Age-Related Susceptibility of Turkeys to Enteric Viruses.

Several different enteric viruses have been identified as the causes of gastrointestinal infections in poultry. Enteric virus infections are well characterized in poults, but limited studies have been conducted in older birds. The susceptibility of 2-, 7-, 12-, 30-, and 52-wk-old turkeys to turkey coronavirus (TCoV) and turkey astrovirus (TAstV) was evaluated, as well as the effect of combined infection of TAstV and TCoV in 2-wk-old poults and turkey hens. From cloacal swabs and intestines, TCoV was consistently detected by reverse transcriptase-PCR throughout the experimental period (1-21 days postinoculation [DPI]) from all age groups. In contrast, the last detection point of TAstV gradually decreased to 21, 16, and 12 DPI in birds inoculated at 2, 7, and 12 wk of age, respectively, and viral RNA was rarely detected from cloacal swabs or intestinal contents in turkey hens within 3 DPI. Infection with TAstV alone did not affect body weight in poults or egg production in hens. The combined infection of TAstV and TCoV did not induce more severe clinical signs and pathology than the TCoV infection alone. However, a severe prolonged decrease in egg production (about 50%) was observed in turkey hens in the combined infection group compared with a transient egg production drop in the TCoV-infected hens alone. The underlying mechanism regarding the age-related TAstV susceptibility and the pathogenesis of the TAstV and TCoV coinfection in layer hens needs to be further elucidated.

Persistence and Tissue Distribution of Infectious Bursal Disease Virus Serotypes 1 and 2 in Turkeys.

Two experiments were conducted to determine the persistence and tissue distribution of serotypes 1 and 2 of infectious bursal disease virus (IBDV) in specific-pathogen-free and vaccinated turkeys. In Experiment 1, three groups of 2-wk-old turkey poults, including a negative control group, were used. In groups 1 and 2, 13 poults in each group were challenged with either serotype 1 (STC) or serotype 2 (OH) strains using an inoculum of 10(4) 50% embryo infectious dose (EID50)/0.2 ml/bird. Thymus, bursa, spleen, kidney, lungs, liver, pancreas, caecum, and breast and thigh muscles were sampled at predetermined intervals. The bursal tissues from birds inoculated with either serotype were reverse transcriptase-PCR (RT-PCR) positive up to 21 days postinoculation (DPI). In both groups virus isolation from bursas was possible up to 14 DPI. Except for the bursas and spleens in birds inoculated with serotype 1 and bursas in birds inoculated with serotype 2, all other tissues were RT-PCR negative. In Experiment 2, five groups of turkey poults were used. At 4 wk of age, group 1 was challenged with a serotype 1 STC strain and group 2 with serotype 2 OH strain using an inoculum size of 10(2) EID50/0.2 ml for both serotypes. Groups 3 and 4 were vaccinated at 2 wk of age using an inactivated serotype 1 IBDV vaccine. At 2 wk postvaccination, groups 3 and 4 were challenged with STC and OH strains respectively. From group 1, bursal, spleen, and liver tissues were RT-PCR positive up to 14 DPI; breast muscle and kidney tissues were positive up to 7 DPI; and lungs and pancreatic tissues were positive up to 3 DPI. From group 2, bursal tissues were RT-PCR positive up to 14 DPI and lung tissues up to 3 DPI. All of the tissue samples collected from groups 3, 4, and 5 were RT-PCR negative. Virus could not be isolated from RT-PCR positive bursal homogenate. In this work, it was confirmed that the virus persisted in the bursa longer than in any other tissues. The difference in the results between Experiments 1 and 2 could be due to the age of poults at vaccination and the higher inoculum size used in Experiment 1. This study indicates that turkeys are more resistant to IBDV as compared to chickens. Viruses of serotypes 1 and 2 infect turkeys and persist in bursal tissue for 14 days and RNA was detected up to 21 days.

