Mammalian Influenza Research Articles

Developments and current challenges in the process of cell culture-based seasonal influenza vaccine manufacture in Japan.

Seasonal influenza is an acute respiratory infection primarily caused by influenza A and B viruses, which circulate annually and cause substantial morbidity and mortality worldwide. Annual influenza vaccination is currently the most effective measure for preventing influenza and greatly reduces the risk of disease severity and the incidence of complications and death. Annual seasonal influenza vaccines are traditionally produced in Japan and many other countries using viruses propagated in embryonated chicken eggs. However, at present, the effectiveness of the seasonal influenza vaccines has some significant limitations, partly because of egg-adaptive mutations in the antigenic sites of the influenza virus haemagglutinin, which are caused by the continued evolution of seasonal influenza viruses. To overcome the limitations of egg-based influenza vaccine production, a mammalian cell culture-based influenza vaccine production system has been developed in Japan in the past decade as an alternative to the current production method. In this review, I have summarised the progress in the development of cell-based seasonal influenza vaccines and discussed the technological challenges encountered in the development of influenza vaccines.

Re-emergence of canine influenza in the United States of America; a call for One Health approach; a letter to the editor

Re-emergence of canine influenza in the United States of America; a call for One Health approach; a letter to the editor

Infection Studies with Airway Organoids from Carollia perspicillata Indicate That the Respiratory Epithelium Is Not a Barrier for Interspecies Transmission of Influenza Viruses.

Bats are a natural reservoir for many viruses and are considered to play an important role in the interspecies transmission of viruses. To analyze the susceptibility of bat airway cells to infection by viruses of other mammalian species, we developed an airway organoid culture model derived from airways of Carollia perspicillata. Application of specific antibodies for fluorescent staining indicated that the cell composition of organoids resembled those of bat trachea and lungs as determined by immunohistochemistry. Infection studies indicated that Carollia perspicillata bat airway organoids (AOs) from the trachea or the lung are highly susceptible to infection by two different porcine influenza A viruses. The bat AOs were also used to develop an air-liquid interface (ALI) culture system of filter-grown epithelial cells. Infection of these cells showed the same characteristics, including lower virulence and enhanced replication and release of the H1N1/2006 virus compared to infection with H3N2/2007. These observations agreed with the results obtained by infection of porcine ALI cultures with these two virus strains. Interestingly, lectin staining indicated that bat airway cells only contain a small amount of alpha 2,6-linked sialic acid, the preferred receptor determinant for mammalian influenza A viruses. In contrast, large amounts of alpha 2,3-linked sialic acid, the preferred receptor determinant for avian influenza viruses, are present in bat airway epithelial cells. Therefore, bat airway cells may be susceptible not only to mammalian but also to avian influenza viruses. Our culture models, which can be extended to other parts of the airways and to other species, provide a promising tool to analyze virus infectivity and the transmission of viruses both from bats to other species and from other species to bats. IMPORTANCE We developed an organoid culture system derived from the airways of the bat species Carollia perspicillata. Using this cell system, we showed that the airway epithelium of these bats is highly susceptible to infection by influenza viruses of other mammalian species and thus is not a barrier for interspecies transmission. These organoids provide an almost unlimited supply of airway epithelial cells that can be used to generate well-differentiated epithelial cells and perform infection studies. The establishment of the organoid model required only three animals, and can be extended to other epithelia (nose, intestine) as well as to other species (bat and other animal species). Therefore, organoids promise to be a valuable tool for future zoonosis research on the interspecies transmission of viruses (e.g., bat → intermediate host → human).

Comparative Susceptibility of Madin-Darby Canine Kidney (MDCK) Derived Cell Lines for Isolation of Swine Origin Influenza A Viruses from Different Clinical Specimens.

Madin–Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75–31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5–7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-β, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples.

