Progress 09/01/13 to 08/31/14
Outputs
Target Audience: The target audience reached over this past year includes avian virologists and epidemiologists interested in vaccinology and in the role of wild birds in transmission of avian diseases. Changes/Problems: In restrospect, Aim 1 - even though a valid question, may not have been likely to result in a postive outcome due to the replicaiton of NDV occuring in the cytoplasm and AIV in the nucleus. None the less, the hypothesis was tested. The difficulties in obtaining invasive species (European House Sparrows and European Starlings) were not foreseen. In previous years these birds were obtained easily. Substituting Japanese Quail was a good solution as there are often outbreaks of ND in these birds and these birds are kept with other type of avian species. 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? Almost all of the samples from Aim 2 are completed, but the data needs to confirmed and compiled. The samples from Aim three are being analyzed. Two manuscripts have begun to be put together. The first contains data from Aims 1 and 2 and the second will have data from all 3 species from Aim 3. Once the data is compiled we will also share this data with interested parties at the Center for Veterinary Biologics and also some vaccine companies who are currently developing these sorts of vaccines for commercial use. This data will be in the final report which will be submitted within 90 days of the anniversary start date, which was Sept 01, 2010.
Impacts
What was accomplished under these goals?
Aim 1 was completed prior to the submission of the third year progress report. There was no evidence of a recombinant Newcastle disease virus containing a gene from avian influenza swapping that gene with a wild type avian influenza strain even with matching sequences. Over this past year Aim 2 was completed as written in the Aims portion of the grant. Four strains of Newcastle disease virus were passed in 14 day old embryos to select for a virulent strain. After this passage there was no change in the fusion cleavage site, nor in the mean death times, suggesting that there was no increase in virulence. Ten of these samples based on having the lowest mean death time were used in an intracerebral pathogencity index assay and no increase in virulence was observed. This completed the aim. However, due to the negative data we decided to further test these ten fluids to increase our confidence in the results. These ten fluids were blindly passaged in 14 day old embryos and the fluids were harvest after each passage. The tenth passage was evaluated for mean death time, sequence of the fusion cleavage site and the intracerebral pathogencity index. This data will be completed in the next 30 days. Aim 3 proposed to evaluate viruses in 3 wild bird species. 1.5 of these species was completed prior to the 3rd progress report the 1/2 of the second species was completed this year and the data is being analyzed. The third species proposed was European Starlings. Due to the inabilty to procure any of these birds, Japanese Quail were substituted. These experiments have also been completed and the data is being analyzed. The initial results show that like pigeons, sparrows, too can trasmit the virus to naive contacts. The viruses replicate well in both species.
Publications
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Progress 09/01/10 to 08/31/14
Outputs
Target Audience: Poultry veterinarians, vaccine scientists, and regulators of veterinary vaccines Changes/Problems: In the original proposal we planned to use European Starlings, but none were able to be captured or bought. We used Japanese Quail instead. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest? Recombinant Newcastle disease vaccines: risk for reversion to virulence and spread in non-target species presented at the Southern Conference of Avian Diseases in association with the International Poultry Exposition, January 22-24, 2013, Atlanta, GA. Experimental Risk assessment of recombinant Newcastle disease virus vaccines presented at the American Association of Avian Pathologists meeting in association with the American Veterinary Medical Association, July 19-23, 2013, Chicago, IL. Evaluation of the infection and transmission of wild type and recombinant strains of Newcastle disase virus in Japanese Quail presented at the Southern Conference of Avian Diseases in association with the International Poultry Exposition, January 26-27, 2015, Atlanta, GA. Dynamics of House Sparrows (Passer domesticus) in Newcastle disease virus transmission within the avifaunal-poultry interface: an epidemiological modeling approach presented at the Southern Conference of Avian Diseases in association with the International Poultry Exposition, January 26-27, 2015, Atlanta, GA. