Systems genetics of influenza A virus-infected mice identifies TRIM21 as a critical regulator of pulmonary innate immune response.

Tripartite motif 21 (TRIM21) is a cytosolic Fc receptor that targets antibody-bound, internalized pathogens for destruction. Apart from this intrinsic defense role, TRIM21 is implicated in autoimmune diseases, inflammation, and autophagy. Whether TRIM21 participates in host interactions with influenza A virus (IAV), however, is unknown. By computational modeling of body weight and lung transcriptome data from the BXD parents (C57BL/6 J (B6) and DBA/2 J (D2)) and 41 BXD mouse strains challenged by IAV, we reveal that a Trim21-associated gene network modulates the early host responses to IAV infection. Trim21 transcripts were significantly upregulated in infected mice of both B6 and D2 backgrounds. Its expression was significantly higher in infected D2 than in infected B6 early after infection and significantly correlated with body weight loss. We identified significant trans-eQTL on chromosome 14 that regulates Trim21 expression. Nr1d2 and Il3ra were among the strongest candidate genes. Pathway analysis found Trim21 to be involved in inflammation and immunity related pathways, such as inflammation signaling pathways (TNF, IL-17, and NF-κB), viral detection signaling pathways (NOD-like and RIG-I-like), influenza, and other respiratory viral infections. Knockdown of TRIM21 in human lung epithelial A549 cells significantly augmented IAV-induced expression of IFNB1, IFNL1, CCL5, CXCL10, and IFN-stimulated genes including DDX58 and IFIH1, among others. Our data suggest that a TRIM21-associated gene network is involved in several aspects of inflammation and viral detection mechanisms during IAV infection. We identify and validate TRIM21 as a critical regulator of innate immune responses to IAV in human lung epithelial cells.

Digital PCR to Measure SARS-CoV-2 RNA, Variants, and Outcomes in Youth.

BACKGROUND: The role of SARS-CoV-2 viral load in predicting contagiousness, disease severity, transmissibility, and clinical decision-making continues to be an area of great interest. However, most studies have been in adults and have evaluated SARS-CoV-2 loads using cycle thresholds (Ct) values, which are not standardized preventing consistent interpretation critical to understanding clinical impact and utility. Here, a quantitative SARS-CoV-2 reverse-transcription digital PCR (RT-dPCR) assay normalized to WHO International Units was applied to children at risk of severe disease diagnosed with COVID-19 at St. Jude Children's Research Hospital between March 28, 2020, and January 31, 2022. METHODS: Demographic and clinical information from children, adolescents, and young adults treated at St. Jude Children's Research Hospital were abstracted from medical records. Respiratory samples underwent SARS-CoV-2 RNA quantitation by RT-dPCR targeting N1 and N2 genes, with sequencing to determine the genetic lineage of infecting virus. RESULTS: Four hundred and sixty-two patients aged 0-24 years (median 11 years old) were included during the study period. Most patients were infected by the omicron variant (43.72%), followed by ancestral strain (22.29%), delta (13.20%), and alpha (2.16%). Viral load at presentation ranged from 2.49 to 9.14 log10 IU/mL, and higher viral RNA loads were associated with symptoms (OR 1.32; CI 95% 1.16-1.49) and respiratory disease (OR 1.23; CI 95% 1.07-1.41). Viral load did not differ by SARS-CoV-2 variant, vaccination status, age, or baseline diagnosis. CONCLUSIONS: SARS-CoV-2 RNA loads predict the presence of symptomatic and respiratory diseases. The use of standardized, quantitative methods is feasible, allows for replication, and comparisons across institutions, and has the potential to facilitate consensus quantitative thresholds for risk stratification and treatment.

covidscreen: a web app and R Package for assessing asymptomatic COVID-19 testing strategies.

