Reduced Influenza B-Specific Postvaccination Antibody Cross-reactivity in the B/Victoria Lineage-Predominant 2019/20 Season.

BACKGROUND: The influenza activity of the 2019/20 season remained high and widespread in the United States with type B viruses predominating the early season. The majority of B viruses characterized belonged to B/Victoria (B/Vic) lineage and contained a triple deletion of amino acid (aa) 162-164 in hemagglutinin (3DEL). These 3DEL viruses are antigenically distinct from B/Colorado/06/2017 (CO/06)-the B/Vic vaccine component of the 2018/19 and 2019/20 seasons representing the viruses with a double deletion of aa 162-163 in hemagglutinin (2DEL). METHODS: We performed molecular characterization and phylogenetic analysis of circulating B/Vic viruses. We also conducted hemagglutination inhibition (HAI) assay using archived human postvaccination sera collected from healthy subjects administered with different types of 2018/19 or 2019/20 seasonal vaccines. Their HAI cross-reactivity to representative 3DEL viruses was analyzed. RESULTS: The CO/06-specific human postvaccination sera, after being adjusted for vaccine type, had significantly reduced HAI cross-reactivity toward representative 3DEL viruses, especially the 136E+150K subgroup. The geometric mean titers against 3DEL viruses containing 136E+150K mutations were 1.6-fold lower in all populations (P = .051) and 1.9-fold lower in adults (P = .016) compared with those against the 136E+150N viruses. CONCLUSIONS: Our results indicate that postvaccination antibodies induced by the B/Vic vaccine component of the 2019/20 influenza season had reduced HAI cross-reactivity toward predominant 3DEL viruses in the United States. A close monitoring of the 3DEL 136E+150K subgroup is warranted should this subgroup return and predominate the 2020/21 influenza season.

Differential Effects of Prior Influenza Exposures on H3N2 Cross-reactivity of Human Postvaccination Sera.

BACKGROUND: Effectiveness of seasonal influenza vaccines mainly depends upon how well vaccine strains represent circulating viruses; mismatched strains can lead to reduced protection. Humans have complex influenza exposure histories that increase with age, which may lead to different postvaccination responses to emerging influenza variants. Recent observational studies also suggest that prior vaccination may influence the performance of current seasonal vaccines. METHODS: To elucidate the effects of age and influenza preexposures on cross-reactivity of vaccination-induced human antibodies, we generated antigenic maps based on postvaccination hemagglutination inhibition titers against representative H3 variants circulating during the 2015-2016, 2014-2015, and 2012-2013 influenza seasons. RESULTS: Antigenic maps determined using sera from subjects 18-64 and ≥65 years of age correlated well with each other but poorly with those determined using sera from children. Antigenic maps derived from human postvaccination sera with H1 influenza preexposure also correlated poorly with those derived from sera with neither H1 nor type B influenza preexposure, and the correlation lessened considerably over time. In contrast, antigenic maps derived from human postvaccination sera with only type B influenza preexposure consistently showed good correlation with those derived from sera with neither H1 nor type B influenza preexposure. CONCLUSIONS: Our results suggest an age-specific difference in human postvaccination responses. Our findings also suggest that prior exposure to H1 or type B influenza may differentially affect cross-reactivity of vaccination-induced H3-specific hemagglutination inhibition antibody responses, and consequently might affect vaccine effectiveness. Our study highlights the need to study the impact of prior exposure on influenza vaccine performance.

Comparative Efficacy of Monoclonal Antibodies That Bind to Different Epitopes of the 2009 Pandemic H1N1 Influenza Virus Neuraminidase.