Cellular and humoral immune responses in adults receiving an inactivated mammalian cell-based influenza vaccine

Abstract Current evaluation of influenza vaccines focuses mainly on humoral response to surface hemagglutinin (HA), although both CD4 and CD8 T cell responses have been reported, to both HA and other viral proteins. We conducted a comprehensive functional analysis on the pre-existing and vaccine-induced immune responses to Flucelvax, a tetravalent mammalian cell-based influenza vaccine which in addition to HA contains additional viral proteins (NA, M1, M2, NP, NEP, NS1, PA, PB1, and PB2). Longitudinal samples of peripheral blood mononuclear cells from a cohort of 10 participants, were stimulated by pools of peptides overlapping individual influenza viral protein components. We examined both humoral and cellular responses, intra-vaccine interference, type-specific immunodominance patterns of responses and cytokine polarization. In addition to serological responses, our results demonstrated vaccine-induced CD4 and CD8 responses that were still present after 90 days post vaccination, a balanced immunogenicity across all 4 included viral strains, as well as unchanged type-specific immunodominance patterns and cytokine polarization post-vaccination. Our results demonstrated distinct CD4 reactivities mostly directed to HA/NA (neuraminidase) proteins in Influenza B strains, but similar CD8 responses to both influenza A and B viruses preferentially targeting the more conserved core viral proteins. The present study suggests that Flucelvax induces, both humoral, CD4 and CD8 cellular immunity, balanced across four different influenza strains and targeting HA and multiple additional viral antigens.

Mammalian and Avian Host Cell Influenza A Restriction Factors

The threat of a new influenza pandemic is real. With past pandemics claiming millions of lives, finding new ways to combat this virus is essential. Host cells have developed a multi-modular system to detect incoming pathogens, a phenomenon called sensing. The signaling cascade triggered by sensing subsequently induces protection for themselves and their surrounding neighbors, termed interferon (IFN) response. This response induces the upregulation of hundreds of interferon-stimulated genes (ISGs), including antiviral effectors, establishing an antiviral state. As well as the antiviral proteins induced through the IFN system, cells also possess a so-called intrinsic immunity, constituted of antiviral proteins that are constitutively expressed, creating a first barrier preceding the induction of the interferon system. All these combined antiviral effectors inhibit the virus at various stages of the viral lifecycle, using a wide array of mechanisms. Here, we provide a review of mammalian and avian influenza A restriction factors, detailing their mechanism of action and in vivo relevance, when known. Understanding their mode of action might help pave the way for the development of new influenza treatments, which are absolutely required if we want to be prepared to face a new pandemic.

A unique feature of swine ANP32A provides susceptibility to avian influenza virus infection in pigs.

Both the replication and transcription of the influenza virus are catalyzed by the viral polymerase complex. The polymerases of most avian influenza A viruses have poor performance in mammalian cells, which is considered to be one of the important species barriers. Pigs have been long considered as important intermediate hosts for interspecies transmission of the avian influenza virus, because of their susceptibility to infection with both avian and mammalian influenza viruses. However, the molecular basis of influenza polymerase adaptation in pigs remains largely unknown. ANP32A and ANP32B proteins have been identified as playing fundamental roles in influenza virus replication and host range determination. In this study, we found that swine ANP32A (swANP32A), unlike swine ANP32B or other mammalian ANP32A or B, shows stronger supporting activity to avian viral polymerase. Knockout of ANP32A in pig cells PK15 dramatically reduced avian influenza polymerase activity and viral infectivity, suggesting a unique feature of swANP32A in supporting avian influenza viral polymerase. This species-specific activity is mapped to two key sites, 106V and 156S, in swANP32A. Interestingly, the amino acid 106V is unique to pigs among all the vertebrate species studied, and when combined with 156S, exhibits positive epistasis in pigs. Mutation of 106V and 156S to the signature found in ANP32As from other mammalian species weakened the interaction between swANP32A and chicken viral polymerase, and reduced polymerase activity. Understanding the molecular basis of ANP32 proteins may help to discover new antiviral targets and design avian influenza resistant genome edited pigs.

Avian Influenza Virus Isolation, Propagation, and Titration in Embryonated Chicken Eggs.

