Characterizing fitness and immune escape of SARS-CoV-2 EG.5 sublineage using elderly serum and nasal organoid.

SARS-CoV-2 Omicron variant has evolved into sublineages. Here, we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However, there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population.

Development and Evaluation of an In-House Real-Time RT-PCR Targeting nsp10 Gene for SARS-CoV-2 Detection.

The emergence of SARS-CoV-2 mutations poses significant challenges to diagnostic tests, as these mutations can reduce the sensitivity of commonly used RT-PCR assays. Therefore, there is a need to design diagnostic assays with multiple targets to enhance sensitivity. In this study, we identified a novel diagnostic target, the nsp10 gene, using nanopore sequencing. Firstly, we determined the analytical sensitivity and specificity of our COVID-19-nsp10 assay. The COVID-19-nsp10 assay had a limit of detection of 74 copies/mL (95% confidence interval: 48-299 copies/mL) and did not show cross-reactivity with other respiratory viruses. Next, we determined the diagnostic performance of the COVID-19-nsp10 assay using 261 respiratory specimens, including 147 SARS-CoV-2-positive specimens belonging to the ancestral strain and Alpha, Beta, Gamma, Delta, Mu, Eta, Kappa, Theta and Omicron lineages. Using a LightMix E-gene RT-PCR assay as the reference method, the diagnostic sensitivity and specificity of the COVID-19-nsp10 assay were found to be 100%. The median Cp values for the LightMix E-gene RT-PCR and our COVID-19-nsp10 RT-PCR were 22.48 (range: 12.95-36.60) and 25.94 (range 16.37-36.87), respectively. The Cp values of the COVID-19-nsp10 RT-PCR assay correlated well with those of the LightMix E-gene RT-PCR assay (Spearman's ρ = 0.968; p < 0.0001). In conclusion, nsp10 is a suitable target for a SARS-CoV-2 RT-PCR assay.

Humoral and cellular immunity against different SARS-CoV-2 variants in patients with chronic kidney disease.

Chronic kidney disease (CKD) patients are at higher risk of severe COVID-19. Humoral and cellular immunity from prior infection or vaccination are important for protection, but the neutralizing antibody (nAb) response against SARS-CoV-2 variants is impaired. We investigated the variant-specific nAb and T cell immunity among CKD patients. Adult CKD patients were recruited between August and October 2022. nAb against the SARS-CoV-2 (ancestral strains and four Omicron sublineages) and T cell response were measured using the live virus neutralization assay and interferon-gamma release assay (IGRA). The correlation between nAb/T-cell response and subsequent infection after recruitment were also determined. Among the 88 recruited patients, 95.5% had prior infection or had completed the primary vaccine series. However, only 77.3% had detectable nAb against at least one SARS-CoV-2 strains, 59.1% tested positive in IGRA, and 52.3% had detectable nAb and tested positive in the IGRA. The nAb geometic mean titers (GMTs) against XBB.1, BA.5 and BA.2.3.20 were significantly lower than those against BA.2 and ancestral strain. Prior SARS-CoV-2 infection was associated with elevated nAb and T cell response. More kidney transplant recipients (KTRs) showed absent nAb and T cell response (36.8% vs. 10.1%), despite a higher prevalence of vaccine booster in this population (94.7% vs. 50.7%). Lower levels of nAb titer and T cell response were significantly associated with subsequent infection. A considerable proportion of CKD patients, especially KTRs, showed absence of humoral and cellular protective immunity against SARS-CoV-2. Strategies to improve immunogenicity in this population are urgently needed.

Global prevalence of SARS-CoV-2 3CL protease mutations associated with nirmatrelvir or ensitrelvir resistance.