UNLABELLED: Antibodies against the neuraminidase (NA) of influenza virus correlate with resistance against disease, but the effectiveness of antibodies against different NA epitopes has not been compared. In the present study, we evaluated the in vitro and in vivo efficacies of four monoclonal antibodies (MAbs): HF5 and CD6, which are specific to two different epitopes in the NA of 2009 pandemic H1N1 (pH1N1) virus, and 4E9 and 1H5, which are specific to a conserved epitope in the NA of both H1N1 and H5N1 viruses. In the in vitro assays, HF5 and CD6 inhibited virus spread and growth more effectively than 4E9 and 1H5, with HF5 being the most effective inhibitor. When administered prophylactically at 5 mg/kg of body weight, HF5 and CD6 protected ~90 to 100% of DBA/2 mice against lethal wild-type pH1N1 virus challenge; however, at a lower dose (1 mg/kg), HF5 protected ~90% of mice, whereas CD6 protected only 25% of mice. 4E9 and 1H5 were less effective than HF5 and CD6, as indicated by the partial protection achieved even at doses as high as 15 mg/kg. When administered therapeutically, HF5 protected a greater proportion of mice against lethal pH1N1 challenge than CD6. However, HF5 quickly selected pH1N1 virus escape mutants in both prophylactic and therapeutic treatments, while CD6 did not. Our findings confirm the important role of NA-specific antibodies in immunity to influenza virus and provide insight into the properties of NA antibodies that may serve as good candidates for therapeutics against influenza. IMPORTANCE: Neuraminidase (NA) is one of the major surface proteins of influenza virus, serving as an important target for antivirals and therapeutic antibodies. The impact of NA-specific antibodies on NA activity and virus replication is likely to depend on where the antibody binds. Using in vitro assays and the mouse model, we compared the inhibitory/protective efficacy of four mouse monoclonal antibodies (MAbs) that bind to different sites within the 2009 pandemic H1N1 (pH1N1) virus NA. The ability of each MAb to protect mice against lethal pH1N1 infection corresponded to its ability to inhibit NA activity in vitro; however, the MAb that was the most effective inhibitor of NA activity selected pH1N1 escape variants in vivo. One of the tested MAbs, which binds to a conserved region in the NA of pH1N1 virus, inhibited NA activity but did not result in escape variants, highlighting its suitability for development as a therapeutic agent.

Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus.

We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here, we showed that the same mutations improved the growth kinetics of a virus containing the A/Vietnam/1203/2004 (H5N1) haemagglutinin and neuraminidase (NA). Total protein yield and NA activity were increased when a chimeric NA was included. These increases indicated that the synergistic effect was due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.

RNA dimerization plays a role in ribosomal frameshifting of the SARS coronavirus.

Messenger RNA encoded signals that are involved in programmed -1 ribosomal frameshifting (-1 PRF) are typically two-stemmed hairpin (H)-type pseudoknots (pks). We previously described an unusual three-stemmed pseudoknot from the severe acute respiratory syndrome (SARS) coronavirus (CoV) that stimulated -1 PRF. The conserved existence of a third stem-loop suggested an important hitherto unknown function. Here we present new information describing structure and function of the third stem of the SARS pseudoknot. We uncovered RNA dimerization through a palindromic sequence embedded in the SARS-CoV Stem 3. Further in vitro analysis revealed that SARS-CoV RNA dimers assemble through 'kissing' loop-loop interactions. We also show that loop-loop kissing complex formation becomes more efficient at physiological temperature and in the presence of magnesium. When the palindromic sequence was mutated, in vitro RNA dimerization was abolished, and frameshifting was reduced from 15 to 5.7%. Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. These results suggest that the homodimeric RNA complex formed by the SARS pseudoknot occurs in the cellular environment and that loop-loop kissing interactions involving Stem 3 modulate -1 PRF and play a role in subgenomic and full-length RNA synthesis.

5' copyback defective viral genomes are major component in clinical and non-clinical influenza samples.

Clinical samples from people with influenza disease have been analyzed to assess the presence and abundance of Defective Viral Genomes (DVGs), but these have not been assessed using the same bioinformatic pipeline. The type of DVG most described for influenza infections (deletion DVGs) differs from the most commonly described DVGs from non-segmented negative stranded viruses (5' copyback). This could be attributed to either differences between viruses or the tools used to detect and characterize DVGs. Here we analyze several NGS datasets from people infected with different types of influenza virus using the same bioinformatic pipeline. We observe that 5' copyback DVGs are prevalent in all human clinical samples but not in the cultured samples. To address this discrepancy between clinical and laboratory cultures, we infected cell culture and ferrets with an H5N8 influenza A virus (FLUAV) and analyzed the DVG composition. The results demonstrate that the DVG population is skewed toward 5' copyback DVGs in the in vivo infections and deletion DVGs in the in vitro infections. This demonstrates that there are differences in vivo genome production and in vitro genome production, and this has implications for how the role of DVGs in clinical disease is studied. We also investigate the role the host cofactor ANP32B has in DVG production.

