Progress 04/01/23 to 03/31/24
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, the data fromthe stakeholder interviews into the resposne to HPAI will be submitted for publicaiton. Work will continue on evolution of the H5N1 clade 2.3.4.4. NGS samples will be examined to look at mutations of the virus from various host species.
Impacts
What was accomplished under these goals?
The project has work three packages: (1) Modelling evolution of AIV across scales, (2) Immune-driven evolution of AIV and (3) Assessing Risk, Predictions and Science-Policy Interface. Our focus is on work package 2 examining immune response and evolution of AIV. We examined immune response of dendritic cells (DCs) to AIV infection. DCs are professional antigen-presenting cells, which are key components of the immune system and involved in the early immune response. DCs are specialized in capturing, processing, and presenting antigens to facilitate immune interactions. Chickens infected with (AIV) demonstrate a wide range of clinical symptoms, based on pathogenicity of the virus. Low pathogenic avian influenza (LPAI) viruses typically induce mild clinical signs, whereas high pathogenic avian influenza (HPAI) induce more severe disease, which can lead to death within days. For this study, chicken bone marrow-derived DC were produced and infected with high and low pathogenic avian influenza viruses of H5N2 or H7N3 subtypes to characterize innate immune responses, study effect on cell morphology, and evaluate virus replication. A strong proinflammatory response, including chicken interleukin-1β, and stimulation of the interferon response pathway were observed at 8 hours post infection. Microscopically, the DCs underwent morphological changes from classic elongated dendrites to a more general rounded shape that eventually led to cell death with the presence of scattered cellular debris. Differences in onset of morphologic changes were observed between H5 and H7 subtypes. Increases in viral titers demonstrated that both HPAI and LPAI are capable of infecting and replicating in DCs. The elevated expression of infected DCs may be indicative with a dysregulation of the immune response typically seen with HPAI infections. We also examined AIV infections in seals. Because recent AIV have crossed species barriers, a retrospective analysis of performed. Genetically diverse AIVs are maintained in wild aquatic birds with increasingly frequent spillover into mammals, yet these represent a small proportion of the overall detections. The isolation of AIV in marine mammals, including seals, has been reported sporadically throughout the last 45 years. Prior to 2016, all reports of AIVs detected from seals were of low pathogenicity AIV (LPAIV). In spite of this, a majority of reported AIV outbreaks caused fatal respiratory diseases, with harbor seals particularly susceptible to infection. The H5 clade 2.3.4.4b highly pathogenic AIV (HPAIV) was detected in seals for the first time in 2016-2017. Globally, many cases of mass seal die-offs have occurred from 2.3.4.4b HPAIV and are attributed to spillover from wild bird species. The potential of seal-to-seal transmission has been considered after massive mortality of southern Elephant seals off the Argentina coast. Close contact between seals and wild birds, the rapid evolution of H5N1 AIVs, and the possibility of efficient mammal-to-mammal transmission, are increasing concerns over the potential for the establishment of a marine mammal reservoir, and public health risks associated with pandemic potential of the virus. This manuscript details the detection of AIV in the seal population, demonstrating interesting features of different subtypes with emphasis on avian-to-mammal-to-mammal transmission. Phylogenetic characterization of the representative seal isolates was performed to demonstrate the relationships within the different virus isolates. Furthermore, we demonstrate that the reassortment events between different LPAIVs occurred before and after the viruses reached the seal population. The reassortment of viral segments plays an important role in the evolution of influenza viruses. Taken together, these data provide information on the impact and isolation of AIV in seal populations Finally, we examined high and low pathogenic avian influenza resistance to the innate immune modulator myxovirus (Mx) resistance gene. The host Mx proteins are key mediators of the antiviral response that block viral replication of several viruses. The effectiveness of chicken Mx (Ck Mx) is controversial, however mouse Mx (Mu Mx1) is a strong antiviral with known effectiveness against AIVs. The mouse Mx1 was stably introduced into a chicken cell line (DF1) to enhance the innate immune response towards low pathogenic avian influenza virus (LPAIV) and highly pathogenic avian influenza virus (HPAIV). Following challenge, titers of both LPAIV and HPAIV were significantly decreased in DF1 cells expressing mouse Mx1 cells at 24 hours post infection. In addition, considerably less cytopathic effect and matrix protein staining was observed in cells expressing mouse Mx1, which suggests mouse Mx1 is broadly effective against multiple AIV subtypes. A pretreatment with chicken interferon-alpha, known to upregulate Mx expression, resulted in an additional 5-10-fold decrease in viral titers compared to the virus grown in mouse Mx1-expressing cells alone. This work provides the foundational studies for using disease resistance to AIV.