Avian influenza virus and some mammalian influenza A viruses can be isolated, propagated, and titrated in embryonated chicken eggs (ECEs). Most sample types can be accommodated in ECE culture with appropriate processing. Isolation may also be accomplished in cell culture, and if a mammalian lineage influenza A is expected to be in an avian sample, for example swine influenza in turkey specimens, mammalian cell may be preferable. Culture in ECEs is highly sensitive but is not specific for influenza A, which may be an advantage because a sample may be screened for several agents at once. Once an agent is isolated in culture, the presence of influenza viruses can be confirmed by hemagglutination inhibition assay, antigen immunoassay, agar gel immunodiffusion assay, or RT-PCR. Finally, ECEs may be used to propagate and titrate an avian influenza virus.

Generation of highly productive and mammalian nonpathogenic recombinant H9N2 avian influenza viruses by optimization of 3’end promoter and NS genome

We developed A/PR/8/34 (PR8) virus-based reverse genetics system in which six internal genes of PR8 and attenuated hemagglutinin and intact neuraminidase genes of field avian influenza viruses (AIVs) have been used for the generation of highly productive recombinant vaccine strains. The 6 + 2 recombinant vaccine strains can induce protective humoral immunity against intended field AIVs; however, the epitopes of B and T cells encoded by internal genes may be important for heterosubtypic protection. Therefore, it is advantageous to use homologous internal genes of field AIVs for recombinant vaccine strains. However, the rescue of recombinant viruses having whole internal genes of field AIVs by the PR8-based reverse genetics system was unsuccessful in some cases. Although partial replacement of an internal gene has been successful for generation of highly productive and mammalian nonpathogenic recombinant viruses, complete replacement of internal genes may be more favorable. In this study, we successfully generated complete recombinant H9N2 AIVs possessing 8 genomes of H9N2 AIVs by optimal combinations of 3’ end promoter sequences of polymerase genomes, and a NS genome. All the generated recombinant viruses showed highly productive and mammalian nonpathogenic traits but some of them showed much higher virus titers in embryonated chicken eggs. Additionally, we found the same mutations of NS1 gene determined pathogenicity of AIVs in chicken embryos as well as mammals. Thus, the 3’ end promoter optimization, and highly productive and mammalian nonpathogenic internal genes may be useful to develop vaccines against AIVs.

Avian influenza: the role of the pig and public health implications

Avian influenza: the role of the pig and public health implications

Successes and Short Comings in Four Years of an International External Quality Assurance Program for Animal Influenza Surveillance.

The US National institutes of Health-Centers of Excellence for Influenza Research and Surveillance is a research consortium that funds numerous labs worldwide to conduct influenza A surveillance in diverse animal species. There is no harmonization of testing procedures among these labs; therefore an external quality assurance (EQA) program was implemented to evaluate testing accuracy among labs in the program in 2012. Accurate detection of novel influenza A variants is crucial because of the broad host range and potentially high virulence of the virus in diverse species. Two molecular detection sample sets and 2 serology sample sets (one with avian origin isolates, and one with mammalian origin isolates each) were made available at approximately six month intervals. Participating labs tested the material in accordance with their own protocols. During a five year period a total of 41 labs from 23 countries ordered a total of 132 avian molecular, 121 mammalian molecular and 90 serology sample sets. Testing was completed by 111 individuals. Detection of type A influenza by RT-PCR was reliable with a pass rate (80% or greater agreement with expected results) of 86.6% for avian and 86.2% for mammalian origin isolates. However, identification of subtype by RT-PCR was relatively poor with 54.1% and 75.9% accuracy for avian and mammalian influenza isolates respectively. Serological testing had an overall pass rate of 86.9% and 22/23 labs used commercial ELISA kits. Based on the results of this EQA program six labs modified their procedures to improve accuracy and one lab identified an unknown equipment problem. These data represent the successful implementation of an international EQA program for an infectious disease; insights into the logistics and test design are also discussed.

