Less neutralization evasion of SARS-CoV-2 BA.2.86 than XBB sublineages and CH.1.1.

The highly mutated BA.2.86, with over 30 spike protein mutations in comparison to Omicron BA.2 and XBB.1.5 variants, has raised concerns about its potential to evade COVID-19 vaccination or prior SARS-CoV-2 infection-elicited immunity. In this study, we employ a live SARS-CoV-2 neutralization assay to compare the neutralization evasion ability of BA.2.86 with other emerged SARS-CoV-2 subvariants, including BA.2-derived CH.1.1, Delta-Omicron recombinant XBC.1.6, and XBB descendants XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1 and FL.1.5.1. Our results show that BA.2.86 is less neutralization evasive than XBB sublineages. XBB descendants XBB.1.16, EG.5.1, and FL.1.5.1 continue to significantly evade neutralization induced by the parental COVID-19 mRNA vaccine and a BA.5 Bivalent booster. Notably, when compared to XBB.1.5, the more recent XBB descendants, particularly EG.5.1, display increased resistance to neutralization. Among all the tested variants, CH.1.1 exhibits the greatest neutralization evasion. In contrast, XBC.1.6 shows a slight reduction but remains comparably sensitive to neutralization when compared to BA.5. Furthermore, a recent XBB.1.5-breakthrough infection significantly enhances the breadth and potency of cross-neutralization. These findings reinforce the expectation that the upcoming XBB.1.5 mRNA vaccine would likely boost the neutralization of currently circulating variants, while also underscoring the critical importance of ongoing surveillance to monitor the evolution and immune evasion potential of SARS-CoV-2 variants.

An Integrated Research-Clinical BSL-2 Platform for a Live SARS-CoV-2 Neutralization Assay.

A reliable and efficient serological test is crucial for monitoring neutralizing antibodies against SARS-CoV-2 and its variants of concern (VOCs). Here, we present an integrated research-clinical platform for a live SARS-CoV-2 neutralization assay, utilizing highly attenuated SARS-CoV-2 (Δ3678_WA1-spike). This strain contains mutations in viral transcription regulation sequences and deletion in the open-reading-frames 3, 6, 7, and 8, allowing for safe handling in biosafety level 2 (BSL-2) laboratories. Building on this backbone, we constructed a genetically stable reporter virus (mGFP Δ3678_WA1-spike) by incorporating a modified green fluorescent protein sequence (mGFP). We also constructed mGFP Δ3678_BA.5-spike and mGFP Δ3678_XBB.1.5-spike by substituting the WA1 spike with variants BA.5 and XBB.1.5 spike, respectively. All three viruses exhibit robust fluorescent signals in infected cells and neutralization titers in an optimized fluorescence reduction neutralization assay that highly correlates with a conventional plaque reduction assay. Furthermore, we established that a streamlined robot-aided Bench-to-Clinics COVID-19 Neutralization Test workflow demonstrated remarkably sensitive, specific, reproducible, and accurate characteristics, allowing the assessment of neutralization titers against SARS-CoV-2 variants within 24 h after sample receiving. Overall, our innovative approach provides a valuable avenue for large-scale testing of clinical samples against SARS-CoV-2 and VOCs at BSL-2, supporting pandemic preparedness and response strategies.

S:D614G and S:H655Y are gateway mutations that act epistatically to promote SARS-CoV-2 variant fitness.

