Virology-the path forward.

In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.

Avian reovirus: a furious and fast evolving pathogen.

Avian reoviruses (ARVs) have a significant economic impact on the poultry industry, affecting commercial and backyard flocks. Spread feco-orally, or vertically, many do not cause morbidity, but pathogenic strains can contribute to several diseases, including tenosynovitis/arthritis, which is clinically the most significant. The last decade has seen a surge in cases in the US, and due to ongoing evolution, seven genotypic clusters have now been identified. Control efforts include strict biosecurity and vaccination with commercial and autogenous vaccines. Research priorities include improving understanding of pathogenesis and developing new vaccines guided by ongoing molecular and serologic surveillance.

CD4+TGFß+ cells infiltrated the bursa of Fabricius following IBDV infection, and correlated with a delayed viral clearance, but did not correlate with disease severity, or immunosuppression.

Introduction: Infectious Bursal Disease Virus (IBDV) causes immunosuppression in chickens. While B-cell destruction is the main cause of humoral immunosuppression, bursal T cells from IBDV-infected birds have been reported to inhibit the mitogenic response of splenocytes, indicating that some T cell subsets in the infected bursa have immunomodulatory activities. CD4+CD25+TGFß+ cells have been recently described in chickens that have immunoregulatory properties and play a role in the pathogenesis of Marek's Disease Virus. Methods: To evaluate if CD4+CD25+TGFß+ cells infiltrated the bursa of Fabricius (BF) following IBDV infection, and influenced the outcome of infection, birds were inoculated at either 2 days or 2 weeks of age with vaccine strain (228E), classic field strain (F52/70), or PBS (mock), and bursal cell populations were quantified by flow cytometry. Results: Both 228E and F52/70 led to atrophy of the BF, a significant reduction of Bu1+-B cells, and a significant increase in CD4+ and CD8α+ T cells in the BF, but only F52/70 caused suppression of immune responses to a test antigen in younger birds, and clinical signs in older birds. Virus was cleared from the BF more rapidly in younger birds than older birds. An infiltration of CD4+CD25+T cells into the BF, and elevated expression of bursal TGFß-1+ mRNA was observed at all time points following infection, irrespective of the strain or age of the birds, but CD4+TGFß+cells and CD4+CD25+TGFß+ cells only appeared in the BF at 28 dpi in younger birds. In older birds, CD4+TGFß+ cells and CD4+CD25+TGFß+ cells were present at earlier time points, from 7dpi following 228E infection, and from 14 and 28 dpi following F52/70 infection, respectively. Discussion: Our data suggest that an earlier infiltration of CD4+TGFß+ cells into the BF correlated with a delayed clearance of virus. However, the influx of CD4+TGFß+ cells and CD4+CD25+TGFß+ into the BF did not correlate with increased pathogenicity, or immunosuppression.

Genetic diversity, recombination and cross-species transmission of a waterbird gammacoronavirus in the wild.

Viruses emerging from wildlife can cause outbreaks in humans and domesticated animals. Predicting the emergence of future pathogens and mitigating their impacts requires an understanding of what shapes virus diversity and dynamics in wildlife reservoirs. In order to better understand coronavirus ecology in wild species, we sampled birds within a coastal freshwater lagoon habitat across 5 years, focussing on a large population of mute swans (Cygnus olor) and the diverse species that they interact with. We discovered and characterised the full genome of a divergent gammacoronavirus belonging to the Goose coronavirus CB17 species. We investigated the genetic diversity and dynamics of this gammacoronavirus using untargeted metagenomic sequencing of 223 faecal samples from swans of known age and sex, and RT-PCR screening of 1632 additional bird samples. The virus circulated persistently within the bird community; virus prevalence in mute swans exhibited seasonal variations, but did not change with swan age-class or epidemiological year. One whole genome was fully characterised, and revealed that the virus originated from a recombination event involving an undescribed gammacoronavirus species. Multiple lineages of this gammacoronavirus co-circulated within our study population. Viruses from this species have recently been detected in aquatic birds from both the Anatidae and Rallidae families, implying that host species habitat sharing may be important in shaping virus host range. As the host range of the Goose coronavirus CB17 species is not limited to geese, we propose that this species name should be updated to 'Waterbird gammacoronavirus 1'. Non-invasive sampling of bird coronaviruses may provide a tractable model system for understanding the evolutionary and cross-species dynamics of coronaviruses.