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Live vaccines for Newcastle disease (ND) are made from strains of Newcastle disease virus (NDV). Since 1999 scientists have been able to make specific changes in the genetic material of these NDV strains in the lab. Not only can these viruses be used to make ND vaccines to control ND in poultry, but foreign genes from other avian pathogens can be inserted into the NDV genome making a recombinant NDV (rNDV) that can be used to make vaccines to control other diseases of poultry. For example, the HA gene of avian influenza virus can be insterted into the NDV and used as a vaccine to control both avian influenza and ND. These rNDV are used as live ND vaccines in Mexico and China. We are testing in a laboratory setting the stability and safety of these rNDV. As a virus passes from one bird to another, the genetic makeup of viruses may change and lead to a virus that causes more illness. The viruses can also change if they infect a host that is already infected with another virus by exchanging genetic material with that other virus. While poultry species (chickens, turkeys) are the intended target for these vaccines, other species that congregate around poultry could easily be infected by them. We are testing in a laboratory setting the stability and safety of these types of viruses in pigeons, sparrows and quail . Poultry are often infected with avian influenza virus and Newcastle disease virus at the same time. AIM 1: To evaluate if genetic material could be swapped between two viruses when a host is infected with both a rNDV containing a HA gene of avian influenza virus and an avain influenza virus. Summary: When a rNDV containing a AIV gene infected a host that was also infected with a wild-type AIV of low virulence there was no indication that the two viruses swapped or exchanged genetic material. Even when conditions were optimized to allow for the best chance of swapping of genetic material, there was no evidence of recombination IMPACT: While you cannot prove a negative, under the experimental conditions of this study, some of which optimized the exchange of genetic material, there was no evidence that a rNDV with an AIV gene would exchange the gene with a wild-type AIV. These data suggest this rNDV-AIV construct is stable and safe to use in poultry. Note that efficacy as a vaccine was not tested. After a viruses passes from one bird to another it may be able to cause more disease and death over time. Aim 2: Pass NDV strains in multiple hosts and evaluate at the end if there was an increase in the viruses ability to cause disease or death. Summary: Two rNDV and two wild-type NDV were passed in older embryos that are able to select for more virulent viruses. Even with ten additional blind passes all but one of the viruses remained as an NDV of low virulence. Further characterization is underway to evaluate if the one sample that has an increase in virulence is merely due to contamination or if a mutation occurred in the virus to change the virulence. IMPACT: Again, it is difficult to prove a negative, but both wild-type NDV and one rNDV remained stable and had not changes in virulence. One rNDV may have collected mutations to allow a change in virulence, but if so it is not at the typical fusion cleavage site. A final conclusion cannot be made until more information is obtained about the sequence of the entire genome of this sample. Birds that mingle with poultry are able to pick up vaccine viruses from the poultry. We wanted to know if the viruses were able to replicate in these types of birds (pigeons, sparrows and quail) and if these birds got sick. Aim 3: rNDV vaccines are able to infect pigeons, sparrows and quail and spread to nearbye contact pigeons, sparrows and quail. Pigeon and quail were equally susceptible to infection with rNDV and rNDV-AIV and shed these viruses up to 14 days after they were infected and transmitted the viruses to contact birds. Pigeons infected with rNDV also shed virus for 10-14 days and contact birds were infected. The sparrows were infected with 20% and 100% when they were infected with two different rNDV strains. These strains transmitted to 0% and 100% of the contact birds, respectively. No clinical disease was observed in any of the pigeons, sparrows or quail infected with these rNDV strains under these experimental conditions. While virus isolation is complete for all three experiments, titration of the amounts of virus shed in oropharyngeal and cloacal swabs is on-going. Summary: All of the rNDV strains infected pigeons, sparrows and quail, and all the viruses transmitted to the contact birds. No clinical disease was observed in these birds when they were infected with rNDV. IMPACT: These preliminary findings suggest that our system is suitable to assess the risk associated with recombinant NDV vaccines. While these rNDV of low virulence and rNDV-AIV did not produce disease they did infect and spread to other pigeons, sparrows and quail highlighting the importance of biosecurity. Not only is biosecurity needed to keep wildlife away from poultry to prevent diseases from the wild-birds being transmitted to the poultry, but also the NDV vaccines of poultry have the ability to infect wild birds. Overall Summary: Under these experimental conditions there was no evidence of recombination between NDV containing an AIV gene and wild type AIV (aim 1). A final conclusion is pending on the outcome of aim 2. While the majority of the isolates had no increase in virulence after being passed in an embryo system that selects for more virulent viruses, there is still one rNDV sample being analyzed. For aim 3, Rock Pigeons, European House Sparrows, and Japanese Quail were able to be infected with rNDV and rNDV-AIV and spread these viruses to contact birds of the same type. Each virus behaved slightly different in that different percentages of birds were infected and different amounts of contact birds were infected. However, no signs of ND were observed in any of the birds infected with the rNDV or rNDV-AIV tested in these experiments. Biosecurity to keep poultry and wild-birds separated is critical. Wild-birds have the ability to be infected with vaccine strains used in poultry and they can transmit these viruses to other birds. Impact: The rNDV used in these study seem to be as stable and safe as wild type NDV strains. Final results are pending.
