Cross-species spill-over potential of the H9N2 bat influenza A virus.

In 2017, a novel influenza A virus (IAV) was isolated from an Egyptian fruit bat. In contrast to other bat influenza viruses, the virus was related to avian A(H9N2) viruses and was probably the result of a bird-to-bat transmission event. To determine the cross-species spill-over potential, we biologically characterize features of A/bat/Egypt/381OP/2017(H9N2). The virus has a pH inactivation profile and neuraminidase activity similar to those of human-adapted IAVs. Despite the virus having an avian virus-like preference for α2,3 sialic acid receptors, it is unable to replicate in male mallard ducks; however, it readily infects ex-vivo human respiratory cell cultures and replicates in the lungs of female mice. A/bat/Egypt/381OP/2017 replicates in the upper respiratory tract of experimentally-infected male ferrets featuring direct-contact and airborne transmission. These data suggest that the bat A(H9N2) virus has features associated with increased risk to humans without a shift to a preference for α2,6 sialic acid receptors.

Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

Hemagglutinin stability as a key determinant of influenza A virus transmission via air.

To cause pandemics, zoonotic respiratory viruses need to adapt to replication in and spread between humans, either via (indirect or direct) contact or through the air via droplets and aerosols. To render influenza A viruses transmissible via air, three phenotypic viral properties must change, of which receptor-binding specificity and polymerase activity have been well studied. However, the third adaptive property, hemagglutinin (HA) acid stability, is less understood. Recent studies show that there may be a correlation between HA acid stability and virus survival in the air, suggesting that a premature conformational change of HA, triggered by low pH in the airways or droplets, may render viruses noninfectious before they can reach a new host. We here summarize available data from (animal) studies on the impact of HA acid stability on airborne transmission and hypothesize that the transmissibility of other respiratory viruses may also be impacted by an acidic environment in the airways.

Rapid evolution of A(H5N1) influenza viruses after intercontinental spread to North America.

Highly pathogenic avian influenza A(H5N1) viruses of clade 2.3.4.4b underwent an explosive geographic expansion in 2021 among wild birds and domestic poultry across Asia, Europe, and Africa. By the end of 2021, 2.3.4.4b viruses were detected in North America, signifying further intercontinental spread. Here we show that the western movement of clade 2.3.4.4b was quickly followed by reassortment with viruses circulating in wild birds in North America, resulting in the acquisition of different combinations of ribonucleoprotein genes. These reassortant A(H5N1) viruses are genotypically and phenotypically diverse, with many causing severe disease with dramatic neurologic involvement in mammals. The proclivity of the current A(H5N1) 2.3.4.4b virus lineage to reassort and target the central nervous system warrants concerted planning to combat the spread and evolution of the virus within the continent and to mitigate the impact of a potential influenza pandemic that could originate from similar A(H5N1) reassortants.

Hemagglutinin destabilization in H3N2 vaccine reference viruses skews antigenicity and prevents airborne transmission in ferrets.

During influenza virus entry, the hemagglutinin (HA) protein binds receptors and causes membrane fusion after endosomal acid activation. To improve vaccine efficiency and pandemic risk assessment for currently-dominant H3N2 influenza viruses, we investigated HA stability of 6 vaccine reference viruses and 42 circulating viruses. Recent vaccine reference viruses had destabilized HA proteins due to egg-adaptive mutation HA1-L194P. Virus growth in cell culture was independent of HA stability. In ferrets, the vaccine reference viruses and circulating viruses required a relatively stable HA (activation and inactivation pH < 5.5) for airborne transmissibility. The recent vaccine reference viruses with destabilized HA proteins had reduced infectivity, had no airborne transmissibility unless reversion to HA1-P194L occurred, and had skewed antigenicity away from the studied viruses and circulating H3N2 viruses. Other vaccine reference viruses with stabilized HAs retained infectivity, transmissibility, and antigenicity. Therefore, HA stabilization should be prioritized over destabilization in vaccine reference virus selection to reduce mismatches between vaccine and circulating viruses.

Novel Vaccines and Drugs That Target the Surface Glycoproteins of Influenza Viruses, RSV, Parainfluenza Viruses, and SARS-CoV-2.

Newly emerging and seasonal respiratory viruses have a great impact on public health[...]