The Formation and Function of Birnaviridae Virus Factories.

The use of infectious bursal disease virus (IBDV) reverse genetics to engineer tagged reporter viruses has revealed that the virus factories (VFs) of the Birnaviridae family are biomolecular condensates that show properties consistent with liquid-liquid phase separation (LLPS). Although the VFs are not bound by membranes, it is currently thought that viral protein 3 (VP3) initially nucleates the formation of the VF on the cytoplasmic leaflet of early endosomal membranes, and likely drives LLPS. In addition to VP3, IBDV VFs contain VP1 (the viral polymerase) and the dsRNA genome, and they are the sites of de novo viral RNA synthesis. Cellular proteins are also recruited to the VFs, which are likely to provide an optimal environment for viral replication; the VFs grow due to the synthesis of the viral components, the recruitment of other proteins, and the coalescence of multiple VFs in the cytoplasm. Here, we review what is currently known about the formation, properties, composition, and processes of these structures. Many open questions remain regarding the biophysical nature of the VFs, as well as the roles they play in replication, translation, virion assembly, viral genome partitioning, and in modulating cellular processes.

An Infectious Bursal Disease Virus (IBDV) Reverse Genetics Rescue System and Neutralization Assay in Chicken B Cells.

Infectious bursal disease virus (IBDV) is a major threat to the productivity of the poultry industry due to morbidity, mortality, and immunosuppression that exacerbates secondary infections and reduces the efficacy of vaccination programs. Field strains of IBDV have a preferred tropism for chicken B cells, the majority of which reside in the bursa of Fabricius (BF). IBDV adaptation to adherent cell culture is associated with mutations altering amino acids in the hypervariable region (HVR) of the capsid protein, which affects immunogenicity and virulence. Until recently, this has limited both the application of reverse genetics systems for engineering molecular clones, and the use of in vitro neutralization assays, to cell-culture-adapted strains of IBDV. Here, we describe the rescue of molecular clones of IBDV containing the HVR from diverse field strains, along with a neutralization assay to quantify antibody responses against the rescued viruses, both using chicken B cells. These methods are readily adaptable to any laboratory with molecular biology expertise and negate the need to obtain wild-type strains. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: A chicken B-cell rescue system for IBDV Basic Protocol 2: A chicken B-cell neutralization assay for IBDV.

Evaluating the Breadth of Neutralizing Antibody Responses Elicited by Infectious Bursal Disease Virus Genogroup A1 Strains Using a Novel Chicken B-Cell Rescue System and Neutralization Assay.

Eight infectious bursal disease virus (IBDV) genogroups have been identified based on the sequence of the capsid hypervariable region (HVR) (A1 to A8). Given reported vaccine failures, there is a need to evaluate the ability of vaccines to neutralize the different genogroups. To address this, we used a reverse genetics system and the chicken B-cell line DT40 to rescue a panel of chimeric IBDVs and perform neutralization assays. Chimeric viruses had the backbone of a lab-adapted strain (PBG98) and the HVRs from diverse field strains as follows: classical F52-70 (A1), U.S. variant Del-E (A2), Chinese variant SHG19 (A2), very virulent UK661 (A3), M04/09 distinct (A4), Italian ITA-04 (A6), and Australian variant Vic-01/94 (A8). Rescued viruses showed no substitutions at amino acid positions 253, 284, or 330, previously found to be associated with cell-culture adaptation. Sera from chickens inoculated with wild-type (wt) (F52-70) or vaccine (228E) A1 strains had the highest mean virus neutralization (VN) titers against the A1 virus (log2 15.4 and 12.7) and the lowest against A2 viruses (log2 7.4 to 7.9; P = 0.0001 to 0.0274), consistent with A1 viruses being most antigenically distant from A2 strains, which correlated with the extent of differences in the predicted HVR structure. VN titers against the other genogroups ranged from log2 9.3 to 13.3, and A1 strains were likely more closely antigenically related to genogroups A3 and A4 than A6 and A8. Our data are consistent with field observations and validate the new method, which can be used to screen future vaccine candidates for breadth of neutralizing antibodies and evaluate the antigenic relatedness of different genogroups. IMPORTANCE There is a need to evaluate the ability of vaccines to neutralize diverse IBDV genogroups and to better understand the relationship between HVR sequence, structure, and antigenicity. Here, we used a chicken B-cell line to rescue a panel of chimeric IBDVs with the HVR from seven diverse IBDV field strains and to conduct neutralization assays and protein modeling. We evaluated the ability of sera from vaccinated or infected birds to neutralize the different genogroups. Our novel chicken B-cell rescue system and neutralization assay can be used to screen IBDV vaccine candidates, platforms, and regimens for the breadth of neutralizing antibody responses elicited, evaluate the antigenic relatedness of diverse IBDV strains, and when coupled with structural modeling, elucidate immunodominant and conserved epitopes to strategically design novel IBDV vaccines in the future.

