Influenza viruses typically cause the most severe disease in children and elderly individuals. However, H1N1 viruses disproportionately affected middle-aged adults during the 2013-2014 influenza season. Although H1N1 viruses recently acquired several mutations in the hemagglutinin (HA) glycoprotein, classic serological tests used by surveillance laboratories indicate that these mutations do not change antigenic properties of the virus. Here, we show that one of these mutations is located in a region of HA targeted by antibodies elicited in many middle-aged adults. We find that over 42% of individuals born between 1965 and 1979 possess antibodies that recognize this region of HA. Our findings offer a possible antigenic explanation of why middle-aged adults were highly susceptible to H1N1 viruses during the 2013-2014 influenza season. Our data further suggest that a drifted H1N1 strain should be included in future influenza vaccines to potentially reduce morbidity and mortality in this age group. influenza | antigenic drift | hemagglutinin | antibody | vaccine

Summary Influenza vaccines must be updated regularly because influenza viruses continuously acquire mutations in antibody binding sites of hemagglutinin (HA). The majority of H3N2 strains circulating in the Northern Hemisphere during the 2014–2015 season are antigenically mismatched to the A/Texas/50/2012 H3N2 vaccine strain. Recent H3N2 strains possess several new HA mutations and it is unknown which of these mutations contribute to the 2014–2015 vaccine mismatch. Here, we use reverse-genetics to demonstrate that mutations in HA antigenic site B are primarily responsible for the current mismatch. Sera isolated from vaccinated humans and infected ferrets and sheep had reduced hemagglutination-inhibition and in vitro neutralization titers against reverse-genetics derived viruses possessing mutations in the HA antigenic site B. These data provide an antigenic explanation for the low influenza vaccine efficacy observed during the 2014–2015 influenza season. Further, our data support the World Health Organization’s decision to update the H3N2 component of future vaccine formulations.

The trans-translation pathway for protein tagging and ribosome release plays a critical role for viability and virulence in a wide range of pathogens but is not found in animals. To explore the use of trans-translation as a target for antibiotic development, a highthroughput screen and secondary screening assays were used to identify small molecule inhibitors of the pathway. Compounds that inhibited protein tagging and proteolysis of tagged proteins were recovered from the screen. One of the most active compounds, KKL-35, inhibited the trans-translation tagging reaction with an IC 50 = 0.9 μM. KKL-35 and other compounds identified in the screen exhibited broad-spectrum antibiotic activity, validating trans-translation as a target for drug development. This unique target could play a key role in combating strains of pathogenic bacteria that are resistant to existing antibiotics.antibiotic target | tmRNA | non-stop translation T he increasing prevalence of antibiotic-resistant bacterial pathogens has spurred a search for new pathways that can be targeted for antibiotic development (1, 2). One pathway that has not been exploited is the trans-translation pathway, which resolves nonstop translation complexes. The components of trans-translation have been identified in every sequenced bacterial genome, and mutations in these components affect viability or virulence in a wide range of bacteria (3, 4), suggesting that inhibitors of trans-translation might be effective broad-spectrum antibiotics. In addition, the trans-translation pathway is not found in animals, so specific inhibitors are expected to have few side effects on the host.The purpose of trans-translation is to remove nonstop translation complexes, i.e., translation reactions in which the ribosome has reached the 3′ end of the mRNA without terminating at a stop codon (4-6). These complexes are prevalent in bacteria because bacterial ribosomes do not require any information from the 3′ end of the mRNA to initiate translation, and bacteria lack most of the mechanisms for mRNA proofreading found in eukaryotes (7). Because hydrolysis of peptidyl-tRNA by release factors requires a stop codon in the A site, normal translation termination cannot occur when the ribosome reads to the 3′ end of the mRNA and there is no in-frame stop codon. Transtranslation resolves nonstop translation complexes using a ribonucleoprotein complex containing transfer-messenger RNA (tmRNA) and the small protein SmpB (4-6). tmRNA-SmpB recognizes nonstop translation complexes and enters the ribosomal A site mimicking a tRNA (8). The nascent polypeptide is transferred to tmRNA, and a specialized reading frame within tmRNA is inserted into the mRNA channel (4-6). Translation resumes using this sequence as a message and terminates at a stop codon at the end of the reading frame, releasing the ribosome and a tagged protein (4-6). The tag sequence is recognized by proteases, and the tagged protein is rapidly degraded (9-12). The net reaction of trans-translation is the removal of all components o...

Influenza virus infections are responsible for more than 250,000 deaths annually. Influenza virus isolation, propagation, and characterization protocols are critical for completing reproducible basic research studies and for generating vaccine seed stocks. Detailed protocols for the isolation and identification of influenza virus have been recently reported (Eisfeld et al., 2014). However, there are few standardized protocols focused on the propagation and characterization of viral isolates, and as a result, viruses propagated in different conditions in different laboratories often have distinct in vitro and in vivo characteristics. Here, we focus on influenza A virus propagation and characterization in the laboratory taking into consideration the overall quality and composition of the virus stock to achieve consistency in virus yield, virulence, and immunostimulatory activity.