Publications
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Progress 09/01/12 to 08/31/13
Outputs
Target Audience: The target audience for this reporting period inlcuded poultry veterinarians, poultry scientists and poultry producers. Data were presented at the American Association of Avian Pathologists annual meeting thatwas held in conjunction with the annual American Veterinary Medical Association conference in July of 2013 in Chicago, Illinois. 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? Data were presented in July of 2013 at the American Association of Avian Pathologists in an oral presentation and also in a poster format at a local, regional meeting Southern Conference of Animal Diseases in January of 2013. What do you plan to do during the next reporting period to accomplish the goals? Aim 1 is complete. For Aim 2 the ICPI will be completed by September 2013. The blind passages will be completed by Oct 2013 and the sequencing of the fusion cleavage site of the samples that are passaged blindly will be completed by November 2013. Aim 3. Procurement of European Starlings and House sparrows has been difficult. There are only a fewpeople with permits in the surrounding areaallowing them to catch these birds. The people who originally agreed to obtain the birds for us are not able to obtain them for us, but I have found another permited person who is in the process of obtaining House sparrows.
Impacts
What was accomplished under these goals?
Aim 1 data demonstrated that under optimal experimental conditions, when a strain of avian influenza virus (AIV) and a recombinant Newcastle disease virus that contained a portion of the AIV sequence are incubated together in an embryonating egg, there was no evidence of homologous or non-homologous re-combinantion. Aim 2 data demonstrated that rNDV with fusion cleavages sites of low virulence incubated in embryonating eggs after one passage are not likely to revert to a virulent status under the experimental conditions of this proposal. Aim 3 data demonstrated that infected and contact pigeons replicated and shed low levels of rNDV.
Publications
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: Specific aim 1: Co-infection of 14 day-old (n=900) embryonating eggs with a recombinant LaSota vaccine virus containing the HA subtype H5 (rLaSota-AI/H5) gene of avian influenza virus and wild type avian influenza viruses of subtypes H5, H6 or H9 has been completed. The rLaSota-AI/H5 was co-infected with AIV subtype H5, H6 and H9 (300 eggs/co-infection). Allantoic fluid from 800 embryonating eggs that died between 24 and 72 h post-infection was collected and is currently being tested for cytopathic effects and plaque formation in cell culture. Specific aim 2: Assessing the potential of recombinant Newcastle disease viruses (attenuated fusion cleavage sites) to revert back to virulence in a host organism has also been completed. Nine hundred 14 day-old embryonating eggs were inoculated with five different Newcastle disease viruses (Wild type Lasota, rLaSota, rM, rZJ1-Lento, and Wild-type Australia). 106 allantoic fluid samples positive for virus from the inoculated eggs have been tested for cytopathic effects in cell culture. 70 of these 106 samples produced cytopathic effects. 20 of these 70 have been sequenced. The remaining viruses are currently being sequences and the fusion cleavage site will be analyzed. Specific aim 3: Recombinant Newcastle disease virus vaccines are able to infect pigeons and spread to contact birds has been partially completed. Infection and transmission experiments in pigeons have been completed. Two groups of sparrows have been infected with two recombinant NDV vaccines. Oropharyngeal and cloacal swabs were collected from all birds on days 2, 4, 6, 8, and 10 pi and samples are stored at -80 degrees for further processing (virus isolation and titration). PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Samples for Specific Aim 1 are undergoing analysis. However, the high mortality rates observed in eggs co-infected with rNDV-LaSota and AIV of subtypes H5, H6 and H9 are likely resultant of AIV replication, as all three strains are known to cause embryo death. Future screening of allantoic fluids from these eggs in cultures of MDCK cells that do not support NDV infection without the addition of trypsin will discern if viruses with increased virulence is present. Of the 20 out of 70 samples that have sequenced for Specific Aim 2, no reversion to virulence or changes in the fusion cleavage site have been observed. The mortality rates observed in these 70 embryonating chicken eggs inoculated with rNDV vaccines are somewhat expected for low virulence NDV isolates. Screening of these allantoic fluids in cell cultures without addition of trypsin suggested potential increase in virulence; however, the definitive answer is obtained by sequencing of the F gene cleavage site. The remaining 50 samples will be evaluated. For Specific Aim 3, none of the pigeons showed clinical signs after being infected with the Newcastle disease strains, however, the birds shed virus and uninfected pigeons placed in contact with infected pigeons were infected and shed virus for two or more days. The sparrow samples are yet to be analyzed. These preliminary findings suggest that our system is suitable to assess the risk associated with recombinant NDV vaccines.