Development of broadly reactive H5N1 vaccine against different Egyptian H5N1 viruses.

The H5N1 highly pathogenic avian influenza (HPAI) virus was isolated for the first time in Egypt in 2006, since then, the virus has become endemic causing a significant threat to the poultry industry and humans. H5N1 HPAI outbreaks continue to occur despite extensive vaccination programs that have been implemented nationwide in different poultry species. Several studies showed that the co-circulating H5N1 viruses in Egypt are genetically and antigenically distant raising a question on the cross protective efficacy of commercial vaccines. In this study, we introduced mutations at the antigenic sites of the hemagglutinin (HA) to broaden reactivity of the Egyptian H5N1 virus. A reverse genetically created variant H5N1 virus (A/chicken/Egypt/1063/2010) with five amino acid mutations (G140R, Y144F, I190L, K192Q, D43N) in the HA gene showed enhanced cross reactivity. This virus showed up to 16 fold increase in reactivity to the classic-lineageH5N1viruses measured by hemagglutination inhibition (HI) assay while maintaining similar level of reactivity with the variant-lineage viruses compared to wild-type virus. In addition, a single amino acid substitution (N165H), which removes potential glycosylation site at the HA globular head of two classic strains (A/chicken/Egypt/527/2012 and A/chicken/Egypt/102d/2010) broadened the reactivity to antisera generated against H5N1 viruses from different clusters. The broadened reactivity of the mutant viruses were also confirmed by testing reactivity of antisera prepared from the mutant viruses against reference viruses from both classic and variant clades. The virus neutralization test using selected antisera and viruses further confirmed the cross HI results. This study highlights that targeted mutation in the HA may be effectively used as a tool to develop broadly reactive influenza vaccines to cope with the continuous antigenic evolution of viruses.

The global one health paradigm: challenges and opportunities for tackling infectious diseases at the human, animal, and environment interface in low-resource settings.

Zoonotic infectious diseases have been an important concern to humankind for more than 10,000 years. Today, approximately 75% of newly emerging infectious diseases (EIDs) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. These interrelated driving forces make it difficult to predict and to prevent zoonotic EIDs. Although significant improvements in environmental and medical surveillance, clinical diagnostic methods, and medical practices have been achieved in the recent years, zoonotic EIDs remain a major global concern, and such threats are expanding, especially in less developed regions. The current Ebola epidemic in West Africa is an extreme stark reminder of the role animal reservoirs play in public health and reinforces the urgent need for globally operationalizing a One Health approach. The complex nature of zoonotic diseases and the limited resources in developing countries are a reminder that the need for implementation of Global One Health in low-resource settings is crucial. The Veterinary Public Health and Biotechnology (VPH-Biotec) Global Consortium launched the International Congress on Pathogens at the Human-Animal Interface (ICOPHAI) in order to address important challenges and needs for capacity building. The inaugural ICOPHAI (Addis Ababa, Ethiopia, 2011) and the second congress (Porto de Galinhas, Brazil, 2013) were unique opportunities to share and discuss issues related to zoonotic infectious diseases worldwide. In addition to strong scientific reports in eight thematic areas that necessitate One Health implementation, the congress identified four key capacity-building needs: (1) development of adequate science-based risk management policies, (2) skilled-personnel capacity building, (3) accredited veterinary and public health diagnostic laboratories with a shared database, and (4) improved use of existing natural resources and implementation. The aim of this review is to highlight advances in key zoonotic disease areas and the One Health capacity needs.

Insights into potential pathogenesis mechanisms associated with Campylobacter jejuni-induced abortion in ewes.