Birnaviridae Virus Factories Show Features of Liquid-Liquid Phase Separation and Are Distinct from Paracrystalline Arrays of Virions Observed by Electron Microscopy.

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs colocalized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 s, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower colocalization of the VF GFP signal with the capsid protein VP2 (Manders' coefficient [MC] 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. IMPORTANCE Members of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.

Modeling Infectious Bursal Disease Virus (IBDV) Antigenic Drift In Vitro.

Infectious bursal disease virus (IBDV) vaccines do not induce sterilizing immunity, and vaccinated birds can become infected with field strains. Vaccine-induced immune selection pressure drives the evolution of antigenic drift variants that accumulate amino acid changes in the hypervariable region (HVR) of the VP2 capsid, which may lead to vaccine failures. However, there is a lack of information regarding how quickly mutations arise, and the relative contribution different residues make to immune escape. To model IBDV antigenic drift in vitro, we serially passaged a classical field strain belonging to genogroup A1 (F52/70) ten times, in triplicate, in the immortalized chicken B cell line, DT40, in the presence of sub-neutralizing concentrations of sera from birds inoculated with IBDV vaccine strain 2512, to generate escape mutants. This assay simulated a situation where classical strains may infect birds that have suboptimal vaccine-induced antibody responses. We then sequenced the HVR of the VP2 capsid at passage (P) 5 and 10 and compared the sequences to the parental virus (P0), and to the virus passaged in the presence of negative control chicken serum that lacked IBDV antibodies. Two escape mutants at P10 had the same mutations, D279Y and G281R, and a third had mutations S251I and D279N. Furthermore, at P5, the D279Y mutation was detectable, but the G281R mutation was not, indicating the mutations arose with different kinetics.

Transcriptomic Analysis of Inbred Chicken Lines Reveals Infectious Bursal Disease Severity Is Associated with Greater Bursal Inflammation In Vivo and More Rapid Induction of Pro-Inflammatory Responses in Primary Bursal Cells Stimulated Ex Vivo.

In order to better understand differences in the outcome of infectious bursal disease virus (IBDV) infection, we inoculated a very virulent (vv) strain into White Leghorn chickens of inbred line W that was previously reported to experience over 24% flock mortality, and three inbred lines (15I, C.B4 and 0) that were previously reported to display no mortality. Within each experimental group, some individuals experienced more severe disease than others but line 15I birds experienced milder disease based on average clinical scores, percentage of birds with gross pathology, average bursal lesion scores and average peak bursal virus titre. RNA-Seq analysis revealed that more severe disease in line W was associated with significant up-regulation of pathways involved in inflammation, cytoskeletal regulation by Rho GTPases, nicotinic acetylcholine receptor signaling, and Wnt signaling in the bursa compared to line 15I. Primary bursal cell populations isolated from uninfected line W birds contained a significantly greater percentage of KUL01+ macrophages than cells isolated from line 15I birds (p < 0.01) and, when stimulated ex vivo with LPS, showed more rapid up-regulation of pro-inflammatory gene expression than those from line 15I birds. We hypothesize that a more rapid induction of pro-inflammatory cytokine responses in bursal cells following IBDV infection leads to more severe disease in line W birds than in line 15I.

The Stronger Downregulation of in vitro and in vivo Innate Antiviral Responses by a Very Virulent Strain of Infectious Bursal Disease Virus (IBDV), Compared to a Classical Strain, Is Mediated, in Part, by the VP4 Protein.

