CONTROL OF EMERGING AND RE-EMERGING POULTRY RESPIRATORY DISEASES IN THE UNITED STATES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0219886
• Project No.: ARK02263
• Multistate No.: NC-_OLD1180
• Project Start Date: Oct 1, 2009
• Project End Date: Sep 30, 2014

Project Director
Kong, BY, CA.

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
Poultry Sciences

Non Technical Summary
Avian viral disease, such as avian influenza, Newcastle disease, infectious bronchitis, pneumovirus, Marek?s disease, or laryngotracheitis cause economic losses in poultry industries by decrease productivities. Effective detection and prevention of avian viruses are of the utmost importance to maintain the US leadership in world poultry product markets. Development of more rapidly produced and more effective vaccine is required to minimize the loss in poultry production. This project for optimal vaccine production will address a stable and safer baseline for protection of avian infectious viral pathogens. By doing so, the improvement in sustainability of US poultry may be realized by the much safer control of long-term threat of virus spread (as either an epidemic or even pandemic).

Animal Health Component

50%

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3299	1040	30%
311	3299	1090	20%
311	3299	1101	50%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1101 - Virology;

Keywords

infectious

viruses

vaccine

Goals / Objectives
Identify reservoirs of infectious respiratory disease agents in wild birds and poultry. Develop improved diagnostic capabilities including real time PCR as well as other rapid on-farm tests for economically important respiratory diseases. Investigate the pathogenesis and polymicrobial interactions of specific infectious agents associated with poultry respiratory diseases (this includes interactions with underlying immunosuppressive agents). Develop new prevention and control strategies for poultry respiratory diseases.

Project Methods
Various regulatory factors will be modulated by genetic engineering technologies in primary chicken cells to establish continuously growing cell lines. Negatively regulating factors in both cell cycle and cell lifespan will be down-regulated using gene knock-down technology, while positively regulating factors will be overexpressed using various eukaryotic expression vectors containing strong promoter. For reverse genetic system, RNA genome of specific avian virus will be converted to cDNA by reverse transcriptase, and double stranded genome will be assembled into plasmid vector containing specialized promoter. Further recombinant virus propagation will be perform in continuously growing avian cell substrates. Mutagenesis will be performed on specific regions of the viral genes to be able to determine if these changes are detrimental to the infectivity/virulency of RNA virus. To construct the virulency defective-, but infectious DNA viruses, various viral genes including surface glycoproteins will be removed using homologous recombination between purified viral DNA genome and various reporter plasmids, that contain selectable markers. In the entire processes, the immortal chicken cell lines will be used as cellular substrates for virus propagation and diagnostic uses to detect viruses.