BACKGROUND: Campylobacter jejuni is commonly found in the gastrointestinal tract of many food-animals including sheep without causing visible clinical symptoms of disease. However, C. jejuni has been implicated in ovine abortion cases worldwide. Specifically, in the USA, the C. jejuni sheep abortion (SA) clone has been increasingly associated with sheep abortion. In vivo studies in sheep (the natural host) are needed to better characterize the virulence potential and pathogenesis of this clone. RESULTS: Pregnant ewes intravenously (IV) or orally inoculated with ovine or bovine abortion-associated C. jejuni SA clones exhibited partial or complete uterine prolapse with retained placenta, and abortion or stillbirth, whereas delivery of healthy lambs occurred in pregnant ewes inoculated with C. jejuni 81-176 or in the uninfected group. In sheep inoculated with the SA clone, histopathological lesions including suppurative necrotizing placentitis and/or endometritis coincided with: 1) increased apoptotic death of trophoblasts, 2) increased expression of the host genes (e.g. genes encoding interleukin IL-6 and IL-15) related to cellular necrosis and pro-inflammatory responses in uterus, and 3) decreased expression of the genes encoding GATA binding protein 6, chordin, and insulin-like 3 (INSL3) that account for embryonic development in uterus. Immunohistochemistry revealed localization of bacterial antigens in trophoblasts lining the chorioallantoic membrane of ewes inoculated with the C. jejuni SA clone. CONCLUSIONS: The results showed that C. jejuni SA clones are capable of causing abortion or stillbirth in experimentally infected sheep. Furthermore, down- or up-regulation of specific genes in the uterus of infected pregnant ewes might implicate host genes in facilitating the disease progression. Since the C. jejuni SA strains share genotypic similarities with clones that have been isolated from human clinical cases of gastroenteritis, these strains might represent a potential public health risk.

The impairment of methylmenaquinol:fumarate reductase affects hydrogen peroxide susceptibility and accumulation in Campylobacter jejuni.

The methylmenaquinol:fumarate reductase (Mfr) of Campylobacter jejuni is a periplasmic respiratory (redox) protein that contributes to the metabolism of fumarate and displays homology to succinate dehydrogenase (Sdh). Since chemically oxidized redox-enzymes, including fumarate reductase and Sdh, contribute to the generation of oxidative stress in Escherichia coli, we assessed the role of Mfr in C. jejuni after exposure to hydrogen peroxide (H2 O2 ). Our results show that a Mfr mutant (∆mfrA) strain was less susceptible to H2 O2 as compared to the wildtype (WT). Furthermore, the H2 O2 concentration in the ∆mfrA cultures was significantly higher than that of WT after exposure to the oxidant. In the presence of H2 O2 , catalase (KatA) activity and katA expression were significantly lower in the ∆mfrA strain as compared to the WT. Exposure to H2 O2 resulted in a significant decrease in total intracellular iron in the ∆mfrA strain as compared to WT, while the addition of iron to the growth medium mitigated H2 O2 susceptibility and accumulation in the mutant. The ∆mfrA strain was significantly more persistent in RAW macrophages as compared to the WT. Scanning electron microscopy showed that infection with the ∆mfrA strain caused prolonged changes to the macrophages' morphology, mainly resulting in spherical-shaped cells replete with budding structures and craters. Collectively, our results suggest a role for Mfr in maintaining iron homeostasis in H2 O2 stressed C. jejuni, probably via affecting the concentrations of intracellular iron.

Effect of coronavirus infection on reproductive performance of turkey hens.

