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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

2020 Annual Report

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.

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
Under Objective 4, virulent Newcastle disease virus (NDV) was eradicated in California after almost 2 years of circulation in the United States. Research has been completed looking at the pathogenesis of the virus and looking for ways to improve control of the virus. One of the important studies completed in FY2020 was a vaccine study that looked at the viral vectored herpesvirus of turkeys (HVT) virus that expressed the fusion gene of a Newcastle disease virus. This is a licensed vaccine in the U.S. and the HVT-NDV vaccines are being more commonly used in U.S. poultry, so documenting that this vaccine was protective for the virus circulating in California was important considering the genetic diversity found with NDV viruses. The virus was also compared to an attenuated live vaccine strain, and the HVT-NDV vaccine performed as good or better than the commonly used live vaccine. The HVT-NDV vaccine has the advantage that it can be administered at day of hatch and possibly by in ovo administration. Several other studies on vaccines were performed with the goal of improved vaccines that can be given by mass administration routes. Studies using attenuated NDV strains for in ovo use and different vaccine vectors continue to be a focus of the laboratory. Under Objective 3, improved diagnostic testing of field samples using next-generation sequencing (NGS) continues with notable achievements. Next-generation sequencing, using a random amplification approach, can identify a wide variety of viral, bacterial, and protozoal pathogens in the same test if the pathogen is in high concentration. Current efforts are designed to improve the sensitivity by decreasing the amount of host genome that is sequenced with current methods. Improvements in sensitivity are being made, but even using the older methods, many poultry pathogens have been identified and sequenced. For example, full-length sequence from three different lineages of infectious bronchitis virus was determined, as well as sequencing of the entire genome of a reference isolate of avian metapneumovirus. Under Objective 1, progress was also made in the area of molecular diagnostics. The most commonly used molecular test for Newcastle disease viruses (NDV) is a real-time reverse transcriptase-PCR (rRT-PCR) test that was developed in 2004 by the Southeast Poultry Research Laboratory in Athens, Georgia, that targeted the matrix protein. Although this test is widely used, there have been documented cases of reduced sensitivity or false negatives using this test. Because of the greatly increased amount of sequence information on NDV, an effort was made to identify highly conserved regions of the NDV genome and design new primers. Using an empiric approach and testing multiple variants, three rRT- PCR tests were developed that, in general, performed better and were more specific than the matrix test for Class II NDV. These tests provide bench validated alternatives to the matrix test to identify NDV from clinical samples if the matrix test is not performing as expected. These alternative tests have been particularly useful in testing from Africa because of the better performance characteristics.

1. A vaccine study was conducted that showed both traditional live attenuated vaccines and viral-vectored vaccines were protective for the California 2018 virulent challenge. The herpes virus of turkeys (HVT) is a commonly used vaccine vector for different pathogens including Newcastle disease virus (NDV). ARS researchers in Athens, Georgia, performed a study to determine if traditional live attenuated vaccines or the viral vectored HVT-NDV vectored vaccine alone or in combination provided adequate protection from a virulent California 2018 NDV challenge. The challenge conducted was representative of the 2018-20 U.S. outbreak virus. The HVT-NDV vaccine, administered at day 1 of age provided comparable protection as the attenuated vaccine virus with decreases in viral shedding at a challenge day of 21 or 28 days after vaccination. This study, the first with the California 2018 challenge virus, supports the use of both vaccines for routine protection from the challenge virus and will help support control efforts.

2. Three new real-time reverse transcription–polymerase chain reaction (rRT-PCR) tests were developed for the detection of Class II Newcastle disease virus (NDV) isolates. Newcastle disease viruses have 2 unique classes, I and II, and considerable genetic diversity. In particular, more genetic diversity is found in Class II viruses. Because of this variation, the most commonly used rRT-PCR tests can have lower sensitivity and specificity and on some occasions can miss the detection of NDV isolates. To fill this gap, ARS researchers in Athens, Georgia, developed three new rRT-PCR tests in conserved areas of the genome. Comparison testing showed improved specificity when evaluating representative genotypes, and overall the sensitivity was as good or better than the currently used test.

3. Sequence analysis of pathogens detected through random sequencing using next generation sequencing. As part of ongoing surveillance on historic samples from the U.S., ARS researchers in Athens, Georgia, have detected multiple infectious bronchitis viruses (IBV) where full length or close to full length sequence was determined using next-generation sequencing. This work has provided the first complete genomes for several different lineages of IBV. This information provides an opportunity for more in-depth sequence analysis of viruses that circulate widely in the United States poultry population.

4. Full genome sequencing and analysis of an avian metapneumovirus subtype B virus. As part of ongoing surveillance studies, ARS researchers in Athens, Georgia, detected several avian metapneumovirus. As part of the analysis of these field samples, a common reference isolate of subtype B virus was fully sequenced for the first time. The availability of full genome sequence provides greater understanding of the phylogenetics of avian metapneumoviruses and improved understanding of the origin of the field strains.

5. Detection and isolation of virulent Newcastle disease virus samples from live bird markets from Tanzania. As part of a surveillance project in Tanzania, oropharyngeal and cloacal swabs, ARS researchers in Athens, Georgia, sampled from chickens from multiple live bird markets from Tanzania. Numerous positive samples were identified by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) and confirmation by virus isolation. The viruses were determined to be virulent based on sequence analysis and selected viruses were shown to be virulent in animals. Two different genotypes were found in Tanzania. This supports previous studies that Tanzania is widely endemic for virulent Newcastle disease virus which creates issues for poultry production in the country.

