Research Project: Intervention Strategies to Predict, Prevent and Control Disease Outbreaks Caused by Emerging Strains of Virulent Newcastle Disease Viruses

Location: Exotic & Emerging Avian Viral Diseases Research

2019 Annual Report

Objectives
Objective 1. Identification of circulating and emerging Newcastle disease viruses, including conducting prevalence studies for NDV in poultry and in synanthropic birds from countries where virulent NDV strains are endemic to determine the presence of variant and emerging viruses in vaccinated poultry and in wild birds, and developing rapid identification assays for variant NDV strains. Objective 2. Identify agents that may cause NDV vaccine failures in endemic countries, including NDV variants and co-infecting agents that may immuno-compromise animals or enhance disease in vaccinated poultry flocks. Objective 3. Develop predictive biology strategies for risk assessment of virus evolution, including developing predictive biology strategies using NexGen (next generation) sequencing to evaluate the rate of change in different virulent NDV strains from unvaccinated, sub-optimally vaccinated, and well-vaccinated poultry. Objective 4. Develop improved NDV vaccines platforms, including identifying and evaluating effective and user friendly NDV vaccine platforms for in ovo or one-day old broilers, and identifying and characterizing protective immune responses for new vaccines platforms that are effective in ovo or in one-day old broilers.

Approach
Identification and characterization of new variants will be addressed by conducting active surveillance, and characterization of new isolates, and by developing rapid diagnostic assays that assures appropriate detection of these exotic samples (objective 1). Identifying immune suppressing agents and Newcastle disease viruses (NDV) variants that may cause disease in vaccinated animals will address the inadequate efficacy of commercial vaccines in endemic countries (Objective 2). Predictive strategies for identifying vaccines and vaccination practices that cause emergence of variant viruses will be addressed by Next Generation (NexGen) sequencing of viruses that emerge under different vaccination regimes (Objective 3). Reduced efficacy of commercial vaccines in young chickens with maternal antibodies will be addressed by developing an improved vaccine platform based in vector that is unknown to chickens (Objective 4).

Progress Report
Virulent NDV (VNDv) was detected in poultry in the United States (U.S.) in May 2018 for the first time since 2003 in California. The laboratory supported the response effort with epidemiological analysis of sequence information from representative isolates and with several challenge studies to compare the 2018 Newcastle disease virus (NDV) with the earlier 2002 California NDV isolate. The pathogenesis study showed both viruses easily infects and transmits into naive poultry. The research showed that the 2018 California virus was related to the 2002 California virus, but that the 2002 virus was not the source of the 2018 outbreak. Biological comparison also showed a high similarity with the California 2002 virus and a Belize 2008 NDV. By demonstrating that both viruses are highly similar, earlier experimental studies with California 2002 virus can reliably be used to help control the recent outbreak virus. During FY2019 96 fusion gene and 88 complete genome sequences of Newcastle disease virus from Mexico, Pakistan, Kenya, Tanzania, Jordan, and the United States were completed and submitted to GenBank by our lab. International collaborations essential to the study of Newcastle disease virus have continued and an agreement with Boehringer Ingelheim animal health to study avian respiratory diseases that affect the outcome of vaccination across Latin America has begun. Virulent Newcastle disease virus (vNDV) is endemic in many countries commercially linked to the U.S., including Mexico, Peru, Colombia, and Venezuela. Whether in domestic poultry or wild birds, vNDV strains remain a threat to all producers of poultry. The longstanding collaboration between the Agricultural Research Service (ARS), Southeast Poultry Research Laboratory (SEPRL) and Latin American partners was strengthened through those agreements and important epidemiological information was gathered on the movement and evolution of the Newcastle disease virus and other avian respiratory agents including avian influenza and mycoplasma. We have continued to disseminate information at international and national avian meetings attended by veterinarians across Latin America. They have improved our capabilities to rapidly detect, identify, and characterize new strains of viruses by the development of a rapid and sensitive sequencing protocol based on a new generation of portable sequencers (Oxford nanopore, Minion). In the area of epidemiology and diagnostics to address Objectives 1 to 3, our laboratory has contributed to the better characterization and classification of Newcastle disease viruses. A new diagnostic approach based on the analysis of paraffin embedded samples through next generation sequencing has allowed the sequencing of complete genomes which has led to a better epidemiological understanding of outbreaks. We have also developed a new rapid low cost diagnostic test that will be useful to detect mixed infections after a few minutes of sequencing based on the MinION Oxford nanopore sequencing platform. We have established an international collaboration with 23 laboratories that developed and updated the Newcastle disease virus classification system and published those findings. To address Objective 4, development of new vaccine friendly platforms a patent has been issued for the use of an ovo-vaccine containing an interleukin 4 gene that can potentially be used by in ovo inoculation.

