The coronavirus disease 2019 (COVID-19) pandemic has demonstrated a clear need for high-throughput, multiplexed and sensitive assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other respiratory viruses and their emerging variants. Here, we present a cost-effective virus and variant detection platform, called microfluidic Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (mCARMEN), which combines CRISPR-based diagnostics and microfluidics with a streamlined workflow for clinical use. We developed the mCARMEN respiratory virus panel to test for up to 21 viruses, including SARS-CoV-2, other coronaviruses and both influenza strains, and demonstrated its diagnostic-grade performance on 525 patient specimens in an academic setting and 166 specimens in a clinical setting. We further developed an mCARMEN panel to enable the identification of 6 SARS-CoV-2 variant lineages, including Delta and Omicron, and evaluated it on 2,088 patient specimens with near-perfect concordance to sequencing-based variant classification. Lastly, we implemented a combined Cas13 and Cas12 approach that enables quantitative measurement of SARS-CoV-2 and influenza A viral copies in samples. The mCARMEN platform enables high-throughput surveillance of multiple viruses and variants simultaneously, enabling rapid detection of SARS-CoV-2 variants.

The global spread of the highly pathogenic avian influenza (HPAI) A(H5N1) virus poses a serious pandemic threat, necessitating the swift development of effective vaccines. The success of messenger RNA (mRNA) vaccine technology in the COVID-19 pandemic, marked by its rapid development and scalability, demonstrates its potential for addressing other infectious threats, such as HPAI A(H5N1). We therefore evaluated mRNA vaccine candidates targeting panzootic influenza A(H5) clade 2.3.4.4b viruses, which have been shown to infect a range of mammalian species, including most recently being detected in dairy cattle. Ferrets were immunized with mRNA vaccines encoding either hemagglutinin alone or hemagglutinin and neuraminidase, derived from a 2.3.4.4b prototype vaccine virus recommended by the World Health Organization. Kinetics of the immune responses, as well as protection against a lethal challenge dose of A(H5N1) virus, were assessed. Two doses of mRNA vaccination elicited robust neutralizing antibody titers against a 2022 avian isolate and a 2024 human isolate. Further, mRNA vaccination conferred protection from lethal challenge, whereas all unvaccinated ferrets succumbed to infection. It also reduced viral titers in the upper and lower respiratory tracts of infected ferrets. These results underscore the effectiveness of mRNA vaccines against HPAI A(H5N1), showcasing their potential as a vaccine platform for future influenza pandemics.

Background: The 2023–2024 US influenza season was characterized by a predominance of A(H1N1)pdm09 virus circulation with cocirculation of A(H3N2) and B/Victoria viruses. We estimated vaccine effectiveness (VE) in the United States against mild-to-moderate medically attended influenza illness in the 2023–2024 season. Methods: We enrolled outpatients aged ≥8 months with acute respiratory illness in 7 states. Respiratory specimens were tested for influenza type/subtype by reverse-transcriptase polymerase chain reaction. Influenza VE was estimated with a test-negative design comparing odds of testing positive for influenza among vaccinated versus unvaccinated participants. We estimated VE by virus subtype/lineage and A(H1N1)pdm09 genetic subclades. Results: Among 6629 enrolled patients, 1780 (27%) tested positive for influenza, including 806 with A(H1N1)pdm09, 567 with B/Victoria, and 328 with A(H3N2). VE against any influenza illness was 44% (95% confidence interval, 36%–51%): 29% (15%–41%) against influenza A(H1N1)pdm09, 74% (65%–81%) against B/Victoria, and 30% (8%–47%) against A(H3N2). Statistically significant protection against any influenza was found for all age groups except adults aged 50–64 years. Lack of protection in this age group was specific to influenza A–associated illness. We observed differences in VE by birth cohort and A(H1N1)pdm09 virus genetic subclade. Conclusions: Vaccination reduced outpatient medically attended influenza overall by 44% and provided protection overall against circulating influenza A and B viruses. Serologic studies would help inform differences observed by age groups.

Antiviral susceptibility monitoring is integral to influenza surveillance conducted by CDC in collaboration with partners. Here, we outlined the algorithm and methods used for assessing antiviral susceptibility of viruses collected during 2023-2024 season. Virus specimens were provided by public health laboratories in the United States (US) and by laboratories in other countries that belong to the Pan American Health Organization. In the US, antiviral susceptibility surveillance conducted nationally is strengthened by sequence-only analysis of additional viruses collected at a state level. Viral genome sequence analysis was the primary approach to assess susceptibility to M2 blockers (n = 5123), neuraminidase (NA) inhibitors (n = 6874), and a polymerase acidic protein (PA) inhibitor (baloxavir, n = 6567). Over 99 % of type A viruses had M2-S31N that confers resistance to M2 blockers. Although oseltamivir-resistant viruses carrying NA-H275Y (N1 numbering) were rare (0.35 %), a cluster of four such viruses was identified in Haiti. Viruses with other NA mutations conferring reduced inhibition by NA inhibitor(s) were also detected sporadically. This includes a cluster of three influenza B viruses in Texas that shared a new mutation, NA-A245G conferring reduced inhibition by peramivir. Three viruses with reduced baloxavir susceptibility were identified, which had PA-I38T, PA-Y24C or PA-V122A; the latter two new mutations identified through augmented approach to sequence analysis. To monitor baseline susceptibility, supplementary in vitro testing was conducted on approximately 7 % of viruses using NA inhibition assay and cell culture-based assay IRINA. Implementation of Sequence First approach provided comprehensive and high throughput methodology for antiviral susceptibility assessment and reduced redundant phenotypic testing.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages with unique spike mutations and caused multiple epidemics domestically and globally. Although COVID-19 vaccines are available, new variants with the capacity for immune evasion continue to emerge. To understand and characterize the evolution of circulating SARS-CoV-2 variants in the U.S., the Centers for Disease Control and Prevention (CDC) initiated the National SARS-CoV-2 Strain Surveillance (NS3) program and has received thousands of SARS-CoV-2 clinical specimens from across the nation as part of a genotype to phenotype characterization process. Focus reduction neutralization with various antisera was used to antigenically characterize 143 SARS-CoV-2 Delta, Mu and Omicron subvariants from selected clinical specimens received between May 2021 and February 2023, representing a total of 59 unique spike protein sequences. BA.4/5 subvariants BU.1, BQ.1.1, CR.1.1, CQ.2 and BA.4/5 + D420N + K444T; BA.2.75 subvariants BM.4.1.1, BA.2.75.2, CV.1; and recombinant Omicron variants XBF, XBB.1, XBB.1.5 showed the greatest escape from neutralizing antibodies when analyzed against post third-dose original monovalent vaccinee sera. Post fourth-dose bivalent vaccinee sera provided better protection against those subvariants, but substantial reductions in neutralization titers were still observed, especially among BA.4/5 subvariants with both an N-terminal domain (NTD) deletion and receptor binding domain (RBD) substitutions K444M + N460K and recombinant Omicron variants. This analysis demonstrated a framework for long-term systematic genotype to antigenic characterization of circulating and emerging SARS-CoV-2 variants in the U.S., which is critical to assessing their potential impact on the effectiveness of current vaccines and antigen recommendations for future updates.