BACKGROUND: COVID-19 has caused over 305 million infections and nearly 5.5 million deaths globally. With complete eradication unlikely, organizations will need to evaluate their risk and the benefits of mitigation strategies, including the effects of regular asymptomatic testing. We developed a web application and R package that provides estimates and visualizations to aid the assessment of organizational infection risk and testing benefits to facilitate decision-making, which combines internal and community information with malleable assumptions. RESULTS: Our web application, covidscreen, presents estimated values of risk metrics in an intuitive graphical format. It shows the current expected number of active, primarily community-acquired infections among employees in an organization. It calculates and explains the absolute and relative risk reduction of an intervention, relative to the baseline scenario, and shows the value of testing vaccinated and unvaccinated employees. In addition, the web interface allows users to profile risk over a chosen range of input values. The performance and output are illustrated using simulations and a real-world example from the employee testing program of a pediatric oncology specialty hospital. CONCLUSIONS: As the COVID-19 pandemic continues to evolve, covidscreen can assist organizations in making informed decisions about whether to incorporate covid test based screening as part of their on-campus risk-mitigation strategy. The web application, R package, and source code are freely available online (see "Availability of data and materials").

Development of a SARS-CoV-2 Vaccine Candidate Using Plant-Based Manufacturing and a Tobacco Mosaic Virus-like Nano-Particle.

Stable, effective, easy-to-manufacture vaccines are critical to stopping the COVID-19 pandemic resulting from the coronavirus SARS-CoV-2. We constructed a vaccine candidate CoV-RBD121-NP, which is comprised of the SARS-CoV-2 receptor-binding domain (RBD) of the spike glycoprotein (S) fused to a human IgG1 Fc domain (CoV-RBD121) and conjugated to a modified tobacco mosaic virus (TMV) nanoparticle. In vitro, CoV-RBD121 bound to the host virus receptor ACE2 and to the monoclonal antibody CR3022, a neutralizing antibody that blocks S binding to ACE2. The CoV-RBD121-NP vaccine candidate retained key SARS-CoV-2 spike protein epitopes, had consistent manufacturing release properties of safety, identity, and strength, and displayed stable potency when stored for 12 months at 2-8 °C or 22-28 °C. Immunogenicity studies revealed strong antibody responses in C57BL/6 mice with non-adjuvanted or adjuvanted (7909 CpG) formulations. The non-adjuvanted vaccine induced a balanced Th1/Th2 response and antibodies that recognized both the S1 domain and full S protein from SARS2-CoV-2, whereas the adjuvanted vaccine induced a Th1-biased response. Both adjuvanted and non-adjuvanted vaccines induced virus neutralizing titers as measured by three different assays. Collectively, these data showed the production of a stable candidate vaccine for COVID-19 through the association of the SARS-CoV-2 RBD with the TMV-like nanoparticle.

Epigraph hemagglutinin vaccine induces broad cross-reactive immunity against swine H3 influenza virus.

Influenza A virus infection in swine impacts the agricultural industry in addition to its zoonotic potential. Here, we utilize epigraph, a computational algorithm, to design a universal swine H3 influenza vaccine. The epigraph hemagglutinin proteins are delivered using an Adenovirus type 5 vector and are compared to a wild type hemagglutinin and the commercial inactivated vaccine, FluSure. In mice, epigraph vaccination leads to significant cross-reactive antibody and T-cell responses against a diverse panel of swH3 isolates. Epigraph vaccination also reduces weight loss and lung viral titers in mice after challenge with three divergent swH3 viruses. Vaccination studies in swine, the target species for this vaccine, show stronger levels of cross-reactive antibodies and T-cell responses after immunization with the epigraph vaccine compared to the wild type and FluSure vaccines. In both murine and swine models, epigraph vaccination shows superior cross-reactive immunity that should be further investigated as a universal swH3 vaccine.

Histone Deacetylase 6 Knockout Mice Exhibit Higher Susceptibility to Influenza A Virus Infection.

The host innate defence against influenza virus infection is an intricate system with a plethora of antiviral factors involved. We have identified host histone deacetylase 6 (HDAC6) as an anti-influenza virus factor in cultured cells. Consistent with this, we report herein that HDAC6 knockout (KO) mice are more susceptible to influenza virus A/PR/8/1934 (H1N1) infection than their wild type (WT) counterparts. The KO mice lost weight faster than the WT mice and, unlike WT mice, could not recover their original body weight. Consequently, more KO mice succumbed to infection, which corresponded with higher lung viral loads. Conversely, the expression of the critical innate antiviral response genes interferon alpha/beta, CD80, CXCL10 and IL15 was significantly downregulated in KO mouse lungs compared to WT mouse lungs. These data are consistent with the known function of HDAC6 of de-acetylating the retinoic acid inducible gene-I (RIG-I) and activating the host innate antiviral response cascade. Loss of HDAC6 thus leads to a blunted innate response and increased susceptibility of mice to influenza A virus infection.