Accomplishments
1. Identification of co-infecting agents in birds infected with virulent Newcastle disease viruses. To understand causes of vaccine failure in the field, we have conducted random sequencing of nucleic acids obtained from poultry samples isolated in endemic countries. The outcome has been the identification of bacterial and viral mixed infections. ARS researchers in Athens, Georgia, identified for the first time the presence of H9N2 avian influenza viruses in poultry samples containing Newcastle disease viruses and multiple bacterial species, including mycoplasma in Kenya poultry samples. This work is important because it demonstrates that additional factors are yet to be identified that may be responsible for field vaccine failures, while re-assuring the producer on the efficacy of current vaccines. We have identified recent strains of H7 avian influenza in poultry in Mexico.

2. An updated unified phylogenetic classification system and revised nomenclature system for Newcastle disease virus was published. Newcastle disease virus has high sequence variability that often is unique by country or region of the world. Several classification systems have previously been published that have established unique genotypes to describe these differences, but because of a lack of strict rules governing the establishment of new genotypes, a single system has not been widely adopted. ARS researchers in Athens, Georgia led an effort to establish a consortium of 23 reference and research laboratories to develop new and consistent naming rules using the complete fusion gene sequence to establish a consistent naming system, and the consortium published these results. This new nomenclature system is likely to become the de facto standard for genotype naming for Newcastle disease viruses.

3. A comparison of how easily virulent Newcastle disease viruses infects chickens was conducted. This study shows increased transmissibiltiy in viruses that are adapted to chickens as compared to wild bird viruses. ARS researchers in Athens, Georgia, established a transmission model for Newcastle disease virus that looks at different challenge doses for how easy it is to infect chickens and observe if transmission occurs between infected and uninfected chickens. Three chicken adapted strains of different genotypes that easily infected chickens were compared. However, two wild bird origin viruses were shown to require high amounts of virus to infect chickens, and these viruses did not transmit well chicken to chicken. These studies document that wild bird adapted viruses present a low risk of introduction into poultry.

4. Pathology and distribution studies on the velogenic viscerotropic Newcastle Disease Virus from California 2018 viruses were conducted. To better determine the risks presented by the current California virulent Newcastle disease virus, ARS researchers in Athens, Georgia, conducted a pathogenesis study and a transmission study in specific pathogen free chickens. The study included broilers and layers and the effect the virus on clinical disease, survival and transmission was evaluated and compared with other previous viruses, specially the California 2002 Newcastle disease virus which belong to the same genotype. It was concluded that the new viruses present characteristics of highly virulent velogenic viruses that are highly similar from the California 2002 viruses. This study is important to evaluate the risk of the present outbreak and to demonstrate that previous studies conducted by ARS researchers at the Southeast Poultry Research Laboratory in Athens, Georgia, on the California 2002 viruses are still valid.

5. Identification of previously unrecognized genetic diversity in avian paramyxovirus serotype 1 (APMV-1) isolated from wild birds in the U.S. and in poultry in several other countries. To determine the presence of variant and emerging viruses in wild birds and poultry, ARS researchers in Athens, Georgia, genetically characterized samples from Mexico, Kenya, Nigeria, Jordan, and the U.S. and the results published in 4 different articles titled: 1) First genome sequence of Newcastle Disease Virus of Genotype VIIi from Jordan; 2) Whole genome sequencing of genotype VI Newcastle disease viruses from formalin-fixed paraffin-embedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the U.S; 3) Presence of Newcastle disease viruses of sub-genotypes Vc and VIn in chickens and apparently healthy wild birds from Mexico in 2017; and 4) Genomic comparison of Newcastle disease viruses isolated in Nigeria between 2002 and 2015 reveals circulation of highly diverse genotypes and spillover into wild birds. Next generation sequencing provided valuable genetic information on evolutionary relationships among viruses. These studies demonstrate the importance of continuously tracking the evolution and genetic diversity of the virus.

