream transcription of the N gene. We also 24 found that viruses with the deletion have been circulating for at least four weeks. 25 During the SARS-CoV outbreak in 2003, a number of genetic variants were observed in 26 the human population [1], and similar variation has since been observed across SARS-27 related CoVs in humans and bats. Overwhelmingly these viruses had mutations or 28 deletions in ORF8, that have been associated with reduced replicative fitness of the 29 virus [2]. This is also consistent with the observation that towards the end of the 30 outbreak sequences obtained from human SARS cases possessed an ORF8 deletion that 31 may be associated with host adaptation [1]. We therefore hypothesise that the major 32 deletion revealed in this study may lead to an attenuated phenotype of SARS-CoV-2

Sex is pivotal for reproduction, healthcare and evolution. In the fish medaka, the Y-chromosomal dmy (also dmrt1bY) serves the sex determiner, which activates dmrt1 for male sex maintenance. However, how dmy makes the male decision via initiating testicular differentiation has remained unknown. Here we report that autosomal gsdf serves a male sex initiator. Gene addition and deletion revealed that gsdf was necessary and sufficient for maleness via initiating testicular differentiation. We show that gsdf transcription is activated directly by dmy. These results establish the autosomal gsdf as the first male sex initiator. We propose that dmy determines maleness through activating gsdf and dmrt1 without its own participation in developmental processes of sex initiation and maintenance. gsdf may easily become a sex determiner or other autosomal genes can be recruited as new sex determiners to initiate gsdf expression. Our findings offer new insights into molecular mechanisms underlying sex development and evolution of sex-controlling genes in vertebrates.