Publications
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
In Silico Genomic Analysis of Avian Influenza Viruses Isolated From Marine Seal Colonies. Chrzastek K, Kapczynski DR. Pathogens. 2024 Nov 16;13(11):1009. doi: 10.3390/pathogens13111009.
PMID: 39599562
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Mo J, Segovia K, Chrzastek K, Briggs K, Kapczynski DR. Morphologic characterization and cytokine response of chicken bone-marrow derived dendritic cells to infection with high and low pathogenic avian influenza virus. Front Immunol. 2024 Aug 30;15:1374838. doi: 10.3389/fimmu.2024.1374838. PMID: 39281683; PMCID: PMC11401072.
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2024
Citation:
Genetic insertion of mouse Myxovirus-resistance gene 1 increases innate resistance against both high and low pathogenic avian influenza virus by significantly decreasing replication in chicken DF1 cell line.
Briggs K, Chrzastek K, Segovia K, Mo J, Kapczynski DR.
Virology. 2024 Jul;595:110066. doi: 10.1016/j.virol.2024.110066. Epub 2024 Mar 31.PMID: 38574415
- Type:
Peer Reviewed Journal Articles
Status:
Published
Year Published:
2023
Citation:
Briggs K, Kapczynski DR. Comparative analysis of PB2 residue 627E/K/V in H5 subtypes of avian influenza viruses isolated from birds and mammals. Front Vet Sci. 2023 Sep 1;10:1250952. doi: 10.3389/fvets.2023.1250952. PMID: 37720472; PMCID: PMC10502342.
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Progress 04/01/22 to 03/31/23
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Apart from the problems with rescuing the H7N9 virus, we have had facility issues with our new animal BSL3 rooms which will delay animal studies until they are corrected. In addition, we have had problems with our SP chicken flocks that supply our eggs. The flocks have become contaimnated with a fowl ademovirus and we are now having to buy SPF eggs for lab use. This is also slowing us down a bit. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Presentations of the resutls have been presented to the scientific community at the following: World Vaccine Congress, Washington, DC, April 2023 Amercian Association of Avian Pathologists, Jacksonville, FL, June 2023 Live Bird Market Working Group, Tucson, AZ, February 2023 Plant Animal Genome, San Diego, CA, January 2023 American Society for Virology, Athens, GA, June 2023 From the UK side: Additional funding acquired to match USDA timelines of grant. Biotechnology and Biological Sciences Research Council (BBSRC): BB/X006123/1 - Understanding animal health threats from emerging H5 high pathogenicity avian influenza viruses (£ 179866; 2022 - 2023) Public engagement activities: -A press release, press conference or response to a media enquiry/interview - Interview with journalist from Time Magazine about Avian influenza Added by: Dr Samantha Lycett / Date Added: 16 Mar 2023 -A press release, press conference or response to a media enquiry/interview - Interview with Jeremy Howell of the BBC over avian influenza Added by: Professor Paul Digard / Date Added: 15 Mar 2023 -A press release, press conference or response to a media enquiry/interview - Panelist for Science Media Centre media briefing event on avian influenza Added by: Professor Paul Digard / Date Added: 15 Mar 2023 -A press release, press conference or response to a media enquiry/interview - Interview with the Financial Times over avian influenza Added by: Professor Paul Digard / Date Added: 15 Mar 2023 -A press release, press conference or response to a media enquiry/interview - Interview with the Guardian newspaper over avian influenza Added by: Professor Paul Digard / Date Added: 15 Mar 2023 -A press release, press conference or response to a media enquiry/interview - Interview with Financial Times over avian influenza Added by: Professor Paul Digard / Date Added: 07 Mar 2023 -A press release, press conference or response to a media enquiry/interview - Scoping discussion with ITN Productions over a broadcast on vaccines and avian influenza Added by: Professor Paul Digard / Date Added: 05 Mar 2022 Influence on policy: -Participation in a guidance/advisory committee - Defra SAC-ED sub sub group on highly pathogenic avian influenza (2022) Added by: Professor Paul Digard / Date Added: 07 Mar 2023 -Participation in a guidance/advisory committee - Defra Scientific Advisory Committee on Emerging and Exotic Diseases (SAC-ED). (2022) Added by: Professor Paul Digard / Date Added: 07 Mar 2023 -Participation in a guidance/advisory committee - Member of SAC-ED HPAI (2022) Added by: Prof Lonneke Vervelde / Date Added: 02 Mar 2023 -Membership of a guideline committee - Discussion meeting on research gaps in avian influenza (2022) Added by: Professor Paul Digard / Date Added: 09 Mar 2022 -CIEL Report: Living with Bird Flu Added by: Professor Lisa Boden / Date Added: 08 Mar 2023 -Highly pathogenic avian influenza in Great Britain: evaluation and future actions - GOV.UK (www.gov.uk) What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, the data fromt results reported here will be submitted. Work will continue on evolution of the H5N1 virus in face of immune pressure from vaccination. We will begin to test the H7N9 HPAI virus in our in vitro studies. Further NGS samples will be prepared to look at mutations of the virus from chcikens and duck samples.