Analysis of influenza A viruses from gulls: An evaluation of inter-regional movements and interactions with other avian and mammalian influenza A viruses

AbstractBirds, including members of the families Anatidae (waterfowl) and Laridae (gulls and terns), serve as the major reservoir of influenza A viruses (IAVs). The ecogeographic contributions of gulls to global IAV dynamics, in terms of geographic scale and virus movements, are important and are distinct from those of waterfowl. Gulls primarily carry the H13 and H16 subtypes, yet can be infected by additional subtypes. Also, gulls are frequently infected by IAVs that contain mixtures of genes from different geographic phylogenetic lineages (e.g. North American and Eurasian). The present analysis examines a variety of viruses isolated from gulls and terns across the world that exhibit particularly high phylogenetic affinities to viruses found in other hosts. This illustrates the potential for gulls to act as highly pathogenic virus carriers, disseminators of viruses over long distances, and contributors in the genesis of pandemic strains. The historical evolution of an entirely Eurasian gull virus isolate...

Susceptibility of swine to H5 and H7 low pathogenic avian influenza viruses

BackgroundThe ability of pigs to become infected with low pathogenic avian influenza (LPAI) viruses and then generate mammalian adaptable influenza A viruses is difficult to determine. Yet, it is an important link to understanding any relationship between LPAI virus ecology and possible epidemics among swine and/or humans.ObjectivesAssess susceptibility of pigs to LPAI viruses found within the United States and their direct contact transmission potential.MethodsPigs were inoculated with one of ten H5 or H7 LPAI viruses selected from seven different bird species to test infectivity, virulence, pathogenesis, and potential to transmit virus to contact pigs through histological, RRT‐PCR and seroconversion data.ResultsAlthough pigs were susceptible to infection with each of the LPAI viruses, no clinical disease was recognized in any pig. During the acute phase of the infection, minor pulmonary lesions were found in some pigs and one or more pigs in each group were RRT‐PCR‐positive in the lower respiratory tract, but no virus was detected in upper respiratory tract (negative nasal swabs). Except for one group, one or more pigs in each LPAI group developed antibody. No LPAI viruses transmitted to contact pigs.Conclusions LPAI strains from various bird populations within the United States are capable of infecting pigs. Although adaptability and transmission of individual strains seem unlikely, the subclinical nature of the infections demonstrates the need to improve sampling and testing methods to more accurately measure incidence of LPAI virus infection in pigs, and their potential role in human‐zoonotic LPAI virus dynamics.

A North American H7N3 Influenza Virus Supports Reassortment with 2009 Pandemic H1N1 and Induces Disease in Mice without Prior Adaptation.

Reassortment between H5 or H9 subtype avian and mammalian influenza A viruses (IAV) can generate a novel virus that causes disease and transmits between mammals. Such information is currently not available for H7 subtype viruses. We evaluated the ability of a low-pathogenicity North American avian H7N3 virus (A/shorebird/Delaware/22/2006) to reassort with mammalian or avian viruses using a plasmid-based competition assay. In addition to genome segments derived from an avian H7N9 virus, the H7N3 virus reassorted efficiently with the PB2, NA, and M segments from the 2009 pandemic H1N1 (PH1N1) virus.In vitro and in vivo evaluation of the H7N3:PH1N1 (7 + 1) reassortant viruses revealed that the PB2, NA, or M segments from PH1N1 largely do not attenuate the H7N3 virus, whereas the PB1, PA, NP, or NS genome segments from PH1N1 do. Additionally, we assessed the functionality of the H7N3:PH1N1 7 + 1 reassortant viruses by measuring the inflammatory response in vivo We found that infection with wild-type H7N3 resulted in increased inflammatory cytokine production relative to that seen with the PH1N1 strain and that the increase was further exacerbated by substitution of PH1N1 PB2 but not NA or M. Finally, we assessed if any adaptations occurred in the individually substituted segments after in vivo inoculation and found no mutations, suggesting that PH1N1 PB2, NA, and M are genetically stable in the background of this H7N3 virus. Taking the data together, we demonstrate that a North American avian H7N3 IAV is genetically and functionally compatible with multiple gene segments from the 2009 pandemic influenza virus strain without prior adaptation. The 2009 pandemic H1N1 virus continues to circulate and reassort with other influenza viruses, creating novel viruses with increased replication and transmission potential in humans. Previous studies have found that this virus can also reassort with H5N1 and H9N2 avian influenza viruses. We now show that several genome segments of the 2009 H1N1 virus are also highly compatible with a low-pathogenicity avian H7N3 virus and that these reassortant viruses are stable and not attenuated in an animal model. These results highlight the potential for reassortment of H1N1 viruses with avian influenza virus and emphasize the need for continued surveillance of influenza viruses in areas of cocirculation between avian, human, and swine viruses.