Progress 10/01/09 to 09/30/14

Outputs
Target Audience: Poultry Producers; Animal Vaccine Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Three graduate student have performed this research for one doctoral dissertation and two master theses. How have the results been disseminated to communities of interest? Sequence variations among different ILTV strains can provide important knowledge information for the development of improved diagnostic tools to discriminate between wild type and vaccine strains, or among vaccines strains, and further, to identify newly emerging mutant strains. Immortal chicken liver cell line can be used for the continuously grwoing cell substrate for vaccine production against avian infectious viruses. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Sequence Variation in Viral Genomes of Infectious Laryngotracheitis Virus (ILTV) using Next Generation Sequencing: The study was conducted to identify the unique variations in wild type and vaccine strain of ILTV genomes to obtain complete information of mutations within the entire genomes compared to a reference ILTV genome sequence (Gallid herpesvirus 1, complete genome; NCBI Reference Sequence: NC_006623.1). Genomes of one wild type and two vaccine ILTV strains were sequenced using Illumina Genome Analyzer 2X of 36 cycles of single-end read. Analysis of the ILTV genome sequences was carried out using trial version of DNAStar software (including NGen, SeqMan Pro, Gen Vision), Blastx and Microsoft Excel. Results showed many amino acid differences distributed across the viral protein coding regions of the three ILTV genomes compared to the reference genome sequence. The sequence differences demonstrated the spectrum of variability among the wild type and vaccine strains. Amino acid differences between wild-type and vaccines were found in several genes including UL52, ORFB, ORFE, UL19, UL5, UL0, US10 (in IR), US3, and US5. Moreover, the results also revealed 52 genes with complete conservation across the four strains (3 strains plus reference strain). As another project, the expression levels of all seventy-six ORFs in ILTV were determined during lytic replication phase in cultured cells using quantitative reverse transcription PCR (qRT-PCR). Total RNA was extracted from ILTV infected chicken embryo lung cells at different time points of 1, 3, and 5 day post infection (dpi). According to qRT-PCR result, the expression of many ILTV genes were highly increased from 1 dpi, and the expression levels were maintained until 3 dpi. The expression levels of sixteen genes were slightly decreased at 3 dpi, while eight genes showed higher expression levels at 3 dpi compared to 1 dpi. Generally, the expression levels of most genes were decreased at 5 dpi showing massive cell death by cytopathic effect (CPE). Specifically, the expression of both UL22, envelop glycoprotein H acting cell-to-cell spreading, and UL29, which functions in DNA replication, was increased from 1 dpi, while the expression of UL21, UL33, and US6 genes, which function in virion morphogenesis, DNA encapsidation, and envelop glycoprotein D, began to increase at 3 dpi. The UL23 gene encoding thymidine kinase, which is responsible to generate the CPE, was highly expressed at 3 dpi. The ICP4, transcriptional regulator and an immediate early gene, was expressed much earlier dpi time points, and the increased level was maintained until 5 dpi. The study to compare nucleotide sequences of two chicken embryo origin (CEO) vaccines of infectious laryngotracheitis virus (ILTV) genomes were completed and published to Virus Genes. Results indicated that the US CEO strains are practically identical to the Australian Serva CEO strain, which is a European-origin vaccine. The sequence differences demonstrated the spectrum of variability among vaccine strains. Only 8 amino acid differences were found in ILTV proteins including UL54, UL27, UL28, UL20, UL1, ICP4, US8, and US10. Comparison of ILTV genome sequences of two US CEO vaccines This study was conducted to identify unique nucleotide differences in two US chicken embryo origin (CEO) vaccines (LT Blen designated as vaccine 1; Laryngo-Vac designated as vaccine 2) of entire infectious laryngotracheitis virus (ILTV) genomes compared to previously known ILTV genome sequences. Currently, six ILTV genome sequences were deposited into GenBank including 63140/C/08/BR (GenBank accession: HM188407), USDA reference challenge (GenBank accession: HM188408), Serva vaccine (GenBank accession: HQ630064), two Australian-origin CEO vaccines, SA2 (GenBank accession: JN596962) and A20 (GenBank accession: JN596963), and composite ILTV genome sequence (GenBank accession: NC006623). Comparison results revealed that few nucleotide differences were found among two US CEO vaccines, Serva vaccine, and 63140/C/08/BR, and indicate that the US CEO and 63140/C/08/BR strains are practically identical to the Australian Serva CEO strain, which is a European-origin vaccine. Further comparison of US CEO vaccines to several ILTV genome sequences revealed that US CEO vaccines are genetically distinct from the two Australian-origin CEO vaccines, SA2 and A20, which showed close similarity. A continuously growing immortal cell substrate can be utilized for virus propagation, diagnostic purposes, and vaccine production. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. CEL-im cells are rapidly growing cell line. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus (ILTV). CEL-im cells produced high AMPV titer, while very low titers for MDV-1 and ILTV were produced. CEL-im cell line has negative telomerase activity and when compared to the primary passage 2 CEL cell counterpart, mRNA expression of p53, Mdm2, p21WAF and p16INK4 were down-regulated in the CEL-im cell line, while RB, E2F-1, and ARF were up-regulated. The newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: ee J, Foster DN, Bottje WG, Gentles LE, and Kong BW. 2013. Establishment of an immortal chicken embryo liver derived cell line. Poultry Science. 92(6):1604-12.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Chandra YG, Lee JY, and Kong BW. 2012. Genome sequence comparison of two vaccine infectious laryngotracheitis virus (ILTV) strains. Virus Genes. 44(3):470-4
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Chandra YG, Lee JY, and Kong BW. (2011). Sequence variation in viral genomes of infectious laryngotracheitis virus (ILTV) using next generation sequencing. International Poultry Science Forum, Atlanta, GA. January 24-25.
• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Kong BW, Chandra YG, and Lee JY. (2011). Sequencing of Infectious Laryngotracheitis Virus (ILTV) Genomes using Illumina Platform. 30th Annual meeting of American Society for Viology. University of Minnesota, Minneapolis, MN. July 16-20.
• Type: Conference Papers and Presentations Status: Published Year Published: 2010 Citation: Lee JY and Kong BW. (2010). The analysis of gene expression of infectious laryngotracheitis virus during lytic replication phase in cultured cells. 29th Annual meeting of American Society for Viology. Montana State University, Bozeman, Montana. July 17-21.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Poultry Producers; Animal Vaccine Industry Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One graduate student has performed this research for doctoral dissertation. 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? CEL-im cells will be examined for the propagation of additional infectious viruses.