SARS-CoV-2 variants bearing complex combinations of mutations that confer increased transmissibility, COVID-19 severity, and immune escape, were first detected after S:D614G had gone to fixation, and likely originated during persistent infection of immunocompromised hosts. To test the hypothesis that S:D614G facilitated emergence of such variants, S:D614G was reverted to the ancestral sequence in the context of sequential Spike sequences from an immunocompromised individual, and within each of the major SARS-CoV-2 variants of concern. In all cases, infectivity of the S:D614G revertants was severely compromised. The infectivity of atypical SARS-CoV-2 lineages that propagated in the absence of S:D614G was found to be dependent upon either S:Q613H or S:H655Y. Notably, Gamma and Omicron variants possess both S:D614G and S:H655Y, each of which contributed to infectivity of these variants. Among sarbecoviruses, S:Q613H, S:D614G, and S:H655Y are only detected in SARS-CoV-2, which is also distinguished by a polybasic S1/S2 cleavage site. Genetic and biochemical experiments here showed that S:Q613H, S:D614G, and S:H655Y each stabilize Spike on virions, and that they are dispensable in the absence of S1/S2 cleavage, consistent with selection of these mutations by the S1/S2 cleavage site. CryoEM revealed that either S:D614G or S:H655Y shift the Spike receptor binding domain (RBD) towards the open conformation required for ACE2-binding and therefore on pathway for infection. Consistent with this, an smFRET reporter for RBD conformation showed that both S:D614G and S:H655Y spontaneously adopt the conformation that ACE2 induces in the parental Spike. Data from these orthogonal experiments demonstrate that S:D614G and S:H655Y are convergent adaptations to the polybasic S1/S2 cleavage site which stabilize S1 on the virion in the open RBD conformation and act epistatically to promote the fitness of variants bearing complex combinations of clinically significant mutations.

CAPRIN1 Is Required for Control of Viral Replication Complexes by Interferon Gamma.

Replication complexes (RCs), formed by positive-strand (+) RNA viruses through rearrangements of host endomembranes, protect their replicating RNA from host innate immune defenses. We have shown that two evolutionarily conserved defense systems, autophagy and interferon (IFN), target viral RCs and inhibit viral replication collaboratively. However, the mechanism by which autophagy proteins target viral RCs and the role of IFN-inducible GTPases in the disruption of RCs remains poorly understood. Here, using murine norovirus (MNV) as a model (+) RNA virus, we show that the guanylate binding protein 1 (GBP1) is the human GTPase responsible for inhibiting RCs. Furthermore, we found that ATG16L1 mediates the LC3 targeting of MNV RC by binding to WIPI2B and CAPRIN1, and that IFN gamma-mediated control of MNV replication was dependent on CAPRIN1. Collectively, this study identifies a novel mechanism for the autophagy machinery-mediated recognition and inhibition of viral RCs, a hallmark of (+) RNA virus replication. IMPORTANCE Replication complexes provide a microenvironment important for (+) RNA virus replication and shield it from host immune response. Previously we have shown that interferon gamma (IFNG) disrupts the RC of MNV via evolutionarily conserved autophagy proteins and IFN-inducible GTPases. Elucidating the mechanism of targeting of viral RC by ATG16L1 and IFN-induced GTPase will pave the way for development of therapeutics targeting the viral replication complexes. Here, we have identified GBP1 as the sole GBP targeting viral RC and uncovered the novel role of CAPRIN1 in recruiting ATG16L1 to the viral RC.

Reverse genetic systems of SARS-CoV-2 for antiviral research.

Reverse genetic systems are widely used to engineer recombinant viruses with desired mutations. In response to the COVID-19 pandemic, four types of reverse genetic systems have been developed for SARS-CoV-2: (i) a full-length infectious clone that can be used to prepare recombinant SARS-CoV-2 at biosafety level 3 (BSL3), (ii) a trans-complementation system that can be used to produce single-round infectious SARS-CoV-2 at BSL2, (iii) an attenuated SARS-CoV-2 vaccine candidate (with deletions of viral accessory genes) that may be developed for veterinary use as well as for antiviral screening at BSL2, and (iv) replicon systems with deletions of viral structural genes that can be used at BSL2. Each of these genetic systems has its advantages and disadvantages that can be used to address different questions for basic and translational research. Due to the long genomic size and bacteria-toxic sequences of SARS-CoV-2, several experimental approaches have been established to rescue recombinant viruses and replicons, including (i) in vitro DNA ligation, (ii) bacterial artificial chromosome (BAC) system, (iii) yeast artificial chromosome (YAC) system, and (iv) circular polymerase extension reaction (CPER). This review summarizes the current status of SARS-CoV-2 genetic systems and their applications for studying viral replication, pathogenesis, vaccines, and therapeutics.