Turkey coronavirus (TCoV) infection causes enteritis in turkeys of varying ages with high mortality in young birds. In older birds, field evidence indicates the possible involvement of TCoV in egg-production drops in turkey hens. However, no experimental studies have been conducted to demonstrate TCoV pathogenesis in turkey hens and its effect on reproductive performance. In the present study, we assessed the possible effect of TCoV on the reproductive performance of experimentally infected turkey hens. In two separate trials, 29- to 30-wk-old turkey hens in peak egg production were either mock-infected or inoculated orally with TCoV (Indiana strain). Cloacal swabs and intestinal and reproductive tissues were collected and standard reverse-transcription PCR was conducted to detect TCoV RNA. In the cloacal swabs, TCoV was detected consistently at 3, 5, 7, and 12 days postinoculation (DPI) with higher rates of detection after 5 DPI (> 90%). All intestinal samples were also positive for TCoV at 7 DPI, and microscopic lesions consisting of severe enteritis with villous atrophy were observed in the duodenum and jejunum of TCoV-infected hens. In one of the trials TCoV was detected from the oviduct of two birds at 7 DPI; however, no or mild microscopic lesions were present. In both experimental trials an average of 28%-29% drop in egg production was observed in TCoV-infected turkey hens between 4 and 7 DPI. In a separate trial we also confirmed that TCoV can efficiently transmit from infected to contact control hens. Our results show that TCoV infection can affect the reproductive performance in turkey hens, causing a transient drop in egg production. This drop in egg production most likely occurred as consequence of the severe enteritis produced by the TCoV. However, the potential replication of TCoV in the oviduct and its effect on pathogenesis should be considered and further investigated.

Antigenic analysis of H5N1 highly pathogenic avian influenza viruses circulating in Egypt (2006-2012).

The highly pathogenic avian influenza (HPAI) H5N1 in Egypt circulated continuously after its introduction in February 2006 with substantial economic losses and frequent human infections. Phylogenetic analysis of the available HA sequences revealed the presence of two main sublineages; the classic 2.2.1 and the variant 2.2.1.1. The classic 2.2.1 had subdivided into two clusters of viruses; cluster C1 contained the originally introduced virus and isolates from 2006 to 2009 and cluster C2 emerged in 2007 and continues to circulate. The variant 2.2.1.1 represents the isolates mainly from chickens and subdivided into two clusters; cluster V1 contains isolates from 2007 to 2009 and cluster V2 contains isolates from 2008 to 2011. Sequence analysis revealed 28 amino acid mutations in the previously reported antigenic sites and high evolution rate which may be due to selective pressure from vaccination and/or natural infection. Antigenic analysis of 18 H5N1 isolates from 2006 to 2012 that represent different clusters was conducted using hemagglutination inhibition (HI) and virus neutralization (VN) assays using hyperimmune sera produced by immunizing SPF chickens with inactivated whole-virus. Antigenic relatedness of ancestral Egyptian H5N1 isolate (459-3/06) with other isolates ranged from 30.7% to 79.1% indicating significant antigenic drift of the H5N1 viruses from the ancestral strains. The antigenic relatedness between C2 and V2 clusters ranged from 28.9% to 68% supporting the need for vaccine seed strains from both clusters. Interestingly, A/CK/EG/1709-6/2008 H5N1 strain showed a broad cross reactivity against viruses in different H5N1 clusters (antigenic relatedness ranged from 63.9% to 85.8%) demonstrating a potential candidate as a vaccine strain. Antigenic cartography which facilitates a quantitative interpretation and easy visualization of serological data was constructed based on HI results and further demonstrated the several antigenic groups among Egyptian H5N1 viruses. In conclusion, the cross reactivity between the co-circulating H5N1 strains may not be adequate for protection against each other and it is recommended to test vaccines that contain isolates from different antigenic groups in experimental infection trials for the selection of vaccine seed strain. Furthermore, the continuous monitoring for detecting the emerging variants followed by detailed antigenic analysis for updating vaccines is warranted.

Replication of swine and human influenza viruses in juvenile and layer turkey hens.