Publications
- No publications reported this period
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: All six viruses (rAI-Whooper/LP-H5-Mongolia, wtNDV-LaSota, rNDV-LaSota, rNDV-LaSota/AI-H5, rNDV-M, wt LoNDV-Australia) that will be used in the project's experiments were grown in 9-10 days-old embryonated chicken eggs to produce the working stocks. Virus stocks have been titrated in 9-10 days-old, specific pathogen free, embryonating chicken eggs by the EID50 method according to Spearman & Karber. We have initiated the experiments outlined on specific Aim 2 with the strains wt NDV-LaSota and rNDV-LaSota being inoculated in 900 14 days-old embryonated chicken eggs (total = 1800 eggs). Eggs that died between 24 and 72 hours post-inoculation (n= 95; WT-LaSota [n=65]; rLaSota [n=30]) were chilled for 24 h and the allantoic fluid harvested and tested for the presence of NDV by HA tests. Allantoic fluids were stored at -80C and will be screened in the presence or absence of trypsin to determine a potential increase in virulence. 25% of Aim 2 is complete. Experiments to assess the spread of recombinant NDV vaccines in non-target species have been planned and will be executed in November of 2011 (Pigeons) and in January of 2012 (Starlings and Sparrows). We have reserved the BSL3-E rooms for the experiments and have ordered the birds for Aim 3. PARTICIPANTS: Diego G. Diel, DVM, MS, PhD was hired in May, 2011 as a postdoctoral fellow to work in the project. He has received initial training to work with Newcastle and Avian influenza viruses and has initiated the experiments outlined in our project. Patti J. Miller, DVM, PhD is the PI of this NIFA proposal and oversees and plans experiments with Dr. Diel who was hired for this project. We continue to wait for the last of the 3 security clearances to arrive so that Dr. Diel can work unescorted. Once he can work unescorted, his output will increase. Dr. Miller has 7 years of experience working with Newcastle disease virus (NDV). Claudio L. Afonso, PhD is a co-investigator and the lead NDV scientist at southeast poultry. He consults on all aims of the project. David L. Suarez, DVM, PhD is a co-investigator on this project who is an expert in avian influenza virus research. Dr. Suarez collaborates and consults on all of the aims of the project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: We have replaced two of the viruses used for Aim 2 because they are more relevant to the study and are more applicable to real life use. They are better choices for the information we would like to obtain.
Impacts
The fact that low virulence strains of NDV wt-LaSota and rNDV-LaSota were able to kill 14 days-old embryonated chicken eggs (wt-LaSota, n=65/900; and rNDV-LaSota, n=30/900) within 24 to 72 hours post-inoculation are suggestive of an increase in virulence. However, screening of the resultant viruses by plaque assays in the presence or absence of trypsin and sequencing of the Fusion protein cleavage site are needed to confirm this possibility. These findings suggest that our system (14 days old embryonated chicken eggs) is suitable to assess the risk of recombinant NDV vaccines to reverse to virulence and potentially may be used in the future to assess the safety of recombinant NDV vaccines prior to their release in the market.
Publications
- No publications reported this period
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