6. Characterization of the evolution of Newcastle disease virus (NDV). As vaccine failures in Eastern Europe, Asia, and Africa are frequently reported, it is important to determine if these failures are caused by the genetic diversity of the viruses or by other causes. ARS researchers in Athens, Georgia conducted evolutionary and classification studies to determine the relationship among isolates leading to the following published discoveries: 1) Global phylodynamic analysis of avian paramyxovirus-1 provides evidence of inter-host transmission and intercontinental spatial diffusion; and 2) Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples. Understanding the evolution, current spread, and methods of detection of these viruses is important to develop effective diagnostic reagents and vaccines. These studies provided data and tools to characterize the diversity of the viruses and will be useful for the development on new vaccines and diagnostics.

7. Identification of epidemiological risk factors of wild birds. To identify epidemiological risk factors between poultry and synantropic birds (wild birds living close to humans and farms), ARS researchers in Athens, Georgia, conducted field studies in collaboration with a team of Mexican scientist from SENASICA-DGSA Mexico, the Asociación de Avicultores de Tepatitlán, Jalisco, and the Department Avian Medicine from the Universidad Nacional Autónoma de México and Boehringer Ingelhiem. These studies evaluated presence of Newcastle disease virus (NDV) in poultry and synantropic birds in densely productive areas in Mexico. The ability to demonstrate epidemiological connections between wildlife and poultry is important to understand the risk to the poultry industry from wild birds. Because of the proximity with the U.S., these Mexican regions present a high risk of introduction through trade, wild birds and illegal transport of birds and are important to the U.S. poultry industry.