Optimizing T-705 (favipiravir) treatment of severe influenza B virus infection in the immunocompromised mouse model.

BACKGROUND: Influenza B virus infections remain insufficiently studied and antiviral management in immunocompromised patients is not well defined. The treatment regimens for these high-risk patients, which have elevated risk of severe disease-associated complications, require optimization and can be partly addressed via animal models. METHODS: We examined the efficacy of monotherapy with the RNA-dependent RNA polymerase inhibitor T-705 (favipiravir) in protecting genetically modified, permanently immunocompromised BALB scid mice against lethal infection with B/Brisbane/60/2008 (BR/08) virus. Beginning at 24 h post-infection, BALB scid mice received oral T-705 twice daily (10, 50 or 250 mg/kg/day) for 5 or 10 days. RESULTS: T-705 had a dose-dependent effect on survival after BR/08 challenge, resulting in 100% protection at the highest dosages. With the 5 day regimens, dosages of 50 or 250 mg/kg/day reduced the peak lung viral titres within the treatment window, but could not efficiently clear the virus after completion of treatment. With the 10 day regimens, dosages of 50 or 250 mg/kg/day significantly suppressed virus replication in the lungs, particularly at 45 days post-infection, limiting viral spread and pulmonary pathology. No T-705 regimen decreased virus growth in the nasal turbinates of mice, which potentially contributed to the viral dynamics in the lungs. The susceptibility of influenza B viruses isolated from T-705-treated mice remained comparable to that of viruses from untreated control animals. CONCLUSIONS: T-705 treatment is efficacious against lethal challenge with BR/08 virus in immunocompromised mice. The antiviral benefit was greatest when longer T-705 treatment was combined with higher dosages.

Severe Influenza Is Characterized by Prolonged Immune Activation: Results From the SHIVERS Cohort Study.

Background: The immunologic factors underlying severe influenza are poorly understood. To address this, we compared the immune responses of influenza-confirmed hospitalized individuals with severe acute respiratory illness (SARI) to those of nonhospitalized individuals with influenza-like illness (ILI). Methods: Peripheral blood lymphocytes were collected from 27 patients with ILI and 27 with SARI, at time of enrollment and then 2 weeks later. Innate and adaptive cellular immune responses were assessed by flow cytometry, and serum cytokine levels were assessed by a bead-based assay. Results: During the acute phase, SARI was associated with significantly reduced numbers of circulating myeloid dendritic cells, CD192+ monocytes, and influenza virus-specific CD8+ and CD4+ T cells as compared to ILI. By the convalescent phase, however, most SARI cases displayed continued immune activation characterized by increased numbers of CD16+ monocytes and proliferating, and influenza virus-specific, CD8+ T cells as compared to ILI cases. SARI was also associated with reduced amounts of cytokines that regulate T-cell responses (ie, interleukin 4, interleukin 13, interleukin 12, interleukin 10, and tumor necrosis factor ß) and hematopoiesis (interleukin 3 and granulocyte-macrophage colony-stimulating factor) but increased amounts of a proinflammatory cytokine (tumor necrosis factor α), chemotactic cytokines (MDC, MCP-1, GRO, and fractalkine), and growth-promoting cytokines (PDGFBB/AA, VEGF, and EGF) as compared to ILI. Conclusions: Severe influenza cases showed a delay in the peripheral immune activation that likely led prolonged inflammation, compared with mild influenza cases.

G45R on nonstructural protein 1 of influenza A virus contributes to virulence by increasing the expression of proinflammatory cytokines in mice.

Nonstructural protein 1 (NS1) is a multifunctional protein that is a viral replication enhancer and virulence factor. In this study, we investigated the effect of the amino acid substitution G45R on the NS1 of A/Puerto Rico/8/1934 (H1N1) (G45R/NS1) on viral virulence and host gene expression in a mouse model and the human lung cell line A549. The G45R/NS1 virus had increased virulence by inducing an earlier and robust proinflammatory cytokine response in mice. Mice infected with the G45R/NS1 virus lost more body weight and had lower survival rates than mice infected with the wild type (WT/NS1) virus. Replication of the G45R/NS1 virus was higher than that of the WT/NS1 virus in vitro, but the replication of both viruses was similar in mouse lungs. In A549 cells, the majority of G45R/NS1 protein was localized in the cytoplasm whereas the majority of WT/NS1 protein was localized in the nucleus. Microarray analysis revealed that A549 cells infected with the G45R/NS1 virus had higher expression of genes encoding proteins associated with the innate immune response and cytokine activity than cells infected with the WT/NS1 virus. These data agree with cytokine production observed in mouse lungs. Our findings suggest that G45R on NS1 protein contributes to viral virulence by increasing the expression of inflammatory cytokines early in infection.