Early apoptosis of porcine alveolar macrophages limits avian influenza virus replication and pro-inflammatory dysregulation.

Pigs are evidently more resistant to avian than swine influenza A viruses, mediated in part through frontline epithelial cells and alveolar macrophages (AM). Although porcine AM (PAM) are crucial in influenza virus control, their mode of control is unclear. To gain insight into the possible role of PAM in the mediation of avian influenza virus resistance, we compared the host effects and replication of two avian (H2N3 and H6N1) and three mammalian (swine H1N1, human H1N1 and pandemic H1N1) influenza viruses in PAM. We found that PAM were readily susceptible to initial infection with all five avian and mammalian influenza viruses but only avian viruses caused early and extensive apoptosis (by 6 h of infection) resulting in reduced virus progeny and moderated pro-inflammation. Full length viral PB1-F2 present only in avian influenza viruses is a virulence factor that targets AM for mitochondrial-associated apoptotic cell death. With the use of reverse genetics on an avian H5N1 virus, we found that full length PB1-F2 contributed to increased apoptosis and pro-inflammation but not to reduced virus replication. Taken together, we propose that early apoptosis of PAM limits the spread of avian influenza viruses and that PB1-F2 could play a contributory role in the process.

Poly I:C adjuvanted inactivated swine influenza vaccine induces heterologous protective immunity in pigs

Swine influenza is widely prevalent in swine herds in North America and Europe causing enormous economic losses and a public health threat. Pigs can be infected by both avian and mammalian influenza viruses and are sources of generation of reassortant influenza viruses capable of causing pandemics in humans. Current commercial vaccines provide satisfactory immunity against homologous viruses; however, protection against heterologous viruses is not adequate. In this study, we evaluated the protective efficacy of an intranasal Poly I:C adjuvanted UV inactivated bivalent swine influenza vaccine consisting of Swine/OH/24366/07 H1N1 and Swine/CO/99 H3N2, referred as PAV, in maternal antibody positive pigs against an antigenic variant and a heterologous swine influenza virus challenge. Groups of three-week-old commercial-grade pigs were immunized intranasally with PAV or a commercial vaccine (CV) twice at 2 weeks intervals. Three weeks after the second immunization, pigs were challenged with the antigenic variant Swine/MN/08 H1N1 (MN08) and the heterologous Swine/NC/10 H1N2 (NC10) influenza virus. Antibodies in serum and respiratory tract, lung lesions, virus shedding in nasal secretions and virus load in lungs were assessed. Intranasal administration of PAV induced challenge viruses specific-hemagglutination inhibition- and IgG antibodies in the serum and IgA and IgG antibodies in the respiratory tract. Importantly, intranasal administration of PAV provided protection against the antigenic variant MN08 and the heterologous NC10 swine influenza viruses as evidenced by significant reductions in lung virus load, gross lung lesions and significantly reduced shedding of challenge viruses in nasal secretions. These results indicate that Poly I:C or its homologues may be effective as vaccine adjuvants capable of generating cross-protective immunity against antigenic variants/heterologous swine influenza viruses in pigs.

Influenza viruses en route from birds to man.

