Antigenically Diverse Swine Origin H1N1 Variant Influenza Viruses Exhibit Differential Ferret Pathogenesis and Transmission Phenotypes.

Influenza A(H1) viruses circulating in swine represent an emerging virus threat, as zoonotic infections occur sporadically following exposure to swine. A fatal infection caused by an H1N1 variant (H1N1v) virus was detected in a patient with reported exposure to swine and who presented with pneumonia, respiratory failure, and cardiac arrest. To understand the genetic and phenotypic characteristics of the virus, genome sequence analysis, antigenic characterization, and ferret pathogenesis and transmissibility experiments were performed. Antigenic analysis of the virus isolated from the fatal case, A/Ohio/09/2015, demonstrated significant antigenic drift away from the classical swine H1N1 variant viruses and H1N1 pandemic 2009 viruses. A substitution in the H1 hemagglutinin (G155E) was identified that likely impacted antigenicity, and reverse genetics was employed to understand the molecular mechanism of antibody escape. Reversion of the substitution to 155G, in a reverse genetics A/Ohio/09/2015 virus, showed that this residue was central to the loss of hemagglutination inhibition by ferret antisera raised against a prototypical H1N1 pandemic 2009 virus (A/California/07/2009), as well as gamma lineage classical swine H1N1 viruses, demonstrating the importance of this residue for antibody recognition of this H1 lineage. When analyzed in the ferret model, A/Ohio/09/2015 and another H1N1v virus, A/Iowa/39/2015, as well as A/California/07/2009, replicated efficiently in the respiratory tract of ferrets. The two H1N1v viruses transmitted efficiently among cohoused ferrets, but respiratory droplet transmission studies showed that A/California/07/2009 transmitted through the air more efficiently. Preexisting immunity to A/California/07/2009 did not fully protect ferrets from challenge with A/Ohio/09/2015.IMPORTANCE Human infections with classical swine influenza A(H1N1) viruses that circulate in pigs continue to occur in the United States following exposure to swine. To understand the genetic and virologic characteristics of a virus (A/Ohio/09/2015) associated with a fatal infection and a virus associated with a nonfatal infection (A/Iowa/39/2015), we performed genome sequence analysis, antigenic testing, and pathogenicity and transmission studies in a ferret model. Reverse genetics was employed to identify a single antigenic site substitution (HA G155E) responsible for antigenic variation of A/Ohio/09/2015 compared to related classical swine influenza A(H1N1) viruses. Ferrets with preexisting immunity to the pandemic A(H1N1) virus were challenged with A/Ohio/09/2015, demonstrating decreased protection. These data illustrate the potential for currently circulating swine influenza viruses to infect and cause illness in humans with preexisting immunity to H1N1 pandemic 2009 viruses and a need for ongoing risk assessment and development of candidate vaccine viruses for improved pandemic preparedness.

Serial passage in ducks of a low-pathogenic avian influenza virus isolated from a chicken reveals a high mutation rate in the hemagglutinin that is likely due to selection in the host.

A comparative study of the ability of three low-pathogenic avian influenza virus (LPAIV) isolates to be transmitted from duck to duck was performed. Pekin ducks were inoculated with two LPAIV isolates from chickens (A/Ck/PA/13609/93 [H5N2], H5N2-Ck; A/Ck/TX/167280-4/02 [H5N3], H5N3-Ck) and one isolate from a wild bird (A/Mute Swan/ MI/451072/06 [H5N1], H5N1-WB). During the establishment of the passage model, only two viruses (H5N1, H5N2) were able to be transmitted from duck to duck. Transmission of these isolates was dependent on the inoculation dose and route of infection. Analysis of swab samples taken from ducks revealed that the wild-bird isolate, H5N1-WB, was primarily shed via the cloacal route. The chicken isolate, H5N2-Ck, was isolated from cloacal as well as oro-pharyngeal swabs. Analysis of the amino acid sequences of the viral surface glycoproteins showed that the hemagglutinin (HA) of the H5N2-Ck isolate was under a stronger evolutionary pressure than the HA of the H5N1-WB isolate, as indicated by the presence of a larger number of amino acid changes observed during passage. The neuraminidase (NA) of both viruses showed either no (in the case of H5N1-WB) or very few amino acid changes.

Author Correction: Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome.

For the first time, a coding complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabeled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza A virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method with total RNA extracted from the allantoic fluid of influenza rA/Puerto Rico/8/1934 (H1N1) virus infected chicken eggs (EID50 6.8 × 109), we demonstrate successful sequencing of the coding complete influenza A virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza A virus. By utilizing the same methodology one can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle, or other pathogens. This approach also has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

Role of H7 hemagglutinin in murine infectivity of influenza viruses following ocular inoculation.

H7 subtype influenza viruses have demonstrated an ocular tropism in humans, causing conjunctivitis and not respiratory symptoms in many infected individuals. However, the molecular determinants which confer ocular tropism are still poorly understood. Here, we used a murine model of ocular inoculation to demonstrate that H7 influenza viruses are more likely to cause infection following ocular exposure than are non-H7 subtype viruses. We included investigation regarding the potential role of several properties of influenza viruses with murine infectivity following ocular inoculation, including virus lineage, pathogenicity, and HA cleavage site composition. Furthermore, we examined the potential contribution of internal proteins to murine ocular infectivity. These studies establish a link between H7 subtype viruses and the risk of heightened infectivity in a mammalian species following ocular exposure, and support the development of non-traditional inoculation methods and models to best understand the human risk posed by influenza viruses of all subtypes.