Post-vaccination serum cytokines levels correlate with breakthrough influenza infections.

Post-vaccination cytokine levels from 256 young adults who subsequently suffered breakthrough influenza infections were compared with matched controls. Modulation within the immune system is important for eliciting a protective response, and the optimal response differs according to vaccine formulation and delivery. For both inactivated influenza vaccine (IIV) and live attenuated influenza vaccines (LAIV) lower levels of IL-8 were observed in post-vaccination sera. Post-vaccination antibody levels were higher and IFN-γ levels were lower in IIV sera compared to LAIV sera. Subjects who suffered breakthrough infections after IIV vaccination had higher levels of sCD25 compared to the control group. There were differences in LAIV post-vaccination interleukin levels for subjects who subsequently suffered breakthrough infections, but these differences were masked in subjects who received concomitant vaccines. Wide variances, sex-based differences and confounders such as concomitant vaccines thwart the establishment of specific cytokine responses as a correlate of protection, but our results provide real world evidence that the status of the immune system following vaccination is important for successful vaccination and subsequent protection against disease.

Glycosylation of H4 influenza strains with pandemic potential and susceptibilities to lung surfactant SP-D.

We recently reported that members of group 1 influenza A virus (IAV) containing H2, H5, H6, and H11 hemagglutinins (HAs) are resistant to lung surfactant protein D (SP-D). H3 viruses, members of group 2 IAV, have high affinity for SP-D, which depends on the presence of high-mannose glycans at glycosite N165 on the head of HA. The low affinity of SP-D for the group 1 viruses is due to the presence of complex glycans at an analogous glycosite on the head of HA, and replacement with high-mannose glycan at this site evoked strong interaction with SP-D. Thus, if members of group 1 IAV were to make the zoonotic leap to humans, the pathogenicity of such strains could be problematic since SP-D, as a first-line innate immunity factor in respiratory tissues, could be ineffective as demonstrated in vitro. Here, we extend these studies to group 2 H4 viruses that are representative of those with specificity for avian or swine sialyl receptors, i.e., those with receptor-binding sites with either Q226 and G228 for avian or recent Q226L and G228S mutations that facilitate swine receptor specificity. The latter have increased pathogenicity potential in humans due to a switch from avian sialylα2,3 to sialylα2,6 glycan receptor preference. A better understanding of the potential action of SP-D against these strains will provide important information regarding the pandemic risk of such strains. Our glycomics and in vitro analyses of four H4 HAs reveal SP-D-favorable glycosylation patterns. Therefore, susceptibilities to this first-line innate immunity defense respiratory surfactant against such H4 viruses are high and align with H3 HA glycosylation.

Persistent growth of a human plasma-derived hepatitis C virus genotype 1b isolate in cell culture.

HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNA(I) (VA RNA(I)), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-alpha/beta-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2'-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics.

Bias at the third nucleotide of codon pairs in virus and host genomes.

Genomes of different sizes and complexity can be compared using common features. Most genomes contain open reading frames, and most genomes use the same genetic code. Redundancy in the genetic code means that different biases in the third nucleotide position of a codon exist in different genomes. However, the nucleotide composition of viruses can be quite different from host nucleotide composition making it difficult to assess the relevance of these biases. Here we show that grouping codons of a codon-pair according to the GC content of the first two nucleotide positions of each codon reveals patterns in nucleotide usage at the third position of the 1st codon. Differences between the observed and expected biases occur predominantly when the first two nucleotides of the 2nd codon are both S (strong, G or C) or both W (weak, A or T), not a mixture of strong and weak. The data indicates that some codon pairs are preferred because of the strength of the interactions between the codon and anticodon, the adjacent tRNAs and the ribosome. Using base-pairing strength and third position bias facilitates the comparison of genomes of different size and nucleotide composition and reveals patterns not previously described.

Enhanced virulence and waning vaccine-elicited antibodies account for breakthrough infections caused by SARS-CoV-2 delta and beyond.

