Evaluation of the transcriptional status of host cytokines and viral genes in the trachea of vaccinated and non-vaccinated chickens after challenge with the infectious laryngotracheitis virus

<p>Infectious laryngotracheitis is a highly contagious disease of chickens responsible for significant economic losses for the poultry industry worldwide. The disease is caused by <i>Gallid herpesvirus-1</i> (GaHV-1) commonly known as the infectious laryngotracheitis virus. Although characterized by their potential to regain virulence, chicken embryo origin (CEO) vaccines are the most effective vaccines against laryngotracheitis as they significantly reduce the replication of challenge virus in the trachea and conjunctiva. Knowledge on the nature of protective immunity elicited by CEO vaccines is very limited. Therefore, elucidating the origin of the immune responses elicited by CEO vaccination is relevant for development of safer control strategies. In this study the transcription levels of key host immune genes (IFN-γ, IFN-β, IL-1β, IL-6, IL-8, IL-18) and viral genes (ICP4, ICP27, UL46, UL49), as well as viral genome loads in trachea were quantified at 6 and 12 hours post-challenge of CEO vaccinated and non-vaccinated chickens. Immediately after challenge a significant increase in IFN-γ gene expression was followed by a significant reduction in viral replication. In contrast to the rapid induction of IFN-γ, expression of the pro-inflammatory cytokines (IL-1β, IL-6, IL-8) and type I IFN β was either slightly reduced or remained at basal levels. These suggest that the former cytokines may not play important roles during immediate early responses induced by ILTV challenge in either vaccinated or non-vaccinated chickens. Overall, these results suggest that the rapid expression of IFN-γ may induce pathways of antiviral responses necessary for blocking early virus replication.</p

Characterization and evaluation of the effect of co-delivering chIFNγ with a DNA vaccination system.

<p>Plasmids expressing NDV F and chIFNγ genes were developed and characterized for their use as DNA vaccines and adjuvant, respectively. DF-1 cells were transfected with pTriEX, pTriEX-ZJ1-F and pTriEX- IFNγ. Cell culture supernatants were tested by western blotting for the presence of F protein <b>(A)</b> and chIFNγ <b>(B)</b>, respectively. Eighteen-day-old SPF ECEs were inoculated with TE buffer, pTriEX, pTriEX-ZJ1-F, or pTriEX-ZJ1-F plus pTriEX- IFNγ and boosted 2 weeks after hatched. Two weeks after booster vaccination, birds were challenged with vZJ1. Oropharyngeal <b>(C)</b> and cloacal <b>(D)</b> swab samples were collected 3 days after challenge to measure the amount of challenge virus shed into the environment. Viral titers were determined by quantitative real time reverse transcription polymerase chain reaction (qRRT-PCR). A standard was prepared with a vZJ1 virus stock of know concentration, this was included in every plate and was used to obtain viral titers expressed as EID₅₀/mL. Morbidity <b>(E)</b> and mortality <b>(F)</b> were also evaluated. Viral shedding results were analyzed with One-way ANOVA followed by a multiple comparisons Tukey's test. Differences in morbidity among groups were evaluated using a two-tailed Z test for comparison of sample proportions. Survival curves were analyzed using the Long-Rank test. Statistical difference was considered with a <i>P<0</i>.<i>05</i>. Significant differences are denoted by different letters.</p

Effect of chIFNγ on hatchability and survival after <i>in ovo</i> vaccination with rZJ1*L/IFNγ and challenge with vZJ1.

<p>Effect of chIFNγ on hatchability and survival after <i>in ovo</i> vaccination with rZJ1*L/IFNγ and challenge with vZJ1.</p

DNA-vaccine groups and plasmid combinations.

<p>DNA-vaccine groups and plasmid combinations.</p

Presence of Vaccine-Derived Newcastle Disease Viruses in Wild Birds

<div><p>Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order <i>Columbiformes</i> (n = 23), followed in frequency by the order <i>Anseriformes</i> (n = 13). Samples were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, LaSota (n = 28) and Hitchner B1 (n = 19). Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife.</p></div

Collated Isolates from GenBank and SEPRL samples.

<p>A total of 54 isolates from the following taxonomic orders are tabulated below: <i>Accipitriformes</i> (n = 1); <i>Anseriformes</i> (n = 13); <i>Charadriiformes</i> (n = 3); <i>Columbiformes</i> (n = 23); <i>Falconiformes</i> (n = 1); <i>Galliformes</i> (n = 4); <i>Passeriformes</i> (n = 2); <i>Pelecaniformes</i> (n = 1); <i>Phoenicopteriformes</i> (n = 1); <i>Psittaciformes</i> (n = 4); Unknown (n = 1). GenBank accession numbers bolded are strains sequenced from this study.</p

Rock Pigeon virus isolation, HI antibody titer, and intracerebral pathogenicity indices (ICPIs).

<p>Field swabs collected from the oral and cloacal cavities positive for virus are listed with a + symbol, otherwise an N/A is listed. ICPI experimental infection swabs collected in the lab from the oral and cloacal cavities positive for virus are listed below with their corresponding ICPI values, otherwise an N/A is listed.</p

Phylogenetic tree of isolates and their relationship to class II NDV viruses.

<p>Phylogenetic analysis based on the complete nucleotide sequence of the fusion gene of isolates representing NDV class II. The evolutionary history was inferred by using the Maximum Likelihood method based on Tamura 3-parameter model with 500 bootstrap replicates [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162484#pone.0162484.ref070" target="_blank">70</a>]. The tree with the highest log likelihood (-108983.3717) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (4 categories (+G, parameter = 0.0936). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 39.7777% sites). The tree is drawn to scale with branch lengths measured in the number of substitutions per site and the percentage of trees in which the associated taxa clustered together are shown below the branches. The analysis involved 81 nucleotide sequences with a total of 1662 positions in the final dataset. Isolates studied in this work are designated in front of the taxa name as follows: USA—●; Ukraine—○; Brazil—□, Bulgaria—■. Evolutionary analyses were conducted in MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162484#pone.0162484.ref067" target="_blank">67</a>]. The Roman numerals presented in the taxa names in the phylogenetic trees represent the respective genotype for each isolate, followed by the GenBank identification number, host name (if available), country of isolation, strain designation and country of isolation.</p