Swine H1N1 Influenza Virus Variants with Enhanced Polymerase Activity and HA Stability Promote Airborne Transmission in Ferrets.

Understanding how animal influenza A viruses (IAVs) acquire airborne transmissibility in humans and ferrets is needed to prepare for and respond to pandemics. Here, we investigated in ferrets the replication and transmission of swine H1N1 isolates P4 and G15, whose majority population had decreased polymerase activity and poor hemagglutinin (HA) stability, respectively. For both isolates, a minor variant was selected and transmitted in ferrets. Polymerase-enhancing variant PA-S321 airborne-transmitted and propagated in one ferret. HA-stabilizing variant HA1-S210 was selected in all G15-inoculated ferrets and was transmitted by contact and airborne routes. With an efficient polymerase and a stable HA, the purified minor variant G15-HA1-S210 had earlier and higher peak titers in inoculated ferrets and was recovered at a higher frequency after airborne transmission than P4 and G15. Overall, HA stabilization played a more prominent role than polymerase enhancement in the replication and transmission of these viruses in ferrets. The results suggest pandemic risk-assessment studies may benefit from deep sequencing to identify minor variants with human-adapted traits. IMPORTANCE Diverse IAVs circulate in animals, yet few acquire the viral traits needed to start a human pandemic. A stabilized HA and mammalian-adapted polymerase have been shown to promote the adaptation of IAVs to humans and ferrets (the gold-standard model for IAV replication, pathogenicity, and transmissibility). Here, we used swine IAV isolates of the gamma lineage as a model to investigate the importance of HA stability and polymerase activity in promoting replication and transmission in ferrets. These are emerging viruses that bind to both α-2,6- and α-2,3-linked receptors. Using isolates containing mixed populations, a stabilized HA was selected within days in inoculated ferrets. An enhanced polymerase was also selected and propagated after airborne transmission to a ferret. Thus, HA stabilization was a stricter requirement, yet both traits promoted transmissibility. Knowing the viral traits needed for pandemic potential, and the relative importance of each, will help identify emerging viruses of greatest concern.

Relationship between hemagglutinin stability and influenza virus persistence after exposure to low pH or supraphysiological heating.

The hemagglutinin (HA) surface glycoprotein is triggered by endosomal low pH to cause membrane fusion during influenza A virus (IAV) entry yet must remain sufficiently stable to avoid premature activation during virion transit between cells and hosts. HA activation pH and/or virion inactivation pH values less than pH 5.6 are thought to be required for IAV airborne transmissibility and human pandemic potential. To enable higher-throughput screening of emerging IAV strains for "humanized" stability, we developed a luciferase reporter assay that measures the threshold pH at which IAVs are inactivated. The reporter assay yielded results similar to TCID50 assay yet required one-fourth the time and one-tenth the virus. For four A/TN/09 (H1N1) HA mutants and 73 IAVs of varying subtype, virion inactivation pH was compared to HA activation pH and the rate of inactivation during 55°C heating. HA stability values correlated highly with virion acid and thermal stability values for isogenic viruses containing HA point mutations. HA stability also correlated with virion acid stability for human isolates but did not correlate with thermal stability at 55°C, raising doubt in the use of supraphysiological heating assays. Some animal isolates had virion inactivation pH values lower than HA activation pH, suggesting factors beyond HA stability can modulate virion stability. The coupling of HA activation pH and virion inactivation pH, and at a value below 5.6, was associated with human adaptation. This suggests that both virologic properties should be considered in risk assessment algorithms for pandemic potential.

Quantifying dose-, strain-, and tissue-specific kinetics of parainfluenza virus infection.