BACKGROUND: Nirmatrelvir-ritonavir (Paxlovid) and ensitrelvir are 3-chymotrypsin-like cysteine protease (3CLpro) inhibitors which have been approved for the treatment of COVID-19 in 2021 and 2022, respectively. Previous studies have identified 3CLpro mutations that are associated with reduced susceptibility to these antivirals. The aim of the current study was to estimate the global prevalence of 3CLpro inhibitor-resistant SARS-CoV-2 strains. METHODS: We compiled a list of 3CLpro mutations which have been associated with nirmatrelvir or ensitrelvir resistance based on either viral replication or 3CLpro activity assays, and determined their prevalence among 13.4 million sequences deposited in GISAID as of December 14, 2022, about 1 year after the approval of nirmatrelvir-ritonavir. We analyzed the prevalence for different time periods, SARS-CoV-2 lineages and geographical locations. FINDINGS: Overall, 0.5% (67,095/13,446,588) of the sequences contained at least one mutation that was shown to affect the inhibitory activity of nirmatrelvir or ensitrelvir on viral replication or 3CLpro activity. We did not observe any increasing trend of resistance after the widespread clinical use of nirmatrelvir-ritonavir. G15S (2070 per million) and T21I (1386 per million) were the most prevalent mutations, and these mutations were dominant in some SARS-CoV-2 lineages. E166V and S144E, previously shown to affect the inhibitory activity of nirmatrelvir on viral replication or protease activity by > 100-folds, were found in <1 per million sequences. INTERPRETATION: Our data suggest that 3CLpro inhibitor resistance is currently rare. However, continuous global genotypic and phenotypic surveillance would be crucial in the early detection of resistant mutants. FUNDING: Richard and Carol Yu, May Tam Mak Mei Yin, The Shaw Foundation Hong Kong, Michael Tong, Marina Lee, Government Consultancy Service, the Emergency Key Program of Guangzhou Laboratory (See acknowledgements for full list).

Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis.

Accurate and simple diagnostic tests for coronavirus disease 2019 (COVID-19) are essential components of the pandemic response. In this study, we evaluated a one-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay coupled with clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein-mediated endpoint detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical samples. RT-LAMP-CRISPR is fast and affordable, does not require bulky thermocyclers, and minimizes carryover contamination risk. Results can be read either visually or with a fluorometer. RT-LAMP-CRISPR assays using primers targeting a highly expressed nsp8 gene and previously described nucleocapsid (N) gene primers were designed. The analytical characteristics and diagnostic performance of RT-LAMP-CRISPR assays were compared to those of a commercial real-time RT-PCR E gene assay. The limits of detection (LODs) of the nsp8 and N RT-LAMP-CRISPR assays were 750 and 2,000 copies/mL, which were higher than that of the commercial real-time RT-PCR assay (31.3 copies/mL). Despite the higher LOD, RT-LAMP-CRISPR assays showed diagnostic sensitivity and specificity of 98.6% and 100%, respectively, equivalent to those of the real-time RT-PCR assay (P = 0.5). The median fluorescence reading from the nsp8 assay (378.3 raw fluorescence unit [RFU] [range, 215.6 to 592.6]) was significantly higher than that of the N gene assay (342.0 RFU [range, 143.0 to 576.6]) (P < 0.0001). In conclusion, we demonstrate that RT-LAMP-CRISPR assays using primers rationally designed from highly expressed gene targets are highly sensitive, specific, and easy to perform. Such assays are a valuable asset in resource-limited settings. IMPORTANCE Accurate tests for the diagnosis of SARS-CoV-2, the virus causing coronavirus disease 2019 (COVID-19), are important for timely treatment and infection control decisions. Conventional tests such as real-time reverse transcription-PCR (RT-PCR) require specialized equipment and are expensive. On the other hand, rapid antigen tests suffer from a lack of sensitivity. In this study, we describe a novel assay format for the diagnosis of COVID-19 that is based on principles of loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas chemistry. A major advantage of this assay format is that it does not require expensive equipment to perform, and results can be read visually. This method proved to be fast, easy to perform, and inexpensive. The test compared well against an RT-PCR assay in terms of the ability to detect SARS-CoV-2 RNA in clinical samples. No false-positive test results were observed. The new assay format is ideal for SARS-CoV-2 diagnosis in resource-limited settings.