Since the first reported isolation of swine influenza viruses (SIVs) in turkeys in the 1980s, transmission of SIVs to turkeys was frequently documented. Recently, the 2009 pandemic H1N1 virus, that was thought to be of swine origin, was detected in turkeys with a severe drop in egg production. In this study, we assessed the infectivity of different mammalian influenza viruses including swine, pandemic H1N1 and seasonal human influenza viruses in both juvenile and layer turkeys. In addition, we investigated the potential influenza virus dissemination in the semen of experimentally infected turkey toms. Results showed that all mammalian origin influenza viruses tested can infect turkeys. SIVs were detected in respiratory and digestive tracts of both juvenile and layer turkeys. Variations in replication efficiencies among SIVs were observed especially in the reproductive tract of layer turkeys. Compared to SIVs, limited replication of seasonal human H1N1 and no detectable replication of recent human-like swine H1N2, pandemic H1N1 and seasonal human H3N2 viruses was noticed. All birds seroconverted to all tested viruses regardless of their replication level. In turkey toms, we were able to detect swine H3N2 virus in semen and reproductive tract of infected toms by real-time RT-PCR although virus isolation was not successful. These data suggest that turkey hens could be affected by diverse influenza strains especially SIVs. Moreover, the differences in the replication efficiency we demonstrated among SIVs and between SIV and human influenza viruses in layer turkeys suggest a possible use of turkeys as an animal model to study host tropism and pathogenesis of influenza viruses. Our results also indicate a potential risk of venereal transmission of influenza viruses in turkeys.

Influenza virus infects bone marrow mesenchymal stromal cells in vitro: implications for bone marrow transplantation.

Mesenchymal stromal cells (MSCs) have differentiation, immunomodulatory, and self-renewal properties and are, therefore, an attractive tool for regenerative medicine and autoimmune diseases. MSCs may be of great value to treat graft-versus-host disease. Influenza virus causes highly contagious seasonal infection and occasional pandemics. The infection is severe in children, elderly, and immunocompromised hosts including hematopoietic stem cell transplant patients. The objective of this study was to determine if MSCs are permissive to influenza virus replication. We isolated MSCs from the bone marrow of 4- to 6-week-old germ-free pigs. Swine and human influenza virus strains were used to infect MSCs in vitro. MSCs expressed known influenza virus α-2,3 and α-2,6 sialic acid receptors and supported replication of swine and human influenza viruses. Viral infection of MSCs resulted in cell lysis and proinflammatory cytokine production. These findings demonstrate that bone marrow-derived MSCs are susceptible to influenza virus. The data also suggest that transplantation of bone marrow MSCs from influenza virus-infected donors may transmit infection to recipients. Also, MSCs may get infected if infused into a patient with an ongoing influenza virus infection.

Interspecies transmission of influenza a viruses between Swine and poultry.

The special susceptibility of pigs to infection with avian and mammalian influenza viruses, the close proximity of pigs and poultry farms, and applied human practices in raising and trading of farm animals/farm animal products, provide opportunities for genetic exchange and interspecies transmission of influenza A viruses. Although only H1 and H3 influenza subtypes have widely circulated and caused disease in pig populations worldwide, H9 subtype is being continuously detected in pigs in Asia, plus sporadic infections with highly pathogenic H5-avian influenza viruses. On the other hand, swine viruses are continuously isolated from poultry species, especially turkeys, causing economic losses in poultry production. The viral and host factors contributing to influenza transmission between pigs and poultry are poorly defined. In addition, surveillance programs for influenza viruses in both species, especially pigs, are rarely implemented, and thus, leaving many questions about influenza unanswered. In this review, we summarize early and recent findings about influenza transmission between swine and poultry with emphasis on the role of turkeys.

Enhanced replication of swine influenza viruses in dexamethasone-treated juvenile and layer turkeys.