Human parainfluenza viruses (HPIVs) are a leading cause of acute respiratory infection hospitalization in children, yet little is known about how dose, strain, tissue tropism, and individual heterogeneity affects the processes driving growth and clearance kinetics. Longitudinal measurements are possible by using reporter Sendai viruses, the murine counterpart of HPIV 1, that express luciferase, where the insertion location yields a wild-type (rSeV-luc(M-F*)) or attenuated (rSeV-luc(P-M)) phenotype. Bioluminescence from individual animals suggests that there is a rapid increase in expression followed by a peak, biphasic clearance, and resolution. However, these kinetics vary between individuals and with dose, strain, and whether the infection was initiated in the upper and/or lower respiratory tract. To quantify the differences, we translated the bioluminescence measurements from the nasopharynx, trachea, and lung into viral loads and used a mathematical model together a nonlinear mixed effects approach to define the mechanisms distinguishing each scenario. The results confirmed a higher rate of virus production with the rSeV-luc(M-F*) virus compared to its attenuated counterpart, and suggested that low doses result in disproportionately fewer infected cells. The analyses indicated faster infectivity and infected cell clearance rates in the lung and that higher viral doses, and concomitantly higher infected cell numbers, resulted in more rapid clearance. This parameter was also highly variable amongst individuals, which was particularly evident during infection in the lung. These critical differences provide important insight into distinct HPIV dynamics, and show how bioluminescence data can be combined with quantitative analyses to dissect host-, virus-, and dose-dependent effects.

Sendai Virus-Vectored Vaccines That Express Envelope Glycoproteins of Respiratory Viruses.

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza viruses (HPIVs) are leading causes of respiratory disease in young children, the elderly, and individuals of all ages with immunosuppression. Vaccination strategies against these pneumoviruses and paramyxoviruses are vast in number, yet no licensed vaccines are available. Here, we review development of Sendai virus (SeV), a versatile pediatric vaccine that can (a) serve as a Jennerian vaccine against HPIV1, (b) serve as a recombinant vaccine against HRSV, HPIV2, HPIV3, and HMPV, (c) accommodate foreign genes for viral glycoproteins in multiple intergenic positions, (d) induce durable, mucosal, B-cell, and T-cell immune responses without enhanced immunopathology, (e) protect cotton rats, African green monkeys, and chimpanzees from infection, and (f) be formulated into a vaccine cocktail. Clinical phase I safety trials of SeV have been completed in adults and 3-6-year-old children. Clinical testing of SeVRSV, an HRSV fusion (F) glycoprotein gene recombinant, has also been completed in adults. Positive results from these studies, and collaborative efforts with the National Institutes of Health and the Serum Institute of India assist advanced development of SeV-based vaccines. Prospects are now good for vaccine successes in infants and consequent protection against serious viral disease.

Interplay between H1N1 influenza a virus infection, extracellular and intracellular respiratory tract pH, and host responses in a mouse model.

During influenza A virus (IAV) entry, the hemagglutinin (HA) protein is triggered by endosomal low pH to undergo irreversible structural changes that mediate membrane fusion. HA proteins from different isolates vary in the pH at which they become activated in endosomes or become irreversible inactivated if exposed to extracellular acid. Little is known about extracellular pH in the upper respiratory tracts of mammals, how pH may shift during IAV infection, and its impact on replication of viruses that vary in HA activation pH. Here, we inoculated DBA/2J mice intranasally with A/TN/1-560/2009 (H1N1) (activation pH 5.5) or a mutant containing the destabilizing mutation HA1-Y17H (pH 6.0). We measured the kinetics of extracellular pH during infection using an optical pH-sensitive microsensor probe placed in the naris, nasal sinus, soft palate, and trachea. We also measured intracellular pH of single-cell suspensions of live, primary lung epithelial cells with various wavelength pH-sensitive dyes localized to cell membranes, cytosol, endosomes, secretory vesicles, microtubules, and lysosomes. Infection with either virus decreased extracellular pH and increased intracellular pH. Peak host immune responses were observed at 2 days post infection (DPI) and peak pH changes at 5 DPI. Extracellular and intracellular pH returned to baseline by 7 DPI in mice infected with HA1-Y17H and was restored later in wildtype-infected. Overall, IAV infection altered respiratory tract pH, which in turn modulated replication efficiency. This suggests a virus-host pH feedback loop that may select for IAV strains containing HA proteins of optimal pH stability, which may be approximately pH 5.5 in mice but may differ in other species.

Hemagglutinin Stability and Its Impact on Influenza A Virus Infectivity, Pathogenicity, and Transmissibility in Avians, Mice, Swine, Seals, Ferrets, and Humans.