Risk Factors for Slow Viral Decline in COVID-19 Patients during the 2022 Omicron Wave.

Formulating termination of isolation (de-isolation) policies requires up-to-date knowledge about viral shedding dynamics. However, current de-isolation policies are largely based on viral load data obtained before the emergence of Omicron variant. In this retrospective cohort study involving adult patients hospitalised for COVID-19 between January and February 2022, we sought to determine SARS-CoV-2 viral shedding kinetics and to investigate the risk factors associated with slow viral decline during the 2022 Omicron wave. A total of 104 patients were included. The viral load was highest (Ct value was lowest) on days 1 post-symptom-onset (PSO) and gradually declined. Older age, hypertension, hyperlipidaemia and chronic kidney disease were associated with slow viral decline in the univariate analysis on both day 7 and day 10 PSO, while incomplete or no vaccination was associated with slow viral decline on day 7 PSO only. However, older age was the only risk factor that remained statistically significant in the multivariate analysis. In conclusion, older age is an independent risk factor associated with slow viral decline in this study conducted during the Omicron-dominant 2022 COVID-19 wave. Transmission-based precaution guidelines should take age into consideration when determining the timing of de-isolation.

Contribution of low population immunity to the severe Omicron BA.2 outbreak in Hong Kong.

Monitoring population protective immunity against SARS-CoV-2 variants is critical for risk assessment. We hypothesize that Hong Kong's explosive Omicron BA.2 outbreak in early 2022 could be explained by low herd immunity. Our seroprevalence study using sera collected from January to December 2021 shows a very low prevalence of neutralizing antibodies (NAb) against ancestral virus among older adults. The age group-specific prevalence of NAb generally correlates with the vaccination uptake rate, but older adults have a much lower NAb seropositive rate than vaccination uptake rate. For all age groups, the seroprevalence of NAb against Omicron variant is much lower than that against the ancestral virus. Our study suggests that this BA.2 outbreak and the exceptionally high case-fatality rate in the ≥80 year-old age group (9.2%) could be attributed to the lack of protective immunity in the population, especially among the vulnerable older adults, and that ongoing sero-surveillance is essential.

Pathogenicity, transmissibility, and fitness of SARS-CoV-2 Omicron in Syrian hamsters.

The in vivo pathogenicity, transmissibility, and fitness of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron (B.1.1.529) variant are not well understood. We compared these virological attributes of this new variant of concern (VOC) with those of the Delta (B.1.617.2) variant in a Syrian hamster model of COVID-19. Omicron-infected hamsters lost significantly less body weight and exhibited reduced clinical scores, respiratory tract viral burdens, cytokine and chemokine dysregulation, and lung damage than Delta-infected hamsters. Both variants were highly transmissible through contact transmission. In noncontact transmission studies Omicron demonstrated similar or higher transmissibility than Delta. Delta outcompeted Omicron without selection pressure, but this scenario changed once immune selection pressure with neutralizing antibodies-active against Delta but poorly active against Omicron-was introduced. Next-generation vaccines and antivirals effective against this new VOC are therefore urgently needed.

Outbreak investigation of airborne transmission of Omicron (B.1.1.529) - SARS-CoV-2 variant of concern in a restaurant: Implication for enhancement of indoor air dilution.