Replicating Single-Cycle Adenovirus Vectors Generate Amplified Influenza Vaccine Responses.

Head-to-head comparisons of conventional influenza vaccines with adenovirus (Ad) gene-based vaccines demonstrated that these viral vectors can mediate more potent protection against influenza virus infection in animal models. In most cases, Ad vaccines are engineered to be replication-defective (RD-Ad) vectors. In contrast, replication-competent Ad (RC-Ad) vaccines are markedly more potent but risk causing adenovirus diseases in vaccine recipients and health care workers. To harness antigen gene replication but avoid production of infectious virions, we developed "single-cycle" adenovirus (SC-Ad) vectors. Previous work demonstrated that SC-Ads amplify transgene expression 100-fold and produce markedly stronger and more persistent immune responses than RD-Ad vectors in Syrian hamsters and rhesus macaques. To test them as potential vaccines, we engineered RD and SC versions of adenovirus serotype 6 (Ad6) to express the hemagglutinin (HA) gene from influenza A/PR/8/34 virus. We show here that it takes approximately 33 times less SC-Ad6 than RD-Ad6 to produce equal amounts of HA antigen in vitro SC-Ad produced markedly higher HA binding and hemagglutination inhibition (HAI) titers than RD-Ad in Syrian hamsters. SC-Ad-vaccinated cotton rats had markedly lower influenza titers than RD-Ad-vaccinated animals after challenge with influenza A/PR/8/34 virus. These data suggest that SC-Ads may be more potent vaccine platforms than conventional RD-Ad vectors and may have utility as "needle-free" mucosal vaccines. IMPORTANCE: Most adenovirus vaccines that are being tested are replication-defective adenoviruses (RD-Ads). This work describes testing newer single-cycle adenovirus (SC-Ad) vectors that replicate transgenes to amplify protein production and immune responses. We show that SC-Ads generate markedly more influenza virus hemagglutinin protein and require substantially less vector to generate the same immune responses as RD-Ad vectors. SC-Ads therefore hold promise to be more potent vectors and vaccines than current RD-Ad vectors.

Pandemic Seasonal H1N1 Reassortants Recovered from Patient Material Display a Phenotype Similar to That of the Seasonal Parent.

UNLABELLED: We have previously shown that 11 patients became naturally coinfected with seasonal H1N1 (A/H1N1) and pandemic H1N1 (pdm/H1N1) during the Southern hemisphere winter of 2009 in New Zealand. Reassortment of influenza A viruses is readily observed during coinfection of host animals and in vitro; however, reports of reassortment occurring naturally in humans are rare. Using clinical specimen material, we show reassortment between the two coinfecting viruses occurred with high likelihood directly in one of the previously identified patients. Despite the lack of spread of these reassortants in the community, we did not find them to be attenuated in several model systems for viral replication and virus transmission: multistep growth curves in differentiated human bronchial epithelial cells revealed no growth deficiency in six recovered reassortants compared to A/H1N1 and pdm/H1N1 isolates. Two reassortant viruses were assessed in ferrets and showed transmission to aerosol contacts. This study demonstrates that influenza virus reassortants can arise in naturally coinfected patients. IMPORTANCE: Reassortment of influenza A viruses is an important driver of virus evolution, but little has been done to address humans as hosts for the generation of novel influenza viruses. We show here that multiple reassortant viruses were generated during natural coinfection of a patient with pandemic H1N1 (2009) and seasonal H1N1 influenza A viruses. Though apparently fit in model systems, these reassortants did not become established in the wider population, presumably due to herd immunity against their seasonal H1 antigen.

Centralized Consensus Hemagglutinin Genes Induce Protective Immunity against H1, H3 and H5 Influenza Viruses.