Cross-neutralization and viral fitness of SARS-CoV-2 Omicron sublineages.

The rapid evolution of SARS-CoV-2 Omicron sublineages mandates a better understanding of viral replication and cross-neutralization among these sublineages. Here we used K18-hACE2 mice and primary human airway cultures to examine the viral fitness and antigenic relationship among Omicron sublineages. In both K18-hACE2 mice and human airway cultures, Omicron sublineages exhibited a replication order of BA.5 ≥ BA.2 ≥ BA.2.12.1 > BA.1; no difference in body weight loss was observed among different sublineage-infected mice. The BA.1-, BA.2-, BA.2.12.1-, and BA.5-infected mice developed distinguishable cross-neutralizations against Omicron sublineages, but exhibited little neutralization against the index virus (i.e. USA-WA1/2020) or the Delta variant. Surprisingly, the BA.5-infected mice developed higher neutralization activity against heterologous BA.2 and BA.2.12.1 than that against homologous BA.5; serum neutralizing titres did not always correlate with viral replication levels in infected animals. Our results revealed a distinct antigenic cartography of Omicron sublineages and support the bivalent vaccine approach.

Determinants and Mechanisms of the Low Fusogenicity and High Dependence on Endosomal Entry of Omicron Subvariants.

The rapid spread and strong immune evasion of the SARS-CoV-2 Omicron subvariants has raised serious concerns for the global COVID-19 pandemic. These new variants exhibit generally reduced fusogenicity and increased endosomal entry pathway utilization compared to the ancestral D614G variant, the underlying mechanisms of which remain elusive. Here, we show that the C-terminal S1 mutations of the BA.1.1 subvariant, H655Y and T547K, critically govern the low fusogenicity of Omicron. Notably, H655Y also dictates the enhanced endosome entry pathway utilization. Mechanistically, T547K and H655Y likely stabilize the spike trimer conformation as suggested by increased molecular interactions in structural modeling and enhanced S1 shedding of their reversion mutants K547T and Y655H in viral producer cells. Importantly, the H655Y mutation also determines the low fusogenicity and enhanced dependence on the endosomal entry pathway of other Omicron subvariants, including BA.2, BA.2.12.1, BA.4/5, and BA.2.75. Together, these results uncover mechanisms governing Omicron subvariant entry and provide insights into altered Omicron tissue tropism and pathogenesis. IMPORTANCE Omicron has been shown to predominantly use the endosomal entry pathway, resulting in reduced lung tropism and reduced disease severity; however, the underlying mechanism is not fully understood. In addition, whether the most recent Omicron subvariants, including BA.5 and BA.2.75, use the same pathway as their ancestor for entry is currently not known. In this study, we show that T547K and H655Y mutations in the C terminus of the S1 subunit critically determine the enhanced dependence on the endosomal entry pathway as well as the reduced cell-cell fusion activity of Omicron BA.1, BA.1.1, and other subvariants. Further experiments and molecular modeling suggest that H655Y and K547T stabilize the spike trimer conformation, likely contributing to the decreased fusogenicity and endosomal entry. Our work uncovers novel mechanisms underlying the distinct entry pathway of Omicron subvariants and advances our understanding of their biological characteristics.

Low neutralization of SARS-CoV-2 Omicron BA.2.75.2, BQ.1.1 and XBB.1 by parental mRNA vaccine or a BA.5 bivalent booster.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages, including the BA.2-derived BA.2.75.2 and the BA.5-derived BQ.1.1 and XBB.1, have accumulated additional spike mutations that may affect vaccine effectiveness. Here we report neutralizing activities of three human serum panels collected from individuals 23-94 days after dose 4 of a parental mRNA vaccine; 14-32 days after a BA.5 bivalent booster from individuals with 2-4 previous doses of parental mRNA vaccine; or 14-32 days after a BA.5 bivalent booster from individuals with previous SARS-CoV-2 infection and 2-4 doses of parental mRNA vaccine. The results showed that a BA.5 bivalent booster elicited a high neutralizing titer against BA.4/5 measured at 14-32 days after boost; however, the BA.5 bivalent booster did not produce robust neutralization against the newly emerged BA.2.75.2, BQ.1.1 or XBB.1. Previous infection substantially enhanced the magnitude and breadth of BA.5 bivalent booster-elicited neutralization. Our data support a vaccine update strategy that future boosters should match newly emerged circulating SARS-CoV-2 variants.