Airborne transmission of SARS-CoV-2 has been increasingly recognized in the outbreak of COVID-19, especially with the Omicron variant. We investigated an outbreak due to Omicron variant in a restaurant. Besides epidemiological and phylogenetic analyses, the secondary attack rates of customers of restaurant-related COVID-19 outbreak before (Outbreak R1) and after enhancement of indoor air dilution (Outbreak R2) were compared. On 27th December 2021, an index case stayed in restaurant R2 for 98 min. Except for 1 sitting in the same table, six other secondary cases sat in 3 corners at 3 different zones, which were served by different staff. The median exposure time was 34 min (range: 19-98 min). All 7 secondary cases were phylogenetically related to the index. Smoke test demonstrated that the airflow direction may explain the distribution of secondary cases. Compared with an earlier COVID-19 outbreak in another restaurant R1 (19th February 2021), which occurred prior to the mandatory enhancement of indoor air dilution, the secondary attack rate among customers in R2 was significantly lower than that in R1 (3.4%, 7/207 vs 28.9%, 22/76, p<0.001). Enhancement of indoor air dilution through ventilation and installation of air purifier could minimize the risk of SARS-CoV-2 transmission in the restaurants.

Rapid Spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Omicron Subvariant BA.2 in a Single-Source Community Outbreak.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant BA.2 sublineage has increased rapidly in Europe and Asia since January 2022. Here, we report the epidemiological and genomic analysis of a large single-source BA.2 outbreak in a housing estate. METHODS: We analyzed the epidemiological information on a community outbreak of BA.2 (STY outbreak). We performed whole viral genome sequencing using the Oxford Nanopore MinION device. We calculated the doubling time of the outbreak within a housing estate. RESULTS: The STY outbreak involved a total of 768 individuals as of 5 February 2022, including 432 residents, visitors, or staff (56.3%) from a single housing estate (KC Estate). The outbreak at the KC Estate had a short doubling time of 1.28 days (95% confidence interval: .560-1.935). The outbreak was promptly controlled with the lockdown of 3 buildings within the housing estate. Whole-genome sequencing was performed for 133 patients in the STY outbreak, including 106 residents of the KC Estate. All 133 sequences from the STY outbreak belonged to the BA.2 sublineage, and phylogenetic analysis showed that these sequences cluster together. All individuals in the STY cluster had the unique mutation C12525T. CONCLUSIONS: Our study highlights the exceptionally high transmissibility of the Omicron variant BA.2 sublineage in Hong Kong, where stringent measures are implemented as part of the elimination strategy. Continual genomic surveillance is crucial in monitoring the emergence of epidemiologically important Omicron sublineages.

Probable Animal-to-Human Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta Variant AY.127 Causing a Pet Shop-Related Coronavirus Disease 2019 (COVID-19) Outbreak in Hong Kong.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect human and other mammals, including hamsters. Syrian (Mesocricetus auratus) and dwarf (Phodopus sp.) hamsters are susceptible to SARS-CoV-2 infection in the laboratory setting. However, pet shop-related Coronavirus Disease 2019 (COVID-19) outbreaks have not been reported. METHODS: We conducted an investigation of a pet shop-related COVID-19 outbreak due to Delta variant AY.127 involving at least 3 patients in Hong Kong. We tested samples collected from the patients, environment, and hamsters linked to this outbreak and performed whole genome sequencing analysis of the reverse transcription polymerase chain reaction (RT-PCR)-positive samples. RESULTS: The patients included a pet shop keeper (Patient 1), a female customer of the pet shop (Patient 2), and the husband of Patient 2 (Patient 3). Investigation showed that 17.2% (5/29) and 25.5% (13/51) environmental specimens collected from the pet shop and its related warehouse, respectively, tested positive for SARS-CoV-2 RNA by RT-PCR. Among euthanized hamsters randomly collected from the storehouse, 3% (3/100) tested positive for SARS-CoV-2 RNA by RT-PCR and seropositive for anti-SARS-CoV-2 antibody by enzyme immunoassay. Whole genome analysis showed that although all genomes from the outbreak belonged to the Delta variant AY.127, there were at least 3 nucleotide differences among the genomes from different patients and the hamster cages. Genomic analysis suggests that multiple strains have emerged within the hamster population, and these different strains have likely transmitted to human either via direct contact or via the environment. CONCLUSIONS: Our study demonstrated probable hamster-to-human transmission of SARS-CoV-2. As pet trading is common around the world, this can represent a route of international spread of this pandemic virus.