Impacts
What was accomplished under these goals? A continuously growing immortal cell substrate can be utilized for virus propagation, diagnostic purposes, and vaccine production. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. CEL-im cells are rapidly growing cell line. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek’s disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus (ILTV). CEL-im cells produced high AMPV titer, while very low titers for MDV-1 and ILTV were produced. CEL-im cell line has negative telomerase activity and when compared to the primary passage 2 CEL cell counterpart, mRNA expression of p53, Mdm2, p21WAF and p16INK4 were down-regulated in the CEL-im cell line, while RB, E2F-1, and ARF were up-regulated. The newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Lee J, Foster DN, Bottje WG, Gentles LE, and Kong BW. 2013. Establishment of an immortal chicken embryo liver derived cell line. Poultry Science. 92(6):1604-12.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The study to compare nucleotide sequences of two chicken embryo origin (CEO) vaccines of infectious laryngotracheitis virus (ILTV) genomes were completed and published to Virus Genes. Results indicated that the US CEO strains are practically identical to the Australian Serva CEO strain, which is a European-origin vaccine. The sequence differences demonstrated the spectrum of variability among vaccine strains. Only 8 amino acid differences were found in ILTV proteins including UL54, UL27, UL28, UL20, UL1, ICP4, US8, and US10. PARTICIPANTS: Chandra, Y. G.; Completed Master of Science Program at Cell and Molecular Biology Graduate program, University of Arkansas. TARGET AUDIENCES: US poultry industry; Animal/poultry vaccine industry PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This information can be used to discriminate between vaccine ILTV strains and further, to identify newly emerging mutant strains of field isolates.

Publications

• Chandra YG, Lee JY, and Kong BW. 2012. Genome sequence comparison of two vaccine infectious laryngotracheitis virus (ILTV) strains. Virus Genes. 44(3):470-4

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Comparison of ILTV genome sequences of two US CEO vaccines This study was conducted to identify unique nucleotide differences in two US chicken embryo origin (CEO) vaccines (LT Blen designated as vaccine 1; Laryngo-Vac designated as vaccine 2) of entire infectious laryngotracheitis virus (ILTV) genomes compared to previously known ILTV genome sequences. Currently, six ILTV genome sequences were deposited into GenBank including 63140/C/08/BR (GenBank accession: HM188407), USDA reference challenge (GenBank accession: HM188408), Serva vaccine (GenBank accession: HQ630064), two Australian-origin CEO vaccines, SA2 (GenBank accession: JN596962) and A20 (GenBank accession: JN596963), and composite ILTV genome sequence (GenBank accession: NC006623). Comparison results revealed that few nucleotide differences were found among two US CEO vaccines, Serva vaccine, and 63140/C/08/BR, and indicate that the US CEO and 63140/C/08/BR strains are practically identical to the Australian Serva CEO strain, which is a European-origin vaccine. Further comparison of US CEO vaccines to several ILTV genome sequences revealed that US CEO vaccines are genetically distinct from the two Australian-origin CEO vaccines, SA2 and A20, which showed close similarity. PARTICIPANTS: Chandra, Y. G.; Graudate Training at Cell and Molecular Biology Graudate program, University of Arkansas TARGET AUDIENCES: Sequence variations found in the vaccine strains can be used to discriminate between vaccine ILTV strains and further, to identify newly emerging mutant strains of field isolates. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This information can be used to discriminate between vaccine ILTV strains and further, to identify newly emerging mutant strains of field isolates.