Neutralization of SARS-CoV-2 Omicron sublineages by 4 doses of the original mRNA vaccine.

Since the initial emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.1, several Omicron sublineages have emerged, leading to BA.5 as the current dominant sublineage. Here, we report the neutralization of different Omicron sublineages by human sera collected from individuals who had distinct mRNA vaccination and/or BA.1 infection. Four-dose-vaccine sera neutralize the original USA-WA1/2020, Omicron BA.1, BA.2, BA.2.12.1, BA.3, and BA.4/5 viruses with geometric mean titers (GMTs) of 1,554, 357, 236, 236, 165, and 95, respectively; two-dose-vaccine-plus-BA.1-infection sera exhibit GMTs of 2,114, 1,705, 730, 961, 813, and 274, respectively; and three-dose-vaccine-plus-BA.1-infection sera show GMTs of 2,962, 2,038, 983, 1,190, 1,019, and 297, respectively. Thus, the four-dose vaccine elicits the lowest neutralization against BA.5; the two-dose vaccine plus BA.1 infection elicits significantly higher GMTs against Omicron sublineages than the four-dose-vaccine; and the three-dose vaccine plus BA.1 infection elicits slightly higher GMTs (statistically insignificant) than the two-dose vaccine plus BA.1 infection. Finally, the BA.2.75 is more susceptible than BA.5 to four-dose-vaccine-elicited neutralization and three-dose-vaccine-plus-BA.1-infection-elicited neutralization.

Determinants and Mechanisms of the Low Fusogenicity and Endosomal Entry of Omicron Subvariants.

The rapid spread and strong immune evasion of the SARS-CoV-2 Omicron subvariants has raised serious concerns for the global COVID-19 pandemic. These new variants exhibit reduced fusogenicity and increased endosomal entry pathway utilization compared to the ancestral D614G variant, the underlying mechanisms of which remain elusive. Here we show that the C-terminal S1 mutations of the BA.1.1 subvariant, H655Y and T547K, critically govern the low fusogenicity of Omicron. Notably, H655Y also dictates the enhanced endosome entry pathway utilization. Mechanistically, T547K and H655Y likely stabilize the spike trimer conformation, as shown by increased molecular interactions in structural modeling as well as reduced S1 shedding. Importantly, the H655Y mutation also determines the low fusogenicity and high dependence on the endosomal entry pathway of other Omicron subvariants, including BA.2, BA.2.12.1, BA.4/5 and BA.2.75. These results uncover mechanisms governing Omicron subvariant entry and provide insights into altered Omicron tissue tropism and pathogenesis.

Neutralization of SARS-CoV-2 Omicron sublineages by 4 doses of mRNA vaccine.

Since the initial emergence of SARS-CoV-2 Omicron BA.1, several Omicron sublineages have emerged, leading to BA.5 as the current dominant sublineage. Here we report the neutralization of different Omicron sublineages by human sera collected from individuals who had distinct mRNA vaccination and/or BA.1 infection. Four-dose-vaccine sera neutralize the original USA-WA1/2020, Omicron BA.1, BA.2, BA.212.1, BA.3, and BA.4/5 viruses with geometric mean titers (GMTs) of 1554, 357, 236, 236, 165, and 95, respectively; 2-dose-vaccine-plus-BA.1-infection sera exhibit GMTs of 2114, 1705, 730, 961, 813, and 274, respectively; and 3-dose-vaccine-plus-BA.1-infection sera show GMTs of 2962, 2038, 983, 1190, 1019, and 297, respectively. Thus, 4-dose-vaccine elicits the lowest neutralization against BA.5; 2-dose-vaccine-plus-BA.1-infection elicits significantly higher GMTs against Omicron sublineages than 4-dose-vaccine; and 3-dose-vaccine-plus-BA.1-infection elicits slightly higher GMTs (statistically insignificant) than the 2-dose-vaccine-plus-BA.1-infection. Finally, compared with BA.5, the newly emerged BA.2.75 is equally evasive of 4-dose-vaccine-elicited neutralization, but more susceptible to 3-dose-vaccine-plus-BA.1-infection-elicited neutralization.