Frequent transmission of swine influenza viruses (SIVs) to turkeys has been reported since 1980s. Experimental studies also showed that SIVs can infect turkeys with varying replication and transmission efficiency depending on the strain. However, host factors involved in infection/replication efficiency remain unclear. To investigate whether the immune status of turkeys might play a role in the susceptibility of turkeys to SIVs, we studied the replication efficiency of two recent SIVs (human-like H1N2 and triple reassortant (TR) H3N2) in dexamethasone-treated turkeys. The viruses were inoculated intranasally in both dexamethasone-treated and untreated control juvenile and layer turkeys. Amount of virus shedding was monitored at 2, 4, and 7 days post inoculation (DPI). Additionally, passage of both viruses was attempted in dexamethasone-treated 4-week-old turkeys. In both juvenile and layer turkeys, we were able to detect human-like H1N2 SIV only from dexamethasone-treated turkeys and no virus was detected in untreated birds. The virus shedding of the TR H3N2 SIV was also consistently higher (≈ 1 Log(10)EID(50)/ml) in dexamethasone-treated birds in both tracheal and cloacal swabs compared to untreated birds. Virus passage in dexamethasone-treated turkeys was successful up to the second passage and no virus was recovered from the third passage. These results show that potential immunosuppression due to dexamethasone treatment may enhance the transmission and adaptation of SIVs in turkeys through enhancement of virus replication, prolonged virus shedding, and possible decrease of infectious dose required to initiate infection.

Persistence and tissue distribution of infectious bursal disease virus in experimentally infected SPF and commercial broiler chickens.

This study was initiated to determine the persistence, distribution, and quantification of infectious bursal disease virus (IBDV) in lymphoid and nonlymphoid tissues of specific-pathogen-free (SPF) and commercial broiler chickens. Two serotype 1 strains, STC classic and IN variant, were independently used in the experiments. Five separate experiments were conducted using 2- and 4-wk-old SPF chickens, 2- and 4-wk-old in ovo-vaccinated commercial broilers, and 2-wk-old commercial broilers having maternally derived anti-IBDV antibodies. Pooled data from five experiments revealed that SPF chickens had a significantly higher incidence of IBDV-positive reverse transcriptase PCR (RT-PCR) results than commercial chickens (multivariable logistic regression, adjusted odds ratio = 15.28; 95% confidence limits [CL] = 9.53, 24.51, P < 0.0001). In many cases, the viral RNA (vRNA) persisted longer in in ovo-vaccinated commercial broilers bearing maternally derived antibodies compared with similar broilers not vaccinated in ovo. The STC strain was more frequently detected in tissues than the IN strain (chi-square P < 0.0001). In lymphoid tissues, STC and IN strains were detected for the longest duration in bursal tissues followed by spleen, thymus, and bone marrow. In nonlymphoid tissues, STC and IN strains were detected the longest in cecum followed by liver, kidney, pancreas, lungs, thigh, and breast muscles. Compared with bursal tissues, muscle and bone marrow tissues were significantly less likely to yield an IBDV-positive RT-PCR result (P < 0.0001). Although STC vRNA was detected up to 42 days postinoculation (DPI) in bursal homogenates of SPF chickens, virus isolation from bursal homogenates using embryonated chicken eggs was only possible up to 28 DPI. Similarly, STC vRNA was detected up to 42 DPI in bursal tissues of commercial broilers, but infectious virus could be isolated only up to 21 DPI. The IN strain was isolated up to 10 DPI from bursal homogenates of SPF chickens and broilers, but vRNA was detected up to 35 DPI in SPF chickens and 21 DPI in broilers. This study emphasizes that the detection ofvRNA is not indicative of the presence of infectious virus, and virus isolation has to be performed to prove the presence of infectious virus.

Respiratory proteins contribute differentially to Campylobacter jejuni's survival and in vitro interaction with hosts' intestinal cells.