Publications

• Chandra YG, Lee JY, and Kong BW. (2011). Sequence variation in viral genomes of infectious laryngotracheitis virus (ILTV) using next generation sequencing. International Poultry Science Forum, Atlanta, GA. January 24-25.
• Kong BW, Chandra YG, and Lee JY. (2011). Sequencing of Infectious Laryngotracheitis Virus (ILTV) Genomes using Illumina Platform. 30th Annual meeting of American Society for Viology. University of Minnesota, Minneapolis, MN. July 16-20.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Sequence Variation in Viral Genomes of Infectious Laryngotracheitis Virus (ILTV) using Next Generation Sequencing: The study was conducted to identify the unique variations in wild type and vaccine strain of ILTV genomes to obtain complete information of mutations within the entire genomes compared to a reference ILTV genome sequence (Gallid herpesvirus 1, complete genome; NCBI Reference Sequence: NC_006623.1). Genomes of one wild type and two vaccine ILTV strains were sequenced using Illumina Genome Analyzer 2X of 36 cycles of single-end read. Analysis of the ILTV genome sequences was carried out using trial version of DNAStar software (including NGen, SeqMan Pro, Gen Vision), Blastx and Microsoft Excel. Results showed many amino acid differences distributed across the viral protein coding regions of the three ILTV genomes compared to the reference genome sequence. The sequence differences demonstrated the spectrum of variability among the wild type and vaccine strains. Amino acid differences between wild-type and vaccines were found in several genes including UL52, ORFB, ORFE, UL19, UL5, UL0, US10 (in IR), US3, and US5. Moreover, the results also revealed 52 genes with complete conservation across the four strains (3 strains plus reference strain). As another project, the expression levels of all seventy-six ORFs in ILTV were determined during lytic replication phase in cultured cells using quantitative reverse transcription PCR (qRT-PCR). Total RNA was extracted from ILTV infected chicken embryo lung cells at different time points of 1, 3, and 5 day post infection (dpi). According to qRT-PCR result, the expression of many ILTV genes were highly increased from 1 dpi, and the expression levels were maintained until 3 dpi. The expression levels of sixteen genes were slightly decreased at 3 dpi, while eight genes showed higher expression levels at 3 dpi compared to 1 dpi. Generally, the expression levels of most genes were decreased at 5 dpi showing massive cell death by cytopathic effect (CPE). Specifically, the expression of both UL22, envelop glycoprotein H acting cell-to-cell spreading, and UL29, which functions in DNA replication, was increased from 1 dpi, while the expression of UL21, UL33, and US6 genes, which function in virion morphogenesis, DNA encapsidation, and envelop glycoprotein D, began to increase at 3 dpi. The UL23 gene encoding thymidine kinase, which is responsible to generate the CPE, was highly expressed at 3 dpi. The ICP4, transcriptional regulator and an immediate early gene, was expressed much earlier dpi time points, and the increased level was maintained until 5 dpi. PARTICIPANTS: Byung-Whi Kong (UARK) serves as PI. In UARK, Jeong Yoon Lee and Chandra Trihadi are participated as graduate students. TARGET AUDIENCES: US poultry industry; Animal/poultry vaccine industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Sequence variations among different ILTV strains are expected to provide insight into the discrimination between wild type and vaccine strains, or among vaccines strains, and further, to identify newly emerging mutant strains. Further, the conserved coding sequences among various ILTV strains can become promising targets to discover a universal, safer vaccine against ILTV. In addition, results of ILTV gene expression may provide insights into the molecular mechanisms of ILTV genome replication during the cytolytic infection phase and further its pathogenesis.

Publications

• Lee JY and Kong BW. (2010). The analysis of gene expression of infectious laryngotracheitis virus during lytic replication phase in cultured cells. 29th Annual meeting of American Society for Viology. Montana State University, Bozeman, Montana. July 17-21.

Progress 10/01/09 to 12/31/09

Outputs
OUTPUTS: For the genetic attenuation for ILTV, various efforts are being tried to produce mutant strains of ILTV using homologous recombination between transfer vectors containing flanking sequences of target viral genes and ILTV genomic DNA (or live ILTV virus). Genetically attenuated ILTV, which can be utilized as a new type of live vaccine, should be produced from continued research. Uisng functional genomics techniques, such as microarray analysis or next generation sequencing method, a new diagnosic tool is being developed to detect/distinguish poultry respiratory viral pathogens, and host-virus interaction is being studied to explore a optimal disease control strategy. PARTICIPANTS: Byung-Whi Kong (UARK) serves as PI. In UARK, Jeong Yoon Lee and Chandra Trihadi are participated as graduate students. As collaborators, Billy M. Hargis (UARK; [e-mail]) and Douglas N. Foster (UMN; [e-mail]) are participated in this project. TARGET AUDIENCES: US poultry industry; Animal/poultry vaccine industry PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Genetically attenuated ILTV vaccine, which would not raise vaccinal ILT, would become a potentially optimal vaccine to protect poulrty from ILT disease.

Publications

• No publications reported this period