Neutralization of Omicron sublineages and Deltacron SARS-CoV-2 by three doses of BNT162b2 vaccine or BA.1 infection.

Distinct SARS-CoV-2 Omicron sublineages have evolved showing increased fitness and immune evasion than the original Omicron variant BA.1. Here, we report the neutralization activity of sera from BNT162b2 vaccinated individuals or unimmunized Omicron BA.1-infected individuals against Omicron sublineages and "Deltacron" variant (XD). BNT162b2 post-dose 3 immune sera neutralized USA-WA1/2020, Omicron BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and XD-spike SARS-CoV-2s with geometric mean titres (GMTs) of 1335, 393, 298, 315, 216, 103, and 301, respectively; thus, BA.4/5 SARS-CoV-2 spike variant showed the highest propensity to evade vaccine neutralization compared to the original Omicron variants BA.1. BA.1-convalescent sera neutralized USA-WA1/2020, BA.1-, BA.2-, BA.2.12.1-, BA.3-, BA.4/5-, and Deltacron-spike SARS-CoV-2s with GMTs of 15, 430, 110, 109, 102, 25, and 284, respectively. The unique mutation F486V in the BA.4/5 spike contributes to the increased evasion of antibody neutralization by sublineage BA.4/5. The low neutralization titres of vaccinated sera or convalescent sera from BA.1 infected individuals against the emerging and rapidly spreading Omicron BA.4/5 variants provide important results for consideration in the selection of an updated vaccine in the current Omicron wave.Trial registration: ClinicalTrials.gov; identifier: NCT04368728.

A live-attenuated SARS-CoV-2 vaccine candidate with accessory protein deletions.

We report a live-attenuated SARS-CoV-2 vaccine candidate with (i) re-engineered viral transcription regulator sequences and (ii) deleted open-reading-frames (ORF) 3, 6, 7, and 8 (∆3678). The ∆3678 virus replicates about 7,500-fold lower than wild-type SARS-CoV-2 on primary human airway cultures, but restores its replication on interferon-deficient Vero-E6 cells that are approved for vaccine production. The ∆3678 virus is highly attenuated in both hamster and K18-hACE2 mouse models. A single-dose immunization of the ∆3678 virus protects hamsters from wild-type virus challenge and transmission. Among the deleted ORFs in the ∆3678 virus, ORF3a accounts for the most attenuation through antagonizing STAT1 phosphorylation during type-I interferon signaling. We also developed an mNeonGreen reporter ∆3678 virus for high-throughput neutralization and antiviral testing. Altogether, the results suggest that ∆3678 SARS-CoV-2 may serve as a live-attenuated vaccine candidate and a research tool for potential biosafety level-2 use.

Neutralization of Omicron BA.1, BA.2, and BA.3 SARS-CoV-2 by 3 doses of BNT162b2 vaccine.

The newly emerged Omicron SARS-CoV-2 has several distinct sublineages including BA.1, BA.2, and BA.3. BA.1 accounts for the initial surge and is being replaced by BA.2, whereas BA.3 is at a low prevalence at this time. Here we report the neutralization of BNT162b2-vaccinated sera (collected 1 month after dose 3) against the three Omicron sublineages. To facilitate the neutralization testing, we have engineered the complete BA.1, BA.2, or BA.3 spike into an mNeonGreen USA-WA1/2020 SRAS-CoV-2. All BNT162b2-vaccinated sera neutralize USA-WA1/2020, BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s with titers of >20; the neutralization geometric mean titers (GMTs) against the four viruses are 1211, 336, 300, and 190, respectively. Thus, the BA.1-, BA.2-, and BA.3-spike SARS-CoV-2s are 3.6-, 4.0-, and 6.4-fold less efficiently neutralized than the USA-WA1/2020, respectively. Our data have implications in vaccine strategy and understanding the biology of Omicron sublineages.