Impacts
What was accomplished under these goals?
Update on project covering 29 August 2022- 5 May 2023: ?Three major accomplishments have been completed in the last 8 months. First, The H7N9 virus, based on the China A/Chicken/Yunnan/1007/2021 H7N9 highly pathogenic avian influenza virus, has been rescued in my BSL3 laboratory. Delays for its rescue were caused by sequences in the PB1 segment. It took several months to identify the problem and we had to remake and sequence this plasmid. We are currently sequencing all the segments to ensure no unintended mutations occurred. Downstream studies are planned with this virus in the coming year. Secondly, we have completed genetic and serological analysis comparing vaccination of chickens and turkeys with current H5 vaccines against the clade 2.3.4.4b virus that was rescued in these studies (A/Turkey/Poland/2021 H5N1 clade 2.3.4.4b HPAI). During 2014-215, the H5Nx HPAIV belonged to 2.3.4.4c clade of the A/goose/Guangdong/1/1996 (Gs/Gd) H5N1 lineage, while in 2021-current, the viruses belong to clade 2.3.4.4b. At the amino acid level, the 2.3.4.4b and c viruses share approximately 96-98 % sequence identity of hemagglutinin (HA) protein. Compared to the original Gs/Gd virus, the clade 2.3.4.4 viruses share approximately 90-96% sequence identity. Viruses within the subclades of 2.3.4.4 have evolved into a-h designation, demonstrating between 13 and 42 amino acid differences in the HA protein. We also examined the antigenic relatedness based on serological cross reactivity using the hemagglutinin-inhibition (HI) assay. Birds were vaccinated with various H5-based inactivated or recombinant vaccines (with 85-98% relatedness to HA) and challenged against the 2.3.4.4c lineage viruses. Serum was tested against homologous vaccine virus or the 2.3.4.4b and c viruses. As expected, birds vaccinated with inactivated 2.3.4.4c viruses maintained the highest level of cross reactive HI titers to the 2.3.4.4b viruses, with an average of 1log2 drop in titer using prechallenge serum. When birds in these groups were challenged with 2.3.4.4b HPAIV, the serological cross reactivity was near 100% in titer. Conversely, when birds received non-Gs/Gd lineage H5 vaccine, the serum titers were generally 7log2 lower against either of the clade 2.3.4.4 viruses. Serum from birds that received vaccination with a clade 2.3.2 vaccine demonstrated reduced titers of approximately 4-5log2 titers. Taken together there appears to be limited serum cross reactivity from birds not vaccinated with 2.3.4.4 antigen, further strengthening the concept of matching the vaccine antigen to the field strain. A manuscript for these results is currently being prepared. In addition, we have completed NGS preparation of HPAI virus mutations from swab samples recovered in vaccinated chickens and turkeys. In these studies, several existing H5 vaccines or vaccine seed strains with varying genetic relatedness (85-100%) to the 2.3.4.4 HPAI viruses were evaluated for protection in poultry. Birds received a single dose of either an inactivated whole H5 AI vaccine, or a recombinant fowl poxvirus or turkey herpesvirus-vectored vaccines with H5 AI hemagglutinin gene inserts followed by challenge with either a U.S. wild bird H5N8 (A/gyrfalcon/Washington/40188-6/2014) or H5N2 (A/northern pintail/Washington/40964/2014) clade 2.3.4.4 isolate. Swab samples taken from these birds were evaluated for challenge virus. Those samples with virus titers greater than 10^3 EID50/ml received further preparation for NGS analysis to determine how the virus changes in the face of humoral and/or cellular immunity. Raw result reads were received last month and sent to The University of Edinburgh for comparison and incorporation into predicative models. We expect to prepare a manuscript for these data soon. Finally, we have performed an in silico analysis of the viral basic polymerase 2 (PB2) gene with regards to the host barrier switch between avian and mammalian