BACKGROUND: The genetic features that facilitate Campylobacter jejuni's adaptation to a wide range of environments are not completely defined. However, whole genome expression studies showed that respiratory proteins (RPs) were differentially expressed under varying conditions and stresses, suggesting further unidentified roles for RPs in C. jejuni's adaptation. Therefore, our objectives were to characterize the contributions of selected RPs to C. jejuni's i- key survival phenotypes under different temperature (37°C vs. 42°C) and oxygen (microaerobic, ambient, and oxygen-limited/anaerobic) conditions and ii- its interactions with intestinal epithelial cells from disparate hosts (human vs. chickens). RESULTS: C. jejuni mutant strains with individual deletions that targeted five RPs; nitrate reductase (ΔnapA), nitrite reductase (ΔnrfA), formate dehydrogenase (ΔfdhA), hydrogenase (ΔhydB), and methylmenaquinol:fumarate reductase (ΔmfrA) were used in this study. We show that only the ΔfdhA exhibited a decrease in motility; however, incubation at 42°C significantly reduced the deficiency in the ΔfdhA's motility as compared to 37°C. Under all tested conditions, the ΔmfrA showed a decreased susceptibility to hydrogen peroxide (H(2)O(2)), while the ΔnapA and the ΔfdhA showed significantly increased susceptibility to the oxidant as compared to the wildtype. Further, the susceptibility of the ΔnapA to H(2)O(2) was significantly more pronounced at 37°C. The biofilm formation capability of individual RP mutants varied as compared to the wildtype. However, the impact of the deletion of certain RPs affected biofilm formation in a manner that was dependent on temperature and/or oxygen concentration. For example, the ΔmfrA displayed significantly deficient and increased biofilm formation under microaerobic conditions at 37°C and 42°C, respectively. However, under anaerobic conditions, the ΔmfrA was only significantly impaired in biofilm formation at 42°C. Additionally, the RPs mutants showed differential ability for infecting and surviving in human intestinal cell lines (INT-407) and primary chicken intestinal epithelial cells, respectively. Notably, the ΔfdhA and the ΔhydB were deficient in interacting with both cell types, while the ΔmfrA displayed impairments only in adherence to and invasion of INT-407. Scanning electron microscopy showed that the ΔhydB and the ΔfdhA exhibited filamentous and bulging (almost spherical) cell shapes, respectively, which might be indicative of defects in cell division. CONCLUSIONS: We conclude that the RPs contribute to C. jejuni's motility, H(2)O(2) resistance, biofilm formation, and in vitro interactions with hosts' intestinal cells. Further, the impact of certain RPs varied in response to incubation temperature and/or oxygen concentration. Therefore, RPs may facilitate the prevalence of C. jejuni in a variety of niches, contributing to the pathogen's remarkable potential for adaptation.

Oct4+ stem/progenitor swine lung epithelial cells are targets for influenza virus replication.

We isolated stem/progenitor epithelial cells from the lungs of 4- to 6-week-old pigs. The epithelial progenitor colony cells were surrounded by mesenchymal stromal cells. The progenitor epithelial colony cells expressed stem cell markers such as octamer binding transcription factor 4 (Oct4) and stage-specific embryonic antigen 1 (SSEA-1), as well as the epithelial markers pancytokeratin, cytokeratin-18, and occludin, but not mesenchymal (CD44, CD29, and CD90) and hematopoietic (CD45) markers. The colony cells had extensive self-renewal potential and had the capacity to undergo differentiation to alveolar type I- and type II-like pneumocytes. Additionally, these cells expressed sialic acid receptors and supported the active replication of influenza virus, which was accompanied by cell lysis. The lysis of progenitor epithelial cells by influenza virus may cause a marked reduction in the potential of progenitor cells for self renewal and for their ability to differentiate into specialized cells of the lung. These observations suggest the possible involvement of lung stem/progenitor cells in influenza virus infection.

Identification of swine H1N2/pandemic H1N1 reassortant influenza virus in pigs, United States.

