Brief Summary of Minutes of Annual Meeting

The annual meeting was adjourned at 5:00 pm December 3 2005. BRIEF SUMMARY OF MINUTES OF ANNUAL MEETING The annual NC 1019 business meeting was held on Friday December 2 2005 at Davos room in the Sheraton Westport Plaza Hotel, St. Louis, MO. Dr. David Suarez, Chair of NC 1019 opened the meeting at 2:00 pm. He welcomed the committee advisor Dr. Jeff Klausner, new member Dr. Joe Jiambrone from Alabama, and guests Dr. John Glisson from Georgia and Dr. W Lee from Ohio, Dr. J. Sharma who sat in for Dr. M. Njenga from Minnesota, and Dr. C. Keeler from Delaware. Dr. Peter Johnson, USDA/CSREES representative, joined the group around 4 pm. Dr. Klausner commended the group for a successful submission and approval of the new project and gave a special thank to the past Chair Dr. Jack Gelb for the time and effort he spent during the process. Dr. Klausner also reminded the Chair and Secretary that the minutes of the meeting should be submitted to his office within 30 days after the meeting is held. Dr. Peter Johnson briefed the group with the CSREES budget appropriation process. Most categories in the budgets, including the NRI, stayed the same with the exception that a new appropriation of $500,000 for Veterinary Medical Service Act was added. Dr. Johnson shared with the group the research priority list for 2006 NRI. He indicated that CSREES continued their process in soliciting input from federal, state and local partners to guide competitive programs as they increase focus. He requested NC 1019 multi-state committee to also provide consensus feedback on the research priority again for 2007. In response to Dr. Johnsons request, the group discussed and prioritized diseases of importance. While there are many important diseases, three came to the top for this year: ILT>IBDV>APV. Since AI has been in many lists for funding (including CSREES and HHS), the group did not recommend AI. Drs. David Suarez and Ching Ching Wu will prepare a feedback letter to Dr. Johnson reflecting our consensus. In addition, the letter will recommend CSREES to add Emerging and reemerging diseases to b. Foreign Animal Diseases under the Priority for Research 2) Non-species specific, high priority areas. This will allow the inclusion of emerging diseases such as broiler and poult enteritis to the example lists given in this category. The meeting venue for the next year was discussed and it was voted to have our next meeting held at the end of January 2007 in Atlanta in conjunction with the International Poultry Exposition and the AI CAP annual meeting. Drs. Suarez will finalize the detail on meeting place, date, and time. He will inform the members of the specifics as soon as they are available. The members will make a decision in Atlanta as to the future meeting plans. The meeting will remain closed but the Chair and Secretary can invite guests per members suggestion or as needed. The business meeting end at 5:00 pm December 2 2005. Station progress report resumed at 8:00 am Saturday December 3 2005. We had excellent discussion on the clinical aspects, pathogenesis, diagnostics, and vaccine/immunology of various poultry disease research among the stations. Since vvIBDV could pose serious threat to the poultry industry, upon hearing the station report from Indiana on new and sensitive vvIBDV detection assay, Dr. Sharma suggested that Drs. Saif and Wu look into the availability and validation of current available vvIBDV detection assays and recommended the committee to urge NAHLN and HHS to provide funds to support what is needed for the inclusion of this assay to their current panel. The committee will provide a letter in support of this request. At the end of the meeting, Dr. Suarez requested that each station submit the annual report and the collaboration records electronically to him ASAP. Minutes Submitted by C.C. Wu

Objective 1. Determine the pathogenesis and interactions of specific agents. Avian Influenza Virus Quail have previously been suggested as host that can support replication of a number of avian influenza viruses. Quail and other species were examined for the ability of viruses to attach to different tissue types. Quail trachea and intestine appeared capable of binding both avian and human lineage viruses. Human trachea/bronchial epithelial (HTBE) cells were also evaluated for the ability to support influenza replication. These cultured cells included a variety of cell types that were polarized. This cell line allowed avian viruses with human virus receptor specificity to replicate, but avian viruses with normal receptor specificity did not. This appears to be a useful model to examine virus replication and host specificity.(U Maryland) We characterized a highly pathogenic outbreak of H5N1 from poultry from South Korea for its relationship to other outbreaks in the region and its potential for crossing the species barrier. We performed sequence analysis and animal studies of the outbreak in conjunction with the Centers for Disease Control in Atlanta and the Veterinary Research and Quarantine Service in South Korea. The results suggested a multiple point source of introduction which helped shape the regulatory response to the outbreaks. Reporting of low pathogenicity avian influenza outbreaks in poultry in the USA has resulted in trade embargos on chicken meat. Avian Metapneumovirus (AMV) Studies were initiated to examine the ability of Avian metapneumovirus (AMV) to induce mucosal cellular and humoral immunity in the upper respiratory tract (URT). Avian metapneumovirus in turkeys revealed that