Here we interrogate the factors responsible for SARS-CoV-2 breakthrough infections in a K18-hACE2 transgenic mouse model. We show that Delta and the closely related Kappa variant cause viral pneumonia and severe lung lesions in K18-hACE2 mice. Human COVID-19 mRNA post-vaccination sera after the 2nd dose are significantly less efficient in neutralizing Delta/Kappa than early 614G virus in vitro and in vivo. By 5 months post-vaccination, ≥50% of donors lack detectable neutralizing antibodies against Delta and Kappa and all mice receiving 5-month post-vaccination sera die after the lethal challenges. Although a 3rd vaccine dose can boost antibody neutralization against Delta in vitro and in vivo, the mean log neutralization titers against the latest Omicron subvariants are 1/3-1/2 of those against the original 614D virus. Our results suggest that enhanced virulence, greater immune evasion, and waning of vaccine-elicited protection account for SARS-CoV-2 variants caused breakthrough infections.

Revisiting the 1976 "swine flu" vaccine clinical trials: cross-reactive hemagglutinin and neuraminidase antibodies and their role in protection against the 2009 H1N1 pandemic virus in mice.

BACKGROUND: The 2009 H1N1 pandemic viruses are genetically similar to A/New Jersey/76 H1N1 virus (NJ/76), the strain selected for the 1976 "swine flu" vaccines. Approximately 45 million people in the United States were vaccinated against NJ/76 30 years ago, but the impact of this nationwide immunization on the current pandemic is largely unknown. METHODS: Archived human serum samples collected during the 1976 swine flu vaccine trials were assessed for cross-reactive antibody responses to the 2009 H1N1 pandemic viruses. RESULTS: Administration of an NJ/76 monovalent vaccine or the combination of a bivalent vaccine (NJ/76 H1N1 and A/Victoria/75 H3N2) plus a B/Hong Kong/72 monovalent vaccine increased hemagglutinin inhibition (HAI) and neuraminidase inhibition (NAI) antibodies cross-reacting with the 2009 H1N1 pandemic viruses. We showed that cross-reactive human HAI antibodies elicited by the 1976 swine flu vaccination played a dominant role in protecting recipient mice against the wild-type A/California/04/2009. Cross-reactive human NAI antibodies were also protective in recipient mice after a lethal challenge with a hemagglutinin mismatched virus bearing the A/California/04/2009 neuraminidase gene. Transfer of human serum samples with an original HAI titer of 43 or an original NAI titer of 472 was estimated to protect 50% of recipient mice from a lethal infection under the experimental conditions described. CONCLUSIONS: The 1976 swine flu vaccination induced cross-reactive HAI and NAI antibodies that were functionally protective in mice, suggesting that this vaccination campaign might have had a positive impact on older adults (≥50 years) in the United States during the 2009 H1N1 pandemic.

Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

A Codon-Pair Bias Associated With Network Interactions in Influenza A, B, and C Genomes.

A new codon-pair bias present in the genomes of different types of influenza virus is described. Codons with fewer network interactions are more frequency paired together than other codon-pairs in influenza A, B, and C genomes. A shared feature among three different influenza types suggests an evolutionary bias. Codon-pair preference can affect both speed of protein translation and RNA structure. This newly identified bias may provide insight into drivers of virus evolution.

Research Updates for Influenza Virus and Vaccine Development.

While the scientific community has been focusing on combating novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is responsible for the current COVID-19 pandemic, we also want to draw your attention to this Special Issue of Vaccines entitled "Influenza Virus and Vaccine Development" [...].

Insights from the comparison of genomic variants from two influenza B viruses grown in the presence of human antibodies in cell culture.

Understanding the extent and limitation of viral genome evolution can provide insight about potential drug and vaccine targets. Influenza B Viruses (IBVs) infect humans in a seasonal manner and causes significant morbidity and mortality. IBVs are negative-sense single-stranded RNA viruses with a segmented genome and can be divided into two antigenically distinct lineages. The two lineages have been circulating and further evolving for almost four decades. The immune response to IBV infection can lead to antibodies that target the strain causing the infection. Some antibodies are cross-reactive and are able to bind strains from both lineages but, because of antigenic drift and immunodominance, both lineages continue to evolve and challenge human health. Here we investigate changes in the genomes of an IBVs from each lineage after passage in tissue culture in the presence of human sera containing polyclonal antibodies directed toward antigenically and temporally distinct viruses. Our previous analysis of the fourth segment, which encodes the major surface protein HA, revealed a pattern of change in which signature sequences from one lineage mutated to the signature sequences of the other lineage. Here we analyze genes from the other genomic segments and observe that most of the quasispecies' heterogeneity occurs at the same loci in each lineage. The nature of the variants at these loci are investigated and possible reasons for this pattern are discussed. This work expands our understanding of the extent and limitations of genomic change in IBV.