Genetically diverse influenza A viruses (IAVs) circulate in wild aquatic birds. From this reservoir, IAVs sporadically cause outbreaks, epidemics, and pandemics in wild and domestic avians, wild land and sea mammals, horses, canines, felines, swine, humans, and other species. One molecular trait shown to modulate IAV host range is the stability of the hemagglutinin (HA) surface glycoprotein. The HA protein is the major antigen and during virus entry, this trimeric envelope glycoprotein binds sialic acid-containing receptors before being triggered by endosomal low pH to undergo irreversible structural changes that cause membrane fusion. The HA proteins from different IAV isolates can vary in the pH at which HA protein structural changes are triggered, the protein causes membrane fusion, or outside the cell the virion becomes inactivated. HA activation pH values generally range from pH 4.8 to 6.2. Human-adapted HA proteins tend to have relatively stable HA proteins activated at pH 5.5 or below. Here, studies are reviewed that report HA stability values and investigate the biological impact of variations in HA stability on replication, pathogenicity, and transmissibility in experimental animal models. Overall, a stabilized HA protein appears to be necessary for human pandemic potential and should be considered when assessing human pandemic risk.

Safety and immunogenicity of an intranasal sendai virus-based vaccine for human parainfluenza virus type I and respiratory syncytial virus (SeVRSV) in adults.

SeVRSV is a replication-competent Sendai virus (SeV)-based vaccine carrying the respiratory syncytial virus (RSV) fusion protein (F) gene. Unmanipulated, non-recombinant SeV is a murine parainfluenza virus type 1 (PIV-1) and serves as a Jennerian vaccine for human PIV-1 (hPIV-1). SeV protects African green monkeys (AGM) from infection after hPIV-1 challenge. The recombinant SeVRSV additionally targets RSV and protects AGM from lower respiratory infections after RSV challenge. The present study is the first to report on the safety, viral genome detection, and immunogenicity following SeVRSV vaccination of healthy adults. Seventeen and four healthy adults received intranasal SeVRSV and PBS, respectively, followed by six months of safety monitoring. Virus genome (in nasal wash) and vaccine-specific antibodies (in sera) were monitored for two and four weeks, respectively, post-vaccination. The vaccine was well-tolerated with only mild to moderate reactions that were also present in the placebo group. No severe reactions occurred. As expected, due to preexisting immunity toward hPIV-1 and RSV in adults, vaccine genome detection was transient. There were minimal antibody responses to SeV and negligible responses to RSV F. Results encourage further studies of SeVRSV with progression toward a clinical trial in seronegative children. Abbreviations: AE-adverse event; SAE-serious adverse event; SeV-Sendai virus; RSV-respiratory syncytial virus; PIV-1-parainfluenza virus-type 1; hPIV-1-human parainfluenza virus-type 1; F-RSV fusion protein; SeVRSV-recombinant SeV carrying the RSV F gene; Ab-antibody; MSW-medically significant wheezing; NOCMC-new onset chronic medical condition, mITT-modified Intent to Treat; ALRI-acute lower respiratory tract infection.

HA stabilization promotes replication and transmission of swine H1N1 gamma influenza viruses in ferrets.

Pandemic influenza A viruses can emerge from swine, an intermediate host that supports adaptation of human-preferred receptor-binding specificity by the hemagglutinin (HA) surface antigen. Other HA traits necessary for pandemic potential are poorly understood. For swine influenza viruses isolated in 2009-2016, gamma-clade viruses had less stable HA proteins (activation pH 5.5-5.9) than pandemic clade (pH 5.0-5.5). Gamma-clade viruses replicated to higher levels in mammalian cells than pandemic clade. In ferrets, a model for human adaptation, a relatively stable HA protein (pH 5.5-5.6) was necessary for efficient replication and airborne transmission. The overall airborne transmission frequency in ferrets for four isolates tested was 42%, and isolate G15 airborne transmitted 100% after selection of a variant with a stabilized HA. The results suggest swine influenza viruses containing both a stabilized HA and alpha-2,6 receptor binding in tandem pose greater pandemic risk. Increasing evidence supports adding HA stability to pre-pandemic risk assessment algorithms.

Human Metapneumovirus: A Largely Unrecognized Threat to Human Health.