species. Of interest in the 2.3.4.4 subclade is its potential adaption to mammals, as prior pandemics have been caused by avian spillover events. A well-described avian-to-mammalian adaptation in the PB2 protein is contained at residue 627. Traditionally, avian isolates contained a glutamate (E), while mammalian/human isolates contained a lysine (K). It was originally thought that this residue played a role in PB2's ability to replicate at lower avian temperatures and provided an explanation for inefficient replication of AIV in non-avian hosts. While temperature may still play a role, more recently structural studies have determined that PB2 interacts with host protein acidic nuclear phosphoprotein 32 family member A (ANP32A) at residue 627 and this interaction is the driving force behind the E627K mutation.In this work, we investigate the number of isolates containing a PB2 627E or K in H5Nx isolates. We compared the prevalence of isolates with a 627K between non-Gs/GD and Gs/GD lineages, then breakdown the ratio of isolates within the Gs/GD lineage. The American-non-Gs/GD/96 (Am_nonGsGD) lineage had 789 complete PB2 sequences with 627K coverage, and the earliest viruses in the dataset were from Wisconsin, USA in 1975. The subtypes in 1975 included H5N2, H5N6, and H5N1. The species in this lineage consisted of chicken/turkey (220), all other avian (567), mammalian (2), and human (0). Of the 789 sequences examined only 2 had a PB2 627K, the animals were a rhea and emu (both Texas, USA, 1993, H5N2). The percent of PB2 627K isolates for the Am_nonGsGD lineage was 0.25%. For the Eurasian-non-Gs/GD/96 (Ea_nonGsGD) lineage, 390 isolates were examined, the earliest isolates were from Scotland in 1959 (H5N1). In this lineage there were 46 chicken/turkey, 342 other avian, 2 mammals, and 0 human isolates. Of the 390 isolates, 4 had a PB2 627K. All 4 isolates came from ostriches in South Africa in 2011 and 2015. The percentage of PB2 627K isolates from the Ea_nonGsGD lineage was 1.03%. Interestingly, there were only 4 mammalian isolates in the dataset within the two lineages. All 4 were H5N2 isolates from swine, two were from Mexico in 2014/2015 and two were from Korea in 2008. Based on the data available, the American and Eurasian lineages appear to have low mammalian/human spillover events and a low percentage of PB2 627K adaptations. To examine Gs/GS clade 2 more thoroughly, we determined the percent PB2 627K in subclades 2.1-2.5, based on available GISAID sequences. Clade 2.1 had a low proportion of avian isolates (34.6%) available compared to mammalian/human isolates (65.4%). All 19 (8.3%) isolates with a PB2 627K in clade 2.1 were mammalian/human in origin. The isolates from clade 2.1 were obtained between 2003-2015. Clade 2.2 was previously shown to have a high incidence of PB2 627K residues. We found that 92.1% of available PB2 sequences classified as clade 2.2 had a 627K residue. Unexpectedly, 578 isolates with a PB2 627K were from avian species and only 36 were mammalian/human. Clade 2.2 isolated ranged from 1997-2017 in the dataset. Clade 2.2 contributed to 83% (614/738) of the total clade 2 PB2 627K population. Clade 2.3 (2003-present) had the largest total number of isolates available (9,797), but only 105 had PB2 627K. Of the isolates with a 627K, 75 were mammalian/human and 30 were of avian origin. The total percentage of PB2 627K isolates for clade 2.3 was 1.1%. Of note, only clade 2.3 contained PB2 627V sequences. The species containing a PB2 627V were mixed, 8 chicken, 2 other avian, 3 red foxes, 1 tiger, and 1 human. Clades 2.4 and 2.5 had low sample sizes and none the isolates contained a PB2 627K residue. Additionally, the isolates available were only from 2003-2004 and 2003-2006, respectfully. Taken together, these results demonstrate a low level of 627K in AIV. Also, that the majority of PB2 627K isolates are from clade 2.2, not 2.3. Results from these studies are being prepared for manuscript submission.