Immune Pressure on Polymorphous Influenza B Populations Results in Diverse Hemagglutinin Escape Mutants and Lineage Switching.

Mutations arise in the genomes of progeny viruses during infection. Mutations that occur in epitopes targeted by host antibodies allow the progeny virus to escape the host adaptive, B-cell mediated antibody immune response. Major epitopes have been identified in influenza B virus (IBV) hemagglutinin (HA) protein. However, IBV strains maintain a seasonal presence in the human population and changes in IBV genomes in response to immune pressure are not well characterized. There are two lineages of IBV that have circulated in the human population since the 1980s, B-Victoria and B-Yamagata. It is hypothesized that early exposure to one influenza subtype leads to immunodominance. Subsequent seasonal vaccination or exposure to new subtypes may modify subsequent immune responses, which, in turn, results in selection of escape mutations in the viral genome. Here we show that while some mutations do occur in known epitopes suggesting antibody escape, many mutations occur in other parts of the HA protein. Analysis of mutations outside of the known epitopes revealed that these mutations occurred at the same amino acid position in viruses from each of the two IBV lineages. Interestingly, where the amino acid sequence differed between viruses from each lineage, reciprocal amino acid changes were observed. That is, the virus from the Yamagata lineage become more like the Victoria lineage virus and vice versa. Our results suggest that some IBV HA sequences are constrained to specific amino acid codons when viruses are cultured in the presence of antibodies. Some changes to the known antigenic regions may also be restricted in a lineage-dependent manner. Questions remain regarding the mechanisms underlying these results. The presence of amino acid residues that are constrained within the HA may provide a new target for universal vaccines for IBV.

The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A/Hong Kong/4801/2014 vaccine strain.

A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines.

The role of programmed-1 ribosomal frameshifting in coronavirus propagation.

Coronaviruses have the potential to cause significant economic, agricultural and health problems. The severe acute respiratory syndrome (SARS) associated coronavirus outbreak in late 2002, early 2003 called attention to the potential damage that coronaviruses could cause in the human population. The ensuing research has enlightened many to the molecular biology of coronaviruses. A programmed -1 ribosomal frameshift is required by coronaviruses for the production of the RNA dependent RNA polymerase which in turn is essential for viral replication. The frameshifting signal encoded in the viral genome has additional features that are not essential for frameshifting. Elucidation of the differences between coronavirus frameshift signals and signals from other viruses may help our understanding of these features. Here we summarize current knowledge and add additional insight regarding the function of the programmed -1 ribosomal frameshift signal in the coronavirus lifecycle.

A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal.

A wide range of RNA viruses use programmed -1 ribosomal frameshifting for the production of viral fusion proteins. Inspection of the overlap regions between ORF1a and ORF1b of the SARS-CoV genome revealed that, similar to all coronaviruses, a programmed -1 ribosomal frameshift could be used by the virus to produce a fusion protein. Computational analyses of the frameshift signal predicted the presence of an mRNA pseudoknot containing three double-stranded RNA stem structures rather than two. Phylogenetic analyses showed the conservation of potential three-stemmed pseudoknots in the frameshift signals of all other coronaviruses in the GenBank database. Though the presence of the three-stemmed structure is supported by nuclease mapping and two-dimensional nuclear magnetic resonance studies, our findings suggest that interactions between the stem structures may result in local distortions in the A-form RNA. These distortions are particularly evident in the vicinity of predicted A-bulges in stems 2 and 3. In vitro and in vivo frameshifting assays showed that the SARS-CoV frameshift signal is functionally similar to other viral frameshift signals: it promotes efficient frameshifting in all of the standard assay systems, and it is sensitive to a drug and a genetic mutation that are known to affect frameshifting efficiency of a yeast virus. Mutagenesis studies reveal that both the specific sequences and structures of stems 2 and 3 are important for efficient frameshifting. We have identified a new RNA structural motif that is capable of promoting efficient programmed ribosomal frameshifting. The high degree of conservation of three-stemmed mRNA pseudoknot structures among the coronaviruses suggests that this presents a novel target for antiviral therapeutics.