Review Publications
Butt, S.L., Taylor, T.L., Volkening, J.D., Dimitrov, K., Williams Coplin, T.D., Lahmers, K.K., Suarez, D.L., Afonso, C.L., Stanton, J. 2018. Rapid virulence prediction and identification of Newcastle disease virus genotypes using third generation sequencing. Virology Journal. 15:179. https://doi.org/10.1186/s12985-018-1077-5.
Ababneh, M., Ferreira, H., Khalifeh, M., Suarez, D.L., Afonso, C.L. 2018. First genome sequence of Newcastle Disease Virus of Genotype VIIi from Jordan. Microbiology Resource Announcements. 5(23):e01136-18. https://doi.org/10.1128/MRA.01136-18.
He, Y., Taylor, T.L., Dimitrov, K.M., Butt, S.L., Stanton, J.B., Goraichuk, I.V., Fenton, H., Poulson, R., Zhang, J., Brown, C.C., Ip, H.S., Isidoro-Ayza, M., Afonso, C.L. 2018. Whole genome sequencing of genotype VI Newcastle disease viruses from formalin-fixed paraffin-embedded tissues from wild pigeons reveals continuous evolution and previously unrecognized genetic diversity in the U.S. Virology Journal. 15:9. https://doi.org/10.1186/s12985-017-0914-2.
Dimitrov, K.M., Ferreira, H.L., Pantin Jackwood, M.J., Taylor, T.L., Goraichuk, I.V., Crossley, B.M., Killian, M., Bergeson, N., Torchetti, M., Afonso, C.L., Suarez, D.L. 2019. Pathogenicity and transmission of virulent Newcastle disease virus from the 2018-2019 California outbreak and related viruses in young and adult chickens. Virology. 531:203-218. https://doi.org/10.1016/j.virol.2019.03.010.
Bertran, K., Lee, D., Criado, M.F., Balzli, C.L., Killmaster, L.F., Kapczynski, D.R., Swayne, D.E. 2018. Maternal antibody inhibition of recombinant Newcastle disease virus vectored vaccine in a primary or booster avian influenza vaccination program of broiler chickens. Vaccine. 36(43):6361-6372. https://doi.org/10.1016/j.vaccine.2018.09.015.
Hicks, J.T., Dimitrov, K.M., Afonso, C.L., Ramey, A.M., Bahl, J. 2019. Global phylodynamic analysis of avian paramyxovirus-1 provides evidence of inter-host transmission and intercontinental spatial diffusion. BMC Evolutionary Biology. 19:108. https://doi.org/10.1186/s12862-019-1431-2.
Ferreira, H.L., Taylor, T.L., Absalon, A.E., Dimitrov, K.M., Cortes-Espinosa, Butt, S.L., Goraichuk, I.V., Volkening, J.D., Suarez, D.L., Afonso, C.L. 2019. Presence of Newcastle disease viruses of sub-genotypes Vc and VIn in backyard chickens and in apparently healthy wild birds from Mexico in 2017. Virus Genes. 55(4):479-489. https://doi.org/10.1007/s11262-019-01663-1.
Welch, C.N., Shittu, I., Abolnik, C., Ponman, S., Dimirov, K.M., Taylor, T.L., Williams Coplin, T.D., Goraichuk, I.V., Meseko, C.A., Ibu, J.O., Gado, D.A., Joannis, T.M., Afonso, C.L. 2019. Genomic comparison of Newcastle disease viruses isolated in Nigeria between 2002 and 2015 reveals circulation of highly diverse genotypes and spillover into wild birds. Archives of Virology. 164(8):2031-2047. https://doi.org/10.1007/s00705-019-04288-9.
Spatz, S.J., Garcia, M., Riblet, S., Ross, T.A., Volkening, J.D., Taylor, T.L., Kim, T.N., Afonso, C.L. 2019. MinION sequencing to genotype US strains of Infectious Laryngotracheitis Virus. Avian Pathology. 48(3):255-269. https://doi.org/10.1080/03079457.2019.1579298.
Joshi, L.R., Bauermann, F.V., Hain, K.S., Kutish, G.F., Armien, A.G., Lehman, C.P., Neiger, R., Afonso, C.L., Tripathy, D.N., Diego, D.G. 2018. Detection of fowlpox virus carrying distinct genome segments of reticuloendotheliosis virus. Archives of Virology. 260:53-59. https://doi.org/10.1016/j.virusres.2018.10.017.
Absalon, A., Cortes, D., Lucio, E., Miller, P.J., Afonso, C.L. 2019. Epidemiology, control, and prevention of Newcastle disease in endemic regions: Latin America. Veterinary Microbiology. 51(5):1033-1048. https://doi.org/10.1007/s11250-019-01843-z.
Dimitrov, K.M., Abolnik, C., Afonso, C.L., Albina, E., Bahl, J., Berg, M., Briand, F., Brown, I.H., Choi, K., Chvala, I., Durr, P.A., Ferreira, H.L., Fusaro, A., Torcheti, M., Miller, P.J., Suarez, D.L. 2019. Updated unified phylogenetic classification system and revised nomenclature for Newcastle disease virus. Infection, Genetics and Evolution. 74:103917. https://doi.org/10.1016/j.meegid.2019.103917.
Sharma, P., Killmaster, L.F., Volkening, J.D., Cardenas-Garcia, S., Wajid, A., Regmani, S.F., Basharat, A., Miller, P.J., Afonso, C.L. 2018. Draft genome sequence of three novel Ochrobactrum spp. isolated from different avian hosts in Pakistan. Genome Announcements. 6(15):e00269-18. https://doi.org/10.1128/genomeA.00269-18.
Butt, S.L., Dimitrov, K.M., Zhang, J., Wajid, A., Bibi, T., Basharat, A., Brown, C.C., Rehmani, S.F., Stanton, J.B., Afonso, C.L. 2019. Enhanced phylogenetic resolution of Newcastle disease outbreaks using complete viral genome sequences from formalin-fixed paraffin-embedded tissue samples. Virus Genes. 55(4):505-512. https://doi.org/10.1007/s11262-019-01669-9.
Ferreira, H.L., Taylor, T.L., Sabra, M., Dimitrov, K.M., Afonso, C.L., Suarez, D.L. 2019. Virulent newcastle disease viruses from chicken origin are more pathogenic and transmissible than viruses normally maintained in wild birds. Veterinary Microbiology. 235:25-34. https://doi.org/10.1016/j.vetmic.2019.06.004.
Goraichuk, I.V., Msoffe, P.L., Chiwangad, G.H., Dimitrov, K.M., Afonso, C.L., Suarez, D.L. 2019. First complete genome sequences of a subgenotype Vd Newcastle disease virus isolate. Microbiology Resource Announcements. 8(27):e00436-19. https://doi.org/10.1128/MRA.00436-19.