Human metapneumovirus (HMPV) infects most children by five years of age. The virus can cause both upper and lower respiratory tract disease and can be life threatening. High-risk populations include young children who are exposed to virus for the first time and the elderly. Currently, there is no standard treatment nor licensed vaccine for HMPV, although several attractive vaccine candidates have been developed for pre-clinical studies. A raised awareness of the impact of HMPV on public health is needed to drive research, complete vaccine development, and thereby prevent significant virus-associated morbidities and mortalities worldwide.

Hemagglutinin Stability Regulates H1N1 Influenza Virus Replication and Pathogenicity in Mice by Modulating Type I Interferon Responses in Dendritic Cells.

Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated the mechanisms by which a destabilizing HA mutation, Y17H (activation pH, 6.0), attenuates virus replication and pathogenicity in DBA/2 mice compared to wild-type (WT) virus (activation pH, 5.5). The extracellular lung pH was measured to be near neutral (pH 6.9 to 7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate the Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and RAW 264.7 cells when inoculated at a multiplicity of infection (MOI) of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial cell cultures and attenuated virus replication, virus spread, the severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses.IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations.

Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice.

Reporter viruses provide a powerful tool to study infection, yet incorporating a nonessential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus with NanoLuc luciferase inserted into the 5' end of the PA gene segment grew to titers 10-fold less than those of the wild type in MDCK cells and in DBA/2 mice and was less virulent. For 12 rounds, we propagated DBA/2 lung samples with the highest bioluminescence-to-titer ratios. Every three rounds, we compared in vivo replication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino acid mutations and PB2 noncoding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate that directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-like in vivo fitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.IMPORTANCE Influenza viruses contribute to 290,000 to 650,000 deaths globally each year. Infection is studied in mice to learn how the virus causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene that causes infected cells to light up, scientists could see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza virus infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.

Ferrets as Models for Influenza Virus Transmission Studies and Pandemic Risk Assessments.

The ferret transmission model is extensively used to assess the pandemic potential of emerging influenza viruses, yet experimental conditions and reported results vary among laboratories. Such variation can be a critical consideration when contextualizing results from independent risk-assessment studies of novel and emerging influenza viruses. To streamline interpretation of data generated in different laboratories, we provide a consensus on experimental parameters that define risk-assessment experiments of influenza virus transmissibility, including disclosure of variables known or suspected to contribute to experimental variability in this model, and advocate adoption of more standardized practices. We also discuss current limitations of the ferret transmission model and highlight continued refinements and advances to this model ongoing in laboratories. Understanding, disclosing, and standardizing the critical parameters of ferret transmission studies will improve the comparability and reproducibility of pandemic influenza risk assessment and increase the statistical power and, perhaps, accuracy of this model.

Influenza Hemagglutinin Protein Stability, Activation, and Pandemic Risk.

For decades, hemagglutinin (HA) protein structure and its refolding mechanism have served as a paradigm for understanding protein-mediated membrane fusion. HA trimers are in a high-energy state and are functionally activated by low pH. Over the past decade, HA stability (or the pH at which irreversible conformational changes are triggered) has emerged as an important determinant in influenza virus host range, infectivity, transmissibility, and human pandemic potential. Here, we review HA protein structure, assays to measure its stability, measured HA stability values, residues and mutations that regulate its stability, the effect of HA stability on interspecies adaptation and transmissibility, and mechanistic insights into this process. Most importantly, HA stabilization appears to be necessary for adapting emerging influenza viruses to humans.

Vaccines for the Paramyxoviruses and Pneumoviruses: Successes, Candidates, and Hurdles.

Human parainfluenza viruses (family Paramyxoviridae), human metapneumovirus, and respiratory syncytial virus (family Pneumoviridae) infect most infants and children within the first few years of life and are the etiologic agents for many serious acute respiratory illnesses. These virus infections are also associated with long-term diseases that impact quality of life, including asthma. Despite over a half-century of vaccine research, development, and clinical trials, no vaccine has been licensed to date for the paramyxoviruses or pneumoviruses for the youngest infants. In this study, we describe the recent reclassification of paramyxoviruses and pneumoviruses into distinct families by the International Committee on the Taxonomy of Viruses. We also discuss some past unsuccessful vaccine trials and some currently preferred vaccine strategies. Finally, we discuss hurdles that must be overcome to support successful respiratory virus vaccine development for the youngest children.