IBDV is economically important to the poultry industry. Very virulent (vv) strains cause higher mortality rates than other strains for reasons that remain poorly understood. In order to provide more information on IBDV disease outcome, groups of chickens (n = 18) were inoculated with the vv strain, UK661, or the classical strain, F52/70. Birds infected with UK661 had a lower survival rate (50%) compared to F52/70 (80%). There was no difference in peak viral replication in the bursa of Fabricius (BF), but the expression of chicken IFNα, IFNß, MX1, and IL-8 was significantly lower in the BF of birds infected with UK661 compared to F52/70 (p < 0.05) as quantified by RTqPCR, and this trend was also observed in DT40 cells infected with UK661 or F52/70 (p < 0.05). The induction of expression of type I IFN in DF-1 cells stimulated with polyI:C (measured by an IFN-ß luciferase reporter assay) was significantly reduced in cells expressing ectopic VP4 from UK661 (p < 0.05), but was higher in cells expressing ectopic VP4 from F52/70. Cells infected with a chimeric recombinant IBDV carrying the UK661-VP4 gene in the background of PBG98, an attenuated vaccine strain that induces high levels of innate responses (PBG98-VP4UK661) also showed a reduced level of IFNα and IL-8 compared to cells infected with a chimeric virus carrying the F52/70-VP4 gene (PBG98-VP4F52/70) (p < 0.01), and birds infected with PBG98-VP4UK661 also had a reduced expression of IFNα in the BF compared to birds infected with PBG98-VP4F52/70 (p < 0.05). Taken together, these data demonstrate that UK661 induced the expression of lower levels of anti-viral type I IFN and proinflammatory genes than the classical strain in vitro and in vivo and this was, in part, due to strain-dependent differences in the VP4 protein.

Discrete Virus Factories Form in the Cytoplasm of Cells Coinfected with Two Replication-Competent Tagged Reporter Birnaviruses That Subsequently Coalesce over Time.

The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common; however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus coinfection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11 or a tetracysteine (TC) tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98-VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection or stained with a biarsenical derivative of the red fluorophore resorufin (ReAsH) following PBG98-VP1-TC infection, had green or red foci in the cytoplasm, respectively, that colocalized with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 h postinfection (hpi) (P < 0.0001), while the average area increased from 1.24 to 45.01 µm2 (P < 0.0001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57 µm/s at 16 hpi compared to 0.22 µm/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During coinfection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10 to 16 hpi, and we speculate that Birnaviridae reassortment requires VF coalescence.IMPORTANCE Reassortment is common in viruses with segmented double-stranded RNA (dsRNA) genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs during a coinfection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 h postinfection. We hypothesize that VF coalescence is required for the reassortment of the Birnaviridae This study provides new information that adds to our understanding of dsRNA virus VF trafficking.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

Differential gene expression in chicken primary B cells infected ex vivo with attenuated and very virulent strains of infectious bursal disease virus (IBDV).

Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF), causing immunosuppression and morbidity in young chickens. In addition to strains that cause classical Gumboro disease, the so-called 'very virulent' (vv) strain, also in circulation, causes more severe disease and increased mortality. IBDV has traditionally been controlled through the use of live attenuated vaccines, with attenuation resulting from serial passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated phenotypes are poorly understood. In order to address this, we aimed to investigate host cell-IBDV interactions using a recently described chicken primary B-cell model, where chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken CD40L. We demonstrated that these cells could support the replication of IBDV when infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by microarray. We found that key genes involved in B-cell activation and signalling (TNFSF13B, CD72 and GRAP) were down-regulated following infection relative to mock, which we speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the induction was far less pronounced upon infection with UK661, which we speculate could contribute to its virulence.

Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.

Early pathogenesis during infectious bursal disease in susceptible chickens is associated with changes in B cell genomic methylation and loss of genome integrity.

We propose a model by which an increase in the genomic modification, 5-hydroxymethylcytosine (5hmC), contributes to B cell death within the chicken bursa of Fabricus (BF) infected with infectious bursal disease virus (IBDV). Our findings indicate that, following an IBDV infection, Rhode Island Red (RIR) chickens have fewer surviving B cells and higher levels of 5hmC in the BF than the more resistant 15l line of birds. Elevated genomic 5hmC levels within the RIR BF are associated with markers of immune responses: infiltrating T cells and increased expression of CD40L, FasL and iNOS. Such changes correlate with genomic fragmentation and the presence of IBDV capsid protein, VP2. To explore the effects of CD40L, the immature B cell line, DT40, was exposed to recombinant chicken CD40L that resulted in changes in nuclear 5hmC distribution. Collectively, our observations suggest that T cell infiltration exacerbates early immunopathology within the BF during an IBDV infection contributing to B cell genomic instability and death to facilitate viral egress and immunosuppression.