Human and other mammalian influenza viruses emerge from a large gene pool provided by avian influenza viruses. Two recent studies (Watanabe et al., 2014; Linster et al., 2014) show that adaptation to a mammalian host depends on a limited number of mutations that allow airborne transmission, a specific trait of the mammalian viruses.

Comprehensive Sequence Analysis of HA Protein of H7 Subtype Avian Influenza Viruses: An Emphasis on Mutations in Novel H7N9 Viruses

ABSTRACT: Aim: H7 avian influenza viruses pose a major public health threat raising concerns regarding their pandemic potential, especially after the recent outbreak in China of H7N9 subtype viruses. The objective was to gain insight into the geographical and host-wise distribution of H7 subtype viruses, and to understand molecular determinants responsible for their adaptation in humans. Materials & methods: In this study we carried out a global comprehensive analysis of 1749 HA sequences belonging to the H7 subtype available in the Global Initiative on Sharing All Influenza Data (GISAID) EpiFlu™ database. We also analyzed full-genome sequences of the 27 influenza strains belonging to the H7N9 subtype isolated recently from China. Results: Most of the H7 strains were from North America (749) followed by in Europe (659) and Asia (284). The majority of the sequences belonged to the H7N7 subtype (524) followed by H7N3 (440) and H7N2 (411), while 107 belonged to H7N9. Comparison of HA sequences of H7 viruses isolated from humans showed the presence of mutations and determinants that could have played a pivotal role in avian-to-human transmission and adaptability in humans. Mutational analysis of all the segments of the recent H7N9 viruses isolated from humans in China revealed that these viruses possessed several characteristic features of mammalian influenza viruses. Conclusion: H7 viruses, irrespective of being of high or low pathogenicity have a propensity to adapt to humans causing mild to severe infections. These viruses have signature mutations that have been associated with interspecies transmission and human adaptability, raising concerns regarding their pandemic potential.

Avian influenza virus isolation, propagation, and titration in embryonated chicken eggs.

Avian influenza virus and some mammalian influenza A viruses are usually isolated, propagated, and titrated in embryonated chicken eggs (ECE). Most any sample type can be accommodated for culture with appropriate processing. Isolation may also be accomplished in cell culture particularly if mammalian lineage isolates are suspected, for example, swine influenza in turkey specimens. Culture is highly sensitive, but is not specific for influenza A, which may be an advantage because a sample may be screened for several agents at once. Once an agent is isolated in culture, the presence of influenza viruses is confirmed with any of several assays. The methods for propagating virus isolates in ECE are described.

The hemagglutinin: a determinant of pathogenicity.

The hemagglutinin (HA) is a prime determinant of the pathogenicity of influenza A viruses. It initiates infection by binding to cell surface receptors and by inducing membrane fusion. The fusion capacity of HA depends on cleavage activation by host proteases, and it has long been known that highly pathogenic avian influenza viruses displaying a multibasic cleavage site differ in protease sensitivity from low pathogenic avian and mammalian influenza viruses with a monobasic cleavage site. Evidence is increasing that there are also variations in proteolytic activation among the viruses with a monobasic cleavage site, and several proteases have been identified recently that activate these viruses in a natural setting. Differences in protease sensitivity of HA and in tissue specificity of the enzymes are important determinants for virus tropism in the respiratory tract and for systemic spread of infection. Protease inhibitors that interfere with cleavage activation have the potential to be used for antiviral therapy and attenuated viruses have been generated by mutation of the cleavage site that can be used for the development of inactivated and live vaccines. It has long been known that human and avian influenza viruses differ in their specificity for sialic acid-containing cell receptors, and it is now clear that human tissues contain also receptors for avian viruses. Differences in receptor-binding specificity of seasonal and zoonotic viruses and differential expression of receptors for these viruses in the human respiratory tract account, at least partially, for the severity of disease. Receptor binding and fusion activation are modulated by HA glycosylation, and interaction of the glycans of HA with cellular lectins also affects virus infectivity. Interestingly, some of the mechanisms underlying pathogenicity are determinants of host range and transmissibility, as well.