6. A rapid next generation sequencing platform was used to rapidly sequence a Salmonella isolate suitable for determination of serotype, virulence factors, and antimicrobial resistance genes. The Minion unit is a relatively inexpensive sequence system that can generate a large amount of sequence. Using this platform, ARS researchers in Athens, Georgia, produced a whole genome sequence in less than a day and had similar accuracy to other next-generation sequencing platforms that take much longer to process. This technique offers the possibility of rapid analysis to support epidemiologic investigations of bacterial diseases.

Review Publications
Taylor, T.L., Volkening, J.D., Dejesus, E., Simmons, M., Dimitrov, K.M., Glenn, T.E., Suarez, D.L., Afonso, C.L. 2019. Rapid, multiplexed, whole genome and plasmid sequencing of foodborne pathogens using long-read nanopore technology. Scientific Reports. 9:16350.
Goraichuk, I.V., Williams Coplin, T.D., Wibowo, M.H., Durr, P.A., Asmara, W., Artanto, S., Dimitrov, K.M., Afonso, C.L., Suarez, D.L. 2020. Complete genome sequences of 11 Newcastle disease virus isolates of sub-genotype VII.2 from Indonesia. Microbiology Resource Announcements. 09(5):e01519-19.
Goraichuk, I.V., Kulkarni, A.B., Williams Coplin, T.D., Suarez, D.L., Afonso, C.L. 2019. First complete genome sequence of currently circulating infectious bronchitis virus strain DMV/1639 of the GI-17 lineage. Microbiology Resource Announcements. 8(34):e00840-19.
Butt, S.L., Moura, V., Susta, L., Miller, P.J., Hutcheson, J.M., Cardenas, G., Brown, C., West, F., Afonso, C.L., Stanton, J. 2019. Tropism of Newcastle disease virus strains for chicken neurons, astrocytes, oligodendrocytes, and microglia. BMC Veterinary Research. 15:317.
Kariithi, H.M., Welch, C.N., Ferreira, H.L., Pusch, E.A., Ateya, L.O., Binepal, Y.S., Lichoti, J.K., Apopo, A.A., Afonso, C.L., Suarez, D.L., Dulu, T.D. 2019. Genetic characterization and pathogenesis of the first H9N2 low pathogenic avian influenza viruses isolated from chickens in Kenyan live bird markets. Infection, Genetics and Evolution. 78:104074.
Yu, Q., Li, Y., Dimitrov, K., Afonso, C.L., Spatz, S.J., Zsak, L. 2020. Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos. Vaccine. 38(4):925-932.
Apopo, A.A., Kariithi, H.M., Ateya, L.O., Binepal, Y.S., Sirya, J.H., Dulu, T.D., Welch, C.N., Hernandez, S.M., Afonso, C.L. 2019. A retrospective study of Newcastle disease in Kenya. Tropical Animal Health and Production. 52:699-710.
Ayala, A.J., Hernandez, S.M., Olivier, T.L., Welch, C.N., Dimitrov, K., Goraichuk, I.V., Afonso, C.L., Miller, P.J. 2019. Experimental infection and transmission of Newcastle disease vaccine virus in four wild passerines. Avian Diseases. 63(3):389-399.
Youk, S., Lee, D., Ferreira, H.L., Afonso, C.L., Absalon, A.E., Swayne, D.E., Suarez, D.L., Pantin Jackwood, M.J. 2019. Rapid evolution of Mexican H7N3 highly pathogenic avian influenza viruses in poultry. PLoS One. 14(9):e0222457.
Goraichuk, I.V., David, J.F., Kulkarni, A.B., Afonso, C.L., Suarez, D.L. 2020. Complete genome sequence of avian coronavirus strain GA08 (GI-27 Lineage). Microbiology Resource Announcements. 9(9):e00068-20.
Goraichuk, I.V., Kapczynski, D.R., Seal, B., Suarez, D.L. 2020. First complete genome sequence of an avian metapneumovirus subtype B strain from Hungary. Microbiology Resource Announcements. 9:e00177-20.
Msoffe, P.L., Chiwangad, G.H., Cardona, C.J., Miller, P.J., Suarez, D.L. 2019. Isolation and characterization of Newcastle disease virus from live bird markets in Tanzania. Avian Diseases. 63(4):634-640.
Ferreira, H.L., Reilley, A.M., Goldenberg, D., Ortiz, I.R., Gallardo, R.A., Suarez, D.L. 2020. Protection conferred by commercial NDV live attenuated and double recombinant HVT vaccines against virulent California 2018 Newcastle disease virus (NDV) in chickens. Vaccine. 38(34):5507-5515.
Goraichuk, I.V., Davis, J.F., Kulkarni, A.B., Afonso, C.L., Suarez, D.L. 2020. A 25-year-old sample contributes the complete genome sequence of avian coronavirus vaccine strain ArkDPI, reisolated from commercial broilers in the United States. Microbiology Resource Announcements. 9(9):e00067-20.
Ferreira, H., Suarez, D.L. 2019. SNP analysis used to select conserved regions for an improved real-time RT-PCR test specific to Newcastle disease virus. Avian Diseases. 63:625-633.