Correction: Influenza A Virus Assembly Intermediates Fuse in the Cytoplasm.

[This corrects the article DOI: 10.1371/journal.ppat.1003971.].

Evaluation of the attenuation, immunogenicity, and efficacy of a live virus vaccine generated by codon-pair bias de-optimization of the 2009 pandemic H1N1 influenza virus, in ferrets.

Codon-pair bias de-optimization (CPBD) of viruses involves re-writing viral genes using statistically underrepresented codon pairs, without any changes to the amino acid sequence or codon usage. Previously, this technology has been used to attenuate the influenza A/Puerto Rico/8/34 (H1N1) virus. The de-optimized virus was immunogenic and protected inbred mice from challenge. In order to assess whether CPBD could be used to produce a live vaccine against a clinically relevant influenza virus, we generated an influenza A/California/07/2009 pandemic H1N1 (2009 pH1N1) virus with de-optimized HA and NA gene segments (2009 pH1N1-(HA+NA)(Min)), and evaluated viral replication and protein expression in MDCK cells, and attenuation, immunogenicity, and efficacy in outbred ferrets. The 2009 pH1N1-(HA+NA)(Min) virus grew to a similar titer as the 2009 pH1N1 wild type (wt) virus in MDCK cells (∼10(6)TCID50/ml), despite reduced HA and NA protein expression on western blot. In ferrets, intranasal inoculation of 2009 pH1N1-(HA+NA)(Min) virus at doses ranging from 10(3) to 10(5) TCID50 led to seroconversion in all animals and protection from challenge with the 2009 pH1N1 wt virus 28 days later. The 2009 pH1N1-(HA+NA)(Min) virus did not cause clinical illness in ferrets, but replicated to a similar titer as the wt virus in the upper and lower respiratory tract, suggesting that de-optimization of additional gene segments may be warranted for improved attenuation. Taken together, our data demonstrate the potential of using CPBD technology for the development of a live influenza virus vaccine if the level of attenuation is optimized.

Replication of live attenuated cold-adapted H2N2 influenza virus vaccine candidates in non human primates.

The development of an H2N2 vaccine is a priority in pandemic preparedness planning. We previously showed that a single dose of a cold-adapted (ca) H2N2 live attenuated influenza vaccine (LAIV) based on the influenza A/Ann Arbor/6/60 (AA ca) virus was immunogenic and efficacious in mice and ferrets. However, in a Phase I clinical trial, viral replication was restricted and immunogenicity was poor. In this study, we compared the replication of four H2N2 LAIV candidate viruses, AA ca, A/Tecumseh/3/67 (TEC67 ca), and two variants of A/Japan/305/57 (JAP57 ca) in three non-human primate (NHP) species: African green monkeys (AGM), cynomolgus macaques (CM) and rhesus macaques (RM). One JAP57 ca virus had glutamine and glycine at HA amino acid positions 226 and 228 (Q-G) that binds to α2-3 linked sialic acids, and one had leucine and serine that binds to α2-3 and α2-6 linked residues (L-S). The replication of all ca viruses was restricted, with low titers detected in the upper respiratory tract of all NHP species, however replication was detected in significantly more CMs than AGMs. The JAP57 ca Q-G and TEC67 ca viruses replicated in a significantly higher percentage of NHPs than the AA ca virus, with the TEC67 ca virus recovered from the greatest percentage of animals. Altering the receptor specificity of the JAP57 ca virus from α2-3 to both α2-3 and α2-6 linked sialic acid residues did not significantly increase the number of animals infected or the titer to which the virus replicated. Taken together, our data show that in NHPs the AA ca virus more closely reflects the human experience than mice or ferret studies. We suggest that CMs and RMs may be the preferred species for evaluating H2N2 LAIV viruses, and the TEC67 ca virus may be the most promising H2N2 LAIV candidate for further evaluation.