In October and November 2010, novel H1N2 reassortant influenza viruses were identified from pigs showing mild respiratory signs that included cough and depression. Sequence and phylogenetic analysis showed that the novel H1N2 reassortants possesses HA and NA genes derived from recent H1N2 swine isolates similar to those isolated from Midwest. Compared to the majority of reported reassortants, both viruses preserved human-like host restrictive and putative antigenic sites in their HA and NA genes. The four internal genes, PB2, PB1, PA, and NS were similar to the contemporary swine triple reassortant viruses' internal genes (TRIG). Interestingly, NP and M genes of the novel reassortants were derived from the 2009 pandemic H1N1. The NP and M proteins of the two isolates demonstrated one (E16G) and four (G34A, D53E, I109T, and V313I) amino acid changes in the M2 and NP proteins, respectively. Similar amino acid changes were also noticed upon incorporation of the 2009 pandemic H1N1 NP in other reassortant viruses reported in the U.S. Thus the role of those amino acids in relation to host adaptation need to be further investigated. The reassortments of pandemic H1N1 with swine influenza viruses and the potential of interspecies transmission of these reassortants from swine to other species including human indicate the importance of systematic surveillance of swine population to determine the origin, the prevalence of similar reassortants in the U.S. and their impact on both swine production and public health.

Fas/FasL and perforin-granzyme pathways mediated T cell cytotoxic responses in infectious bursal disease virus infected chickens.

Infectious bursal disease (IBD) is a highly contagious disease of chickens which leads to immunosuppression. In our previous study it was demonstrated that, possibly, CD4(+) and CD8(+) T cells may employ perforin and granzyme-A pathway for the clearance of IBDV-infected bursal cells. In this study, we evaluated the cytotoxic T cell responses involving two independently functioning but complementary mechanisms: Fas-Fas ligand and perforin-granzyme pathways in IBDV-infected chickens. As demonstrated previously, infection of chickens with IBDV was accompanied by influx of CD8(+) T cells in the bursa and spleen. There was an upregulation in the gene expression of cytolytic molecules: Fas and Fas ligand (FasL), perforin (PFN) and granzyme-A (Gzm-A) in bursal and in the splenic tissues of IBDV inoculated chickens. Additionally, for the first time, we detected Fas, Fas ligand, Caspase-3 and PFN producing CD8(+) T cells in the bursa and spleen of IBDV-infected chickens. The infiltration and activation of CD8(+) T cells was substantiated by the detection of Th1 cytokine, IFN-γ. These data suggest that T cells may be involved in the clearance of virus from the target organ bursa and peripheral tissues such as spleen. The findings of these studies provide new insights into the pathogenesis of IBD and provide mechanistic evidence that the cytotoxic T cells may act through both Fas-FasL and perforin-granzyme pathways in mediating the clearance of virus-infected cells.

Development of DIVA (differentiation of infected from vaccinated animals) vaccines utilizing heterologous NA and NS1 protein strategies for the control of triple reassortant H3N2 influenza in turkeys.

Since 2003, triple reassortant (TR) swine H3N2 influenza viruses containing gene segments from human, avian, and swine origins have been detected in the U.S. turkey populations. The initial outbreak that occurred involved birds that were vaccinated with the currently available H3 swine- and avian-origin influenza vaccines. Antigenically, all turkey swine-lineage TR H3N2 isolates are closely related to each other but show little or no antigenic cross-reactivity with the avian origin or swine origin influenza vaccine strains that are currently being used in turkey operations. These results call for re-evaluation of currently available influenza vaccines being used in turkey flocks and development of more effective DIVA (differentiation of infected from vaccinated animals) vaccines. In this study, we selected one TR H3N2 strain, A/turkey/OH/313053/04 (H3N2) that showed broad cross reactivity with other recent TR turkey H3N2 isolates, and created NA- and NS-based DIVA vaccines using traditional reassortment as well as reverse genetics methods. Protective efficacy of those vaccines was determined in 2-week-old and 80-week-old breeder turkeys. The reassortant DIVA vaccines significantly reduced the presence of challenge virus in the oviduct of breeder turkeys as well as trachea and cloaca shedding of both young and old breeder turkeys, suggesting that proper vaccination could effectively prevent egg production drop and potential viral contamination of eggs in infected turkeys. Our results demonstrate that the heterologous NA and NS1 DIVA vaccines together with their corresponding serological tests could be useful for the control of TR H3N2 influenza in turkeys.