Co-circulation of two SARS-CoV-2 variant strains within imported pet hamsters in Hong Kong.

During the investigation of a pet shop outbreak of severe acute respiratory coronavirus 2 (SARS-CoV-2) with probable hamster-to-human transmission, the environmental and hamster samples in epidemiologically linked pet shops were found positive for SARS-CoV-2 Delta variant AY.127 strains which are phylogenetically closely related to patients and reported European strains. This interspecies' spill-over has triggered transmission in 58 patients epidemiologically linked to three pet shops. Incidentally, three dwarf hamsters imported from the Netherlands and centralized in a warehouse distributing animals to pet shops were positive for SARS-CoV-2 spike variant phylogenetically related to European B.1.258 strains from March 2020. This B.1.258 strain almost disappeared in July 2021. While no hamster-to-human transmission of B.1.258-like strain was found in this outbreak, molecular docking showed that its spike receptor-binding domain (RBD) has a similar binding energy to human ACE2 compared to that of Delta variant AY.127. Therefore, the potential of this B.1.258-related spike variant for interspecies jumping cannot be ignored. The co-circulation of B.1.258-related spike variants with Delta AY.127, which originated in Europe and was not previously found in Hong Kong, suggested that hamsters in our wholesale warehouse and retail pet shops more likely have acquired these viruses in the Netherlands or stopovers during delivery by aviation than locally. The risk of human-to-hamster reverse zoonosis by multiple SARS-CoV-2 variants leading to further adaptive spike mutations with subsequent transmission back to humans cannot be underestimated as an outbreak source of COVID-19. Testing imported pet animals susceptible to SARS-CoV-2 is warranted to prevent future outbreaks.

Rapid Diagnosis of Mycobacterium marinum Infection by Next-Generation Sequencing: A Case Report.

We present the first report of histology- and culture-proven Mycobacterium marinum infection diagnosed by next-generation sequencing (NGS). It took <2 days to make a microbiological diagnosis using the Oxford Nanopore Technologies' MinION device, compared to 20 days for the mycobacterium to be isolated from the tissue biopsy. NGS is particularly useful for culture-negative and slow-growing microorganism infections, such as mycobacterial, fungal and partially treated pyogenic bacterial infections. Due to its low equipment cost, short turn-around-time and portable size, the Oxford Nanopore Technologies' MinION device is a useful platform for NGS in routine clinical microbiology laboratories.

SARS-CoV-2 Omicron variant shows less efficient replication and fusion activity when compared with Delta variant in TMPRSS2-expressed cells.

The novel SARS-CoV-2 Omicron variant (B.1.1.529), first found in early November 2021, has sparked considerable global concern and it has >50 mutations, many of which are known to affect transmissibility or cause immune escape. In this study, we sought to investigate the virological characteristics of the Omicron variant and compared it with the Delta variant which has dominated the world since mid-2021. Omicron variant replicated more slowly than the Delta variant in transmembrane serine protease 2 (TMPRSS2)-overexpressing VeroE6 (VeroE6/TMPRSS2) cells. Notably, the Delta variant replicated well in Calu3 cell line which has robust TMPRSS2 expression, while the Omicron variant replicated poorly in this cell line. Competition assay showed that Delta variant outcompeted Omicron variant in VeroE6/TMPRSS2 and Calu3 cells. To confirm the difference in entry pathway between the Omicron and Delta variants, we assessed the antiviral effect of bafilomycin A1, chloroquine (inhibiting endocytic pathway), and camostat (inhibiting TMPRSS2 pathway). Camostat potently inhibited the Delta variant but not the Omicron variant, while bafilomycin A1 and chloroquine could inhibit both Omicron and Delta variants. Moreover, the Omicron variant also showed weaker cell-cell fusion activity when compared with Delta variant in VeroE6/TMPRSS2 cells. Collectively, our results suggest that Omicron variant infection is not enhanced by TMPRSS2 but is largely mediated via the endocytic pathway. The difference in entry pathway between Omicron and Delta variants may have an implication on the clinical manifestations or disease severity.

Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant by Sera From BNT162b2 or CoronaVac Vaccine Recipients.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) omicron variant, designated as a variant of concern by the World Health Organization, carries numerous spike mutations that are known to evade neutralizing antibodies elicited by coronavirus disease 2019 (COVID-19) vaccines. A deeper understanding of the susceptibility of omicron variant to vaccine-induced neutralizing antibodies is urgently needed for risk assessment. METHODS: Omicron variant strains HKU691 and HKU344-R346K were isolated from patients using TMPRSS2-overexpressing VeroE6 cells. Whole genome sequence was determined using nanopore sequencing. Neutralization susceptibility of ancestral lineage A virus and the omicron, delta and beta variants to sera from 25 BNT162b2 and 25 CoronaVac vaccine recipients was determined using a live virus microneutralization assay. RESULTS: The omicron variant strain HKU344-R346K has an additional spike R346K mutation, which is present in 8.5% of strains deposited in the GISAID database. Only 20% and 24% of BNT162b2 recipients had detectable neutralizing antibody against the omicron variant HKU691 and HKU344-R346K, respectively, whereas none of the CoronaVac recipients had detectable neutralizing antibody titer against either omicron isolate. For BNT162b2 recipients, the geometric mean neutralization antibody titers (GMTs) of the omicron variant isolates (5.43 and 6.42) were 35.7-39.9-fold lower than that of the ancestral virus (229.4), and the GMTs of both omicron variant isolates were significantly lower than those of the beta and delta variants. There was no significant difference in the GMTs between HKU691 and HKU344-R346K. CONCLUSIONS: Omicron variant escapes neutralizing antibodies elicited by BNT162b2 or CoronaVac. The additional R346K mutation did not affect the neutralization susceptibility. Our data suggest that the omicron variant may be associated with lower COVID-19 vaccine effectiveness.

Impact of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variant-Associated Receptor Binding Domain (RBD) Mutations on the Susceptibility to Serum Antibodies Elicited by Coronavirus Disease 2019 (COVID-19) Infection or Vaccination.

BACKGROUND: Several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages with mutations at the spike protein receptor binding domain (RBD) have reduced susceptibility to antibody neutralization, and have been classified as variants of concern (VOCs) or variants of interest (VOIs). Here we systematically compared the neutralization susceptibility and RBD binding of different VOCs/VOIs, including B.1.617.1 (kappa variant) and P.3 (theta variant), which were first detected in India and the Philippines, respectively. METHODS: The neutralization susceptibility of the VOCs/VOIs (B.1.351, B.1.617.1, and P.3) and a non-VOC/VOI without RBD mutations (B.1.36.27) to convalescent sera from coronavirus disease 2019 (COVID-19) patients or BNT162b2 vaccinees was determined using a live virus microneutralization (MN) assay. Serum immunoglobulin G (IgG) binding to wild-type and mutant RBDs were determined using an enzyme immunoassay. RESULTS: The geometric mean neutralization titers (GMT) of B.1.351, P.3, and B.1.617.1 were significantly lower than that of B.1.36.27 for COVID-19 patients infected with non-VOCs/VOIs (3.4- to 5.7-fold lower) or individuals who have received 2 doses of BNT162b2 vaccine (4.4- to 7.3-fold lower). The GMT of B.1.351 or P.3 were lower than that of B.1.617.1. For the 4 patients infected with B.1.351 or B.1.617.1, the MN titer was highest for their respective lineage. RBD with E484K or E484Q mutation, either alone or in combination with other mutations, showed greatest reduction in serum IgG binding. CONCLUSIONS: P.3 and B.1.617.1 escape serum neutralization induced by natural infection or vaccine. Infection with 1 variant does not confer cross-protection for heterologous lineages. Immunogenicity testing for second generation COVID-19 vaccines should include multiple variant and "nonvariant" strains.