the attenuated and virulent strains of the virus induced IgA+ cells in the respiratory mucosa of the upper respiratory tract (URT). Turkey macrophages were also shown to be susceptible to in vitro infection and activation by AMV. (U Minnesota) The sequence of the Colorado 96 AMV isolate was completed. The virus was similar to a human metapneumovirus and to other type C viruses from Minnesota. The virus was more divergent with the types A and B AMV isolates found in other countries. The g protein was highly variable with up to 19 % sequence divergence with the Minnesota viruses. Using the cloned genes in a reverse genetic system, a complete genome was rescued of the type C AMV virus. The rescued virus biological was the same as the parent virus. (U Maryland) Newcastle Disease Virus Newcastle disease virus is a negative sense single stranded virus infection of poultry. The virus encodes for multiple proteins that are produced as viral constructs separated by short intergenic sequences. In an effort to evaluate the role these intergenic sequences have on viral virulence, reverse genetics was used to modify the length of the sequences. The viruses were rescued and little difference was seen in growth in cell culture, but in general a decrease in virulence was seen when chickens were challenged with the virus. The use of reverse genetics was also evaluated for the examination of the virulence role of the polymerase gene, which is important for virus replication. The polymerase gene was swapped from a virulent virus to a non-virulent virus. These studies suggest a role for the polymerase gene as a possible virulence factor.(U Maryland) Characterization of infection and disease of turkeys with NDV strains of low to high virulence. In general, disease among NDV infected turkeys was found to be less severe than in similarly infected chickens, and turkeys infected with virulent strains shed virus for a longer time and appeared to be subclinical carriers for some of the isolates. (SEPRL) Infectious Bursal Disease Virus Positive sense RNA transcripts of Infectious bursal disease virus (IBDV) genome segments A and B have previously been shown to be infectious. We demonstrated that recovery of IBDV from the transfection of Vero cells with positive sense RNA transcripts of genome segments A and B was enhanced by expression of the viral structural proteins VP2 with VP3 or by expression of viral polyprotein VP243 from DNA plasmids in trans. Expression of individual viral proteins VP2, VP3, or VP4 alone from DNA plasmids did not enhance IBDV recovery. Earliest virus recovery from transfection of positive sense RNA transcripts of genomic segments A and B was at 36 h and mean titers were 101.8 pfu/ml. IBDV was recovered 6 hours after transfection in cells concurrently expressing either VP2 with VP3 or VP243 and mean titers were 108.5 pfu/ml or 109.2 pfu/ml, respectively. Likewise, expression of the viral polyprotein from DNA plasmid increased the permissiveness of Vero cells for infection with non-culture adapted IBDV. The titer of recovered non-culture adapted virus from 103.3pfu/ml to 1010.3pfu/ml with expression of the viral polyprotein. (Purdue U) Objective 2. Surveillance, occurrence and consequences of agents and host variation on disease susceptibility. Avian Influenza Over 5000 poultry samples were tested by real-time RT-PCR (RRT-PCR) and over 3000 blood samples by AGID were tested and found to be negative for avian influenza from live bird markets and backyard flocks in the New England States. (U Connecticut) 60 wild bird samples were collected from Alabama and tested by virus isolation for avian influenza. All the samples were negative. (Auburn U) H3N2 viruses have been isolated from outbreaks in several states from turkey breeder flocks experiencing drops in egg production. The antigenic relatedness of four isolates, the IL isolate, OH isolate, an NC recent turkey isolate, and the swine vaccine used in Illinois, were compared and a high antigenic relatedness was observed between the turkey isolates, but only a 10% relatedness of the swine vaccine to the turkey isolates. The variability of circulating H3N2 viruses may affect how vaccination is used to control this problem. (Ohio State U) Infectious Bursal Disease Virus (IBDV) A comparison of the detection of antibodies against serotypes 1 and 2 IBDV by commercial ELISA kits indicated that currently available commercial ELISA kits detected antibodies elicited by the two serotypes of IBDV. Hence, the prevalence of serotype 2 antibodies in the flocks should be considered while determining antibody profiles of the flocks against serotype 1 viruses. (Ohio State U) Objective 3. Develop new and improved methods for the diagnosis, prevention, and control of avian respiratory diseases. Avian Influenza An Adenovirus-vectored vaccine, originally developed for humans against H5N1, was given to SPF leghorns in ovo and was shown to produce measurable antibody titers. The birds were challenged with a highly pathogenic avian influenza virus and most birds were clinically protected from disease. This vector system is replication restricted virus and therefore it has the safety of a killed vaccine, but the immune response of a live vaccine. The use of in ovo vaccination offers the possibility of mass vaccination of poultry during an avian influenza outbreak. (Auburn U and SEPRL). The use of DNA vaccines for use in the production of reference diagnostic reagents were improved with the addition of the cytokine adjuvants, IL-2 and interferon. DNA vaccines can produce high quality antibodies to specific proteins that are extremely valuable for diagnostic reagents, but the response to DNA vaccines are variable. Alternative methods for adjuvanting or increasing the immune response were conducted for both the H5 and H7 hemagglutinin protein of avian influenza, and it was found that both the inclusion of plasmids with the cytokines IL-2 and type 1 interferon improved the response for H7 vaccines. This work will allow the improved production of reference antibodies in the future that are safer and easier to produce than the current methods.