Pathogenicity testing of influenza candidate vaccine viruses in the ferret model.

The development of influenza candidate vaccine viruses (CVVs) for pre-pandemic vaccine production represents a critical step in pandemic preparedness. The multiple subtypes and clades of avian or swine origin influenza viruses circulating world-wide at any one time necessitates the continuous generation of CVVs to provide an advanced starting point should a novel zoonotic virus cross the species barrier and cause a pandemic. Furthermore, the evolution and diversity of novel influenza viruses that cause zoonotic infections requires ongoing monitoring and surveillance, and, when a lack of antigenic match between circulating viruses and available CVVs is identified, the production of new CVVs. Pandemic guidelines developed by the WHO Global Influenza Program govern the design and preparation of reverse genetics-derived CVVs, which must undergo numerous safety and quality tests prior to human use. Confirmation of reassortant CVV attenuation of virulence in ferrets relative to wild-type virus represents one of these critical steps, yet there is a paucity of information available regarding the relative degree of attenuation achieved by WHO-recommended CVVs developed against novel viruses with pandemic potential. To better understand the degree of CVV attenuation in the ferret model, we examined the relative virulence of six A/Puerto Rico/8/1934-based CVVs encompassing five different influenza A subtypes (H2N3, H5N1, H5N2, H5N8, and H7N9) compared with the respective wild-type virus in ferrets. Despite varied virulence of wild-type viruses in the ferret, all CVVs examined showed reductions in morbidity and viral shedding in upper respiratory tract tissues. Furthermore, unlike the wild-type counterparts, none of the CVVs spread to extrapulmonary tissues during the acute phase of infection. While the magnitude of virus attenuation varied between virus subtypes, collectively we show the reliable and reproducible attenuation of CVVs that have the A/Puerto Rico/9/1934 backbone in a mammalian model.

A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve.

Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of 'universal' influenza vaccine strategies.

Development of influenza A(H7N9) candidate vaccine viruses with improved hemagglutinin antigen yield in eggs.

BACKGROUND: The emergence of avian influenza A(H7N9) virus in poultry causing zoonotic human infections was reported on March 31, 2013. Development of A(H7N9) candidate vaccine viruses (CVV) for pandemic preparedness purposes was initiated without delay. Candidate vaccine viruses were derived by reverse genetics using the internal genes of A/Puerto/Rico/8/34 (PR8). The resulting A(H7N9) CVVs needed improvement because they had titers and antigen yields that were suboptimal for vaccine manufacturing in eggs, especially in a pandemic situation. METHODS: Two CVVs derived by reverse genetics were serially passaged in embryonated eggs to improve the hemagglutinin (HA) antigen yield. The total viral protein and HA antigen yields of six egg-passaged CVVs were determined by the BCA assay and isotope dilution mass spectrometry (IDMS) analysis, respectively. CVVs were antigenically characterized by hemagglutination inhibition (HI) assays with ferret antisera. RESULTS: Improvement of total viral protein yield was observed for the six egg-passaged CVVs; HA quantification by IDMS indicated approximately a twofold increase in yield of several egg-passaged viruses as compared to that of the parental CVV. Several different amino acid substitutions were identified in the HA of all viruses after serial passage. However, HI tests indicated that the antigenic properties of two CVVs remained unchanged. CONCLUSIONS: If influenza A(H7N9) viruses were to acquire sustained human-to-human transmissibility, the improved HA yield of the egg-passaged CVVs generated in this study could expedite vaccine manufacturing for pandemic mitigation.

Identification of Influenza A/PR/8/34 Donor Viruses Imparting High Hemagglutinin Yields to Candidate Vaccine Viruses in Eggs.

One of the important lessons learned from the 2009 H1N1 pandemic is that a high yield influenza vaccine virus is essential for efficient and timely production of pandemic vaccines in eggs. The current seasonal and pre-pandemic vaccine viruses are generated either by classical reassortment or reverse genetics. Both approaches utilize a high growth virus, generally A/Puerto Rico/8/1934 (PR8), as the donor of all or most of the internal genes, and the wild type virus recommended for inclusion in the vaccine to contribute the hemagglutinin (HA) and neuraminidase (NA) genes encoding the surface glycoproteins. As a result of extensive adaptation through sequential egg passaging, PR8 viruses with different gene sequences and high growth properties have been selected at different laboratories in past decades. The effect of these related but distinct internal PR8 genes on the growth of vaccine viruses in eggs has not been examined previously. Here, we use reverse genetics to analyze systematically the growth and HA antigen yield of reassortant viruses with 3 different PR8 backbones. A panel of 9 different HA/NA gene pairs in combination with each of the 3 different lineages of PR8 internal genes (27 reassortant viruses) was generated to evaluate their performance. Virus and HA yield assays showed that the PR8 internal genes influence HA yields in most subtypes. Although no single PR8 internal gene set outperformed the others in all candidate vaccine viruses, a combination of specific PR8 backbone with individual HA/NA pairs demonstrated improved HA yield and consequently the speed of vaccine production. These findings may be important both for production of seasonal vaccines and for a rapid global vaccine response during a pandemic.