With the exception of the live attenuated influenza vaccine there have been no substantial changes in influenza vaccine strategies since the 1940's. Here we report an alternative vaccine approach that uses Adenovirus-vectored centralized hemagglutinin (HA) genes as vaccine antigens. Consensus H1-Con, H3-Con and H5-Con HA genes were computationally derived. Mice were immunized with Ad vaccines expressing the centralized genes individually. Groups of mice were vaccinated with 1 X 1010, 5 X 107 and 1 X 107 virus particles per mouse to represent high, intermediate and low doses, respectively. 100% of the mice that were vaccinated with the high dose vaccine were protected from heterologous lethal challenges within each subtype. In addition to 100% survival, there were no signs of weight loss and disease in 7 out of 8 groups of high dose vaccinated mice. Lower doses of vaccine showed a reduction of protection in a dose-dependent manner. However, even the lowest dose of vaccine provided significant levels of protection against the divergent influenza strains, especially considering the stringency of the challenge virus. In addition, we found that all doses of H5-Con vaccine were capable of providing complete protection against mortality when challenged with lethal doses of all 3 H5N1 influenza strains. This data demonstrates that centralized H1-Con, H3-Con and H5-Con genes can be effectively used to completely protect mice against many diverse strains of influenza. Therefore, we believe that these Ad-vectored centralized genes could be easily translated into new human vaccines.

Mammalian adaptation of influenza A(H7N9) virus is limited by a narrow genetic bottleneck.

Human infection with avian influenza A(H7N9) virus is associated mainly with the exposure to infected poultry. The factors that allow interspecies transmission but limit human-to-human transmission are unknown. Here we show that A/Anhui/1/2013(H7N9) influenza virus infection of chickens (natural hosts) is asymptomatic and that it generates a high genetic diversity. In contrast, diversity is tightly restricted in infected ferrets, limiting further adaptation to a fully transmissible form. Airborne transmission in ferrets is accompanied by the mutations in PB1, NP and NA genes that reduce viral polymerase and neuraminidase activity. Therefore, while A(H7N9) virus can infect mammals, further adaptation appears to incur a fitness cost. Our results reveal that a tight genetic bottleneck during avian-to-mammalian transmission is a limiting factor in A(H7N9) influenza virus adaptation to mammals. This previously unrecognized biological mechanism limiting species jumps provides a measure of adaptive potential and may serve as a risk assessment tool for pandemic preparedness.

New reassortant and enzootic European swine influenza viruses transmit efficiently through direct contact in the ferret model.

The reverse zoonotic events that introduced the 2009 pandemic influenza virus into pigs have drastically increased the diversity of swine influenza viruses in Europe. The pandemic potential of these novel reassortments is still unclear, necessitating enhanced surveillance of European pigs with additional focus on risk assessment of these new viruses. In this study, four European swine influenza viruses were assessed for their zoonotic potential. Two of the four viruses were enzootic viruses of subtype H1N2 (with avian-like H1) and H3N2, and two were new reassortants, one with avian-like H1 and human-like N2 and one with 2009 pandemic H1 and swine-like N2. All viruses replicated to high titres in nasal wash and nasal turbinate samples from inoculated ferrets and transmitted efficiently by direct contact. Only the H3N2 virus transmitted to naïve ferrets via the airborne route. Growth kinetics using a differentiated human bronchial epithelial cell line showed that all four viruses were able to replicate to high titres. Further, the viruses revealed preferential binding to the 2,6-α-silalylated glycans and investigation of the antiviral susceptibility of the viruses revealed that all were sensitive to neuraminidase inhibitors. These findings suggested that these viruses have the potential to infect humans and further underline the need for continued surveillance as well as biological characterization of new influenza A viruses.

A novel genetic locus linked to pro-inflammatory cytokines after virulent H5N1 virus infection in mice.

BACKGROUND: Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus. RESULTS: Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well (r =0.86-0.96, P <0.0001) with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality. CONCLUSION: A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.

Respiratory transmission of an avian H3N8 influenza virus isolated from a harbour seal.

The ongoing human H7N9 influenza infections highlight the threat of emerging avian influenza viruses. In 2011, an avian H3N8 influenza virus isolated from moribund New England harbour seals was shown to have naturally acquired mutations known to increase the transmissibility of highly pathogenic H5N1 influenza viruses. To elucidate the potential human health threat, here we evaluate a panel of avian H3N8 viruses and find that the harbour seal virus displays increased affinity for mammalian receptors, transmits via respiratory droplets in ferrets and replicates in human lung cells. Analysis of a panel of human sera for H3N8 neutralizing antibodies suggests that there is no population-wide immunity to these viruses. The prevalence of H3N8 viruses in birds and multiple mammalian species including recent isolations from pigs and evidence that it was a past human pandemic virus make the need for surveillance and risk analysis of these viruses of public health importance.