(SEPRL) Avian Metapneumovirus (AMV) Turkeys were immunized with adjuvanted rNP and recombinant M protein (rMP) administered intramuscularly and immunized and unimmunized controls were challenged with virulent AMV by the respiratory route. At a dose of 40 ug/bird, rNP protected eight of nine birds. rMP at the same dosage level protected three out of seven birds. At a dose of 80 ug of each rNP and rMP, 100% protection was achieved. This recombinant vaccine shows promise as an improved control measure for AMV. Another study was designed to determine if in vivo passages of AMV would increase virus virulence leading to consistent clinical signs in turkeys. The results of this preliminary study indicate that in vivo passage of AMV in birds may increase virus virulence and the resulting virus could serve as a suitable challenge inoculum for use in vaccination trials. Currently Vero cells are commonly used for the isolation of AMV from clinical samples, but because Vero cells are a mammalian cell line, concern that the virus is changed by passage in this cell line is a concern. Sequence analysis supports this idea, since 11 amino acid differences in the F gene of AMV propagated in turkey cells and that propagated in Vero cells. In an attempt to find a better avian origin cell line for isolation of AMV, alternatives were tested. A non-tumorigenic immortal turkey turbinate cell line was developed that is susceptible to AMV, and may provide a valuable alternative to Vero cells. (U Minnesota) Infectious Bursal Disease Virus (IBDV) IBDV is an immunosuppressive disease found in chickens. A mild form is found in the U.S., but very virulent forms are found in many other countries. A sequence comparison of the virulent and non-virulent forms showed sequence difference that were used as a differential RT-PCR test. A diagnostic assay was developed that reliably differentiated very virulent infectious bursal disease virus (vvIBDV) from non-vvIBDV strains. The availability of of rapid diagnostic tests facilitate identification of field isolates, and allow a rapid response for control of highly virulent IBDV viruses are introduced into the U.S. IBDV also exists as two antigenically different groups, and a RT-PCR test was developed that could rapidly differentiate serotype 1 viruses, serotype 2 viruses, and the vv strains of IBDV. These tools allow the rapid differentiation of the different serotypes to provide rapid characterization of the viruses. (Ohio State U) A real-time RT-PCR assay was developed utilizing dual-labeled fluorescent probes binding to VP4 sequence that are specific to the classical, variant and very virulent strains of Infectious Bursal Disease Virus (IBDV). The assay was highly sensitive and could detect as little as 3 ´ 102 to 3 ´ 103 copies of viral template. The variant sequence-specific probe was found to be highly specific in detecting isolates classified as variant A, D, E, G and GLS-5, and did not react with classical strains. The classical sequence-specific probe also demonstrated high sensitivity and specificity and differentiate between isolates that were variant and classical strains. The very virulent sequence-specific probe positively detected the Holland vvIBDV isolate and did not react with classical or variant strains. (Purdue U) WORK PLANNED FOR THE COMING YEAR University of Connecticut- Surveillance of wild birds for avian influenza collected from tracheas and cloacal samples from Connecticut will be tested. Multiplex PCR (MPCR) for avian influenza and subtypes H5, H7 and H9 is being developed at the Guangxi Veterinary Research Institute Nanning, China. MPCR will be tested on the North American AI isolates to confirm its sensitivity and specificity. Auburn University- The wild bird surveillance for avian influenza will be increased in the next year. Additional studies of antibody response in wild birds will be conducted. The Mx gene will be evaluated for protection against avian influenza in different poultry lines. Comparison of different procedures for poultry litter management will be evaluated to determine if it adequately inactivates Infectious Laryngotracheitis Virus (ILTV) and AIV. The Ohio State University- Surveillance for influenza and pneumoviruses will continue using for serologic and molecular diagnostic tests. Studies will be initiated on the molecular changes in influenza viruses associated with crossing the species barrier. Production and characterization of monoclonal antibodies to the vvIBDV will continue. Purdue University- We will examine the pathogenesis of IBDV using reverse genetically engineered strains for the purpose of understanding the molecular events and mechanisms by which the virus interacts with bursa of Fabricius. We will also study additional chicken cytokine genes on immune response and protection of chickens against IBD by DNA vaccination. We will also study the effect of boosting with transgenic algae expressing IBDV VP2 on DNA vaccination of chickens against IBD.

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