Publications
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Progress 04/01/21 to 03/31/22
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The only major problems associated with the work have been delays caused by the SARS-CoV-2 pandemic. USDA shut down on site research activities at most locations during the pandemic, so we have been delayed from our original timeline. We are hoping to make that up in the next reporting cycle. What opportunities for training and professional development has the project provided?A postdoctoral scientist, Dr. Kelsey Briggs, was hired at USDA in January of 2022. She has been trained to work with avian influenza virus and has just completed her training to work with select agents in BSL3 laboratories. In additon, the new USDA BSL3 laboratory/animal facility recently passed inspection and will be brought onlione later this year. We have taken beneficail occupancy of the building and expexct to begin virus work no later than December 2022. How have the results been disseminated to communities of interest?Two publications associated with this grant have been generated byour collaborators in China and U.K.: Lin R, Lu L, Lycett S, Liu W, Li J. Dealing with Highly Pathogenic Avian Influenza: An Impending Crisis. Innovation (N Y). 2021 Jan 21;2(1):100084. doi: 10.1016/j.xinn.2021.100084. PMID: 34557739; PMCID: PMC8456418. Li J, Zhang C, Cao J, Yang Y, Dong H, Cui Y, Yao X, Zhou H, Lu L, Lycett S, Wang X, Song H, Liu W, Gao GF, Shi W, Bi Y. Re-emergence of H5N8 highly pathogenic avian influenza virus in wild birds, China. Emerg Microbes Infect. 2021 Dec;10(1):1819-1823. doi: 10.1080/22221751.2021.1968317. PMID: 34392820; PMCID: PMC84 What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, the H7 and H9 viruses will be rescued in the laboratory. Once the sequences have been confirmed, they will be tested in mallards, geese and poultry. Samples taken will taken forNGS analysis and based on those results (along with the field data) models of avian influenza evolution will be begin to be generated in wild birds and domestic poultry.
Impacts
What was accomplished under these goals?
Predictive phylogenetics for evolutionary and transmission dynamics of newly emerging avian influenza viruses Update on project 28 August 2022 from USDA. This project is a collaboration between the US, UK and China - involving Roslin Institute and Global Academy, University of Edinburgh (UK), University of Georgia (US), USDA - Southeast Poultry Research Laboratory (SEPRL) (US), and Chinese Academy of Sciences, Beijing (China). It is about integrating avian influenza surveillance data and viral sequences with laboratory and transmission experiments, to understand viral emergence and strain fitness in birds in wild and domestic contexts, and to generate risk maps. The project has work three packages: (1) Modelling evolution of AIV across scales, (2) Immune-driven evolution of AIV and (3) Assessing Risk, Predictions and Science-Policy Interface. The project started in April 2021, and to date we have used an early form of predictive phylogenetics to select appropriate avian influenza strains with which to perform transmission and in vitro experiments - these have been selected for subtypes H5, H7 and H9 and represent recent outbreaks, 'sister' strains (that did not results in large outbreaks) and pre-cursor strains, and the results will form part of the ongoing predictive model. The following virus subtypes are currently being recreated in the USDA BSL 3 laboratory: A/turkey/Poland/H1289-21RS1385-20/2021 H5N8 HPAI (EPI_ISL_3102079) A/chicken/Yunnan/1007/2021 H7N9 HPAI (GISAID ID: EPI_ISL_1495982) A/chicken/Jiangxi/BJ-2021-84/2021 H9N2 (No ref available at this time). Note that these experiments will take place at the in the US, UK, and China from June 2022, and have been designed so that we are not exchanging whole live viruses. To date the H5N1 isolate has been rescued in the laboratory containing the wild type HPAI cleavage site as well as a low pathogenic cleavage H5 variant. In parallel to this we have been analyzing avian influenza sequences from surveillance data, and using phylodynamics to track the origin, route and speed of spread of highly pathogenic H5NX (clade 2.3.4.4) in Asia and Europe. This work includes the analyzing the current highly pathogenic avian influenza outbreaks in Europe and the UK for the 2021/2022 season. This current strain of highly pathogenic avian influenza has previously caused outbreaks in Europe and the UK in the autumn - winter seasons for 2014/15, 2016/17 and 2020/21. It looks to bebeing brought to the UK from migrating wild birds, and this autumn - winter season it again looks to be making incursions into the UK from wild birds. It is possible that highly pathogenic avian influenza may be endemic in wild birds flyways that cross the UK and Europe (i.e. not die out over the summer). We are now working on a predictive phylodynamic model including spatial risk factors such as Biodiversity, Bird flyways, Climate, Elevation, Forest, Land use, Socio-economic, Vegetation andWater. We have also been collaborating with the VEO European Union Horizon 2020 project (includes APHA), and EPIC (Centre of Expertise for Animal Disease Outbreaks, Scottish Government) about advise for the current outbreaks. We are now beginning to include US H5 HPAI (clade 2.3.4.4) outbreaks that started in the December of 2021 and continued into fall of 2022 in our phylodynamic analysis. This was not originally in the work, but is another opportunity to develop and test our models.
Publications
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