Phylodynamic approaches to studying avian influenza virus.

Avian influenza viruses can cause severe disease in domestic and wild birds and are a pandemic threat. Phylodynamics is the study of how epidemiological, evolutionary, and immunological processes can interact to shape viral phylogenies. This review summarizes how phylodynamic methods have and could contribute to the study of avian influenza viruses. Specifically, we assess how phylodynamics can be used to examine viral spread within and between wild or domestic bird populations at various geographical scales, identify factors associated with virus dispersal, and determine the order and timing of virus lineage movement between geographic regions or poultry production systems. We discuss factors that can complicate the interpretation of phylodynamic results and identify how future methodological developments could contribute to improved control of the virus.

H9N2 avian influenza virus dispersal along Bangladeshi poultry trading networks.

Avian influenza virus subtype H9N2 is endemic in Bangladesh's poultry population. The subtype affects poultry production and poses a potential zoonotic risk. Insufficient understanding of how the poultry trading network shapes the dissemination of avian influenza viruses has hindered the design of targeted interventions to reduce their spread. Here, we use phylodynamic analyses of haemagglutinin sequences to investigate the spatial spread and dispersal patterns of H9N2 viruses in Bangladesh's poultry population, focusing on its two largest cities (Dhaka and Chattogram) and their poultry production and distribution networks. Our analyses suggest that H9N2 subtype avian influenza virus lineage movement occurs relatively less frequently between Bangladesh's two largest cities than within each city. H9N2 viruses detected in single markets are often more closely related to viruses from other markets in the same city than to each other, consistent with close epidemiological connectivity between markets. Our analyses also suggest that H9N2 viruses may spread more frequently between chickens of the three most commonly sold types (sunali-a cross-bred of Fayoumi hen and Rhode Island Red cock, deshi-local indigenous, and exotic broiler) in Dhaka than in Chattogram. Overall, this study improves our understanding of how Bangladesh's poultry trading system impacts avian influenza virus spread and should contribute to the design of tailored surveillance that accommodates local heterogeneity in virus dispersal patterns.

A large-scale study of a poultry trading network in Bangladesh: implications for control and surveillance of avian influenza viruses.

BACKGROUND: Since its first report in 2007, avian influenza (AI) has been endemic in Bangladesh. While live poultry marketing is widespread throughout the country and known to influence AI dissemination and persistence, trading patterns have not been described. The aim of this study is to assess poultry trading practices and features of the poultry trading networks which could promote AI spread, and their potential implications for disease control and surveillance. Data on poultry trading practices was collected from 849 poultry traders during a cross-sectional survey in 138 live bird markets (LBMs) across 17 different districts of Bangladesh. The quantity and origins of traded poultry were assessed for each poultry type in surveyed LBMs. The network of contacts between farms and LBMs resulting from commercial movements of live poultry was constructed to assess its connectivity and to identify the key premises influencing it. RESULTS: Poultry trading practices varied according to the size of the LBMs and to the type of poultry traded. Industrial broiler chickens, the most commonly traded poultry, were generally sold in LBMs close to their production areas, whereas ducks and backyard chickens were moved over longer distances, and their transport involved several intermediates. The poultry trading network composed of 445 nodes (73.2% were LBMs) was highly connected and disassortative. However, the removal of only 5.6% of the nodes (25 LBMs with the highest betweenness scores), reduced the network's connectedness, and the maximum size of output and input domains by more than 50%. CONCLUSIONS: Poultry types need to be discriminated in order to understand the way in which poultry trading networks are shaped, and the level of risk of disease spread that these networks may promote. Knowledge of the network structure could be used to target control and surveillance interventions to a small number of LBMs.

VAV1 regulates experimental autoimmune arthritis and is associated with anti-CCP negative rheumatoid arthritis.

Rheumatoid arthritis (RA) patients can be stratified into two subgroups defined by the presence or absence of antibodies against citrullinated circular peptides (anti-CCP) with most of the genetic association found in anti-CCP positive RA. Here we addressed the role of VAV1, previously associated to multiple sclerosis (MS), in the pathogenesis of RA in experimental models and in a genetic association study. Experimental arthritis triggered by pristane or collagen type II was induced in DA rats and in the DA.BN-R25 congenic line that carries a polymorphism in Vav1. Difference in arthritis severity was observed only after immunization with pristane. In a case-control study, 34 SNPs from VAV1 locus were analyzed by Immunochip genotyping in 11475 RA patients (7573 anti-CCP positive and 3902 negative) and 15,870 controls in six cohorts of European Caucasians. A combination of the previous MS-associated haplotype and two additional SNPs was associated with anti-CCP negative RA (alleles G-G-A-A of rs682626-rs2546133-rs2617822-rs12979659, OR=1.13, P=1.27 × 10-5). The same markers also contributed to activity of RA at baseline with the strongest association in the anti-CCP negative group for the rs682626-rs12979659 G-A haplotype (ß=-0.283, P=0.0048). Our study suggests a role for VAV1 and T-cell signaling in the pathology of anti-CCP-negative RA.

Cultural Practices Shaping Zoonotic Diseases Surveillance: The Case of Highly Pathogenic Avian Influenza and Thailand Native Chicken Farmers.

Effectiveness of current passive zoonotic disease surveillance systems is limited by the under-reporting of disease outbreaks in the domestic animal population. Evaluating the acceptability of passive surveillance and its economic, social and cultural determinants appears a critical step for improving it. A participatory rural appraisal was implemented in a rural subdistrict of Thailand. Focus group interviews were used to identify sanitary risks perceived by native chicken farmers and describe the structure of their value chain. Qualitative individual interviews with a large diversity of actors enabled to identify perceived costs and benefits associated with the reporting of HPAI suspicions to sanitary authorities. Besides, flows of information on HPAI suspected cases were assessed using network analysis, based on data collected through individual questionnaires. Results show that the presence of cockfighting activities in the area negatively affected the willingness of all chicken farmers and other actors to report suspected HPAI cases. The high financial and affective value of fighting cocks contradicted the HPAI control policy based on mass culling. However, the importance of product quality in the native chicken meat value chain and the free veterinary services and products delivered by veterinary officers had a positive impact on suspected case reporting. Besides, cockfighting practitioners had a significantly higher centrality than other actors in the information network and they facilitated the spatial diffusion of information. Social ties built in cockfighting activities and the shared purpose of protecting valuable cocks were at the basis of the diffusion of information and the informal collective management of diseases. Building bridges with this informal network would greatly improve the effectiveness of passive surveillance.

Analysis of Swine Movements in a Province in Northern Vietnam and Application in the Design of Surveillance Strategies for Infectious Diseases.

While swine production is rapidly growing in South-East Asia, the structure of the swine industry and the dynamic of pig movements have not been well-studied. However, this knowledge is a prerequisite for understanding the dynamic of disease transmission in swine