Single-dose monomeric HA subunit vaccine generates full protection from influenza challenge.

Recombinant subunit vaccines are an efficient strategy to meet the demands of a possible influenza pandemic, because of rapid and scalable production. However, vaccines made from recombinant hemagglutinin (HA) subunit protein are often of low potency, requiring high dose or boosting to generate a sustained immune response. We have improved the immunogenicity of a plant-made HA vaccine by chemical conjugation to the surface of the Tobacco mosaic virus (TMV) which is non infectious in mammals. We have previously shown that TMV is taken up by mammalian dendritic cells and is a highly effective antigen carrier. In this work, we tested several TMV-HA conjugation chemistries, and compared immunogenicity in mice as measured by anti-HA IgG titers and hemagglutination inhibition (HAI). Importantly, pre-existing immunity to TMV did not reduce initial or boosted titers. Further optimization included dosing with and without alum or oil-in water adjuvants. Surprisingly, we were able to stimulate potent immunogenicity and HAI titers with a single 15 µg dose of HA as a TMV conjugate. We then evaluated the efficacy of the TMV-HA vaccine in a lethal virus challenge in mice. Our results show that a single dose of the TMV-HA conjugate vaccine is sufficient to generate 50% survival, or 100% survival with adjuvant, compared with 10% survival after vaccination with a commercially available H1N1 vaccine. TMV-HA is an effective dose-sparing influenza vaccine, using a single-step process to rapidly generate large quantities of highly effective flu vaccine from an otherwise low potency HA subunit protein.

Isolation of a novel swine influenza virus from Oklahoma in 2011 which is distantly related to human influenza C viruses.

Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.

Characterization of a porcine intestinal epithelial cell line for influenza virus production.

We have developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrate that SD-PJEC is a cell line of epithelial origin that preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly support the growth of human-origin influenza B viruses. Propagation of swine-origin influenza viruses in these cells results in a rapid growth rate within the first 24 h post-infection and the titres ranged from 4 to 8 log(10) TCID(50) ml(-1). The SD-PJEC cell line was further tested as a potential alternative cell line to Madin-Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine-origin influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log(10) TCID(50) ml(-1), respectively. The availability of this swine-specific cell line represents a more relevant substrate for studies and growth of swine-origin influenza viruses.

Fatal outcome of pandemic H1N1 2009 influenza virus infection is associated with immunopathology and impaired lung repair, not enhanced viral burden, in pregnant mice.

Pandemic A (H1N1) 2009 influenza virus (pH1N1) infection in pregnant women can be severe. The mechanisms that affect infection outcome in this population are not well understood. To address this, pregnant and nonpregnant BALB/c mice were inoculated with the wild-type pH1N1 strain A/California/04/09. To determine whether innate immune responses are associated with severe infection, we measured the innate cells trafficking into the lungs of pregnant versus nonpregnant animals. Increased infiltration of pulmonary neutrophils and macrophages strongly correlated with an elevated mortality in pregnant mice. In agreement with this, the product of nitric oxide (nitrite) and several cytokines associated with recruitment and/or function of these cells were increased in the lungs of pregnant animals. Surprisingly, increased mortality in pregnant mice was not associated with higher virus load because equivalent virus titers and immunohistochemical staining were observed in the nasal cavities or lungs of all mice. To determine whether exacerbated inflammatory responses and elevated cellularity resulted in lung injury, epithelial regeneration was measured. The lungs of pregnant mice exhibited reduced epithelial regeneration, suggesting impaired lung repair. Despite these immunologic alterations, pregnant animals demonstrated equivalent percentages of pulmonary influenza virus-specific CD8(+) T lymphocytes, although they displayed elevated levels of T-regulator lymphocytes (Tregs) in the lung. Also, pregnant mice mounted equal antibody titers in response to virus or immunization with a monovalent inactivated pH1N1 A/California/07/09 vaccine. Therefore, immunopathology likely caused by elevated cellular recruitment is an implicated mechanism of severe pH1N1 infection in pregnant mice.