Remdesivir and GS-441524 Retain Antiviral Activity against Delta, Omicron, and Other Emergent SARS-CoV-2 Variants.

Genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern/variants of interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV [VEKLURY]) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here, we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein enzyme-linked immunosorbent assay (ELISA) and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with 50% effective concentration (EC50) values 0.30- to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.13- to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants, with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.

Cross-neutralization of Omicron BA.1 against BA.2 and BA.3 SARS-CoV-2.

The Omicron SARS-CoV-2 has several distinct sublineages, among which sublineage BA.1 is responsible for the initial Omicron surge and is now being replaced by BA.2 worldwide, whereas BA.3 is currently at a low frequency. The ongoing BA.1-to-BA.2 replacement underscores the importance to understand the cross-neutralization among the three Omicron sublineages. Here we test the neutralization of BA.1-infected human sera against BA.2, BA.3, and USA/WA1-2020 (a strain isolated in late January 2020). The BA.1-infected sera neutralize BA.1, BA.2, BA.3, and USA/WA1-2020 SARS-CoV-2s with geometric mean titers (GMTs) of 445, 107, 102, and 16, respectively. Thus, the neutralizing GMTs against heterologous BA.2, BA.3, and USA/WA1-2020 are 4.2-, 4.4-, and 28.4-fold lower than the GMT against homologous BA.1, respectively. These findings have implications in COVID-19 vaccine strategy.

Neutralization and durability of 2 or 3 doses of the BNT162b2 vaccine against Omicron SARS-CoV-2.

Two doses of the BNT162b2 mRNA vaccine are highly effective against SARS-CoV-2. Here, we tested the antibody neutralization against Omicron SARS-CoV-2 after 2 and 3 doses of BNT162b2. Serum from vaccinated individuals was serially tested for its ability to neutralize wild-type SARS-CoV-2 (USA-WA1/2020) and an engineered USA-WA1/2020 bearing the Omicron spike glycoprotein. At 2 or 4 weeks post dose 2, the neutralization geometric mean titers (GMTs) against the wild-type and Omicron-spike viruses were 511 and 20, respectively; at 1 month post dose 3, the neutralization GMTs increased to 1,342 and 336; and at 4 months post dose 3, the neutralization GMTs decreased to 820 and 171. The data support a 3-dose vaccination strategy and provide a glimpse into the durability of the neutralization response against Omicron.

A live-attenuated SARS-CoV-2 vaccine candidate with accessory protein deletions.

We report a live-attenuated SARS-CoV-2 vaccine candidate with (i) re-engineered viral transcriptional regulator sequences and (ii) deleted open-reading-frames (ORF) 3, 6, 7, and 8 (Δ3678). The Δ3678 virus replicates about 7,500-fold lower than wild-type SARS-CoV-2 on primary human airway cultures, but restores its replication on interferon-deficient Vero-E6 cells that are approved for vaccine production. The Δ3678 virus is highly attenuated in both hamster and K18-hACE2 mouse models. A single-dose immunization of the Δ3678 virus protects hamsters from wild-type virus challenge and transmission. Among the deleted ORFs in the Δ3678 virus, ORF3a accounts for the most attenuation through antagonizing STAT1 phosphorylation during type-I interferon signaling. We also developed an mNeonGreen reporter Δ3678 virus for high-throughput neutralization and antiviral testing. Altogether, the results suggest that Δ3678 SARS-CoV-2 may serve as a live-attenuated vaccine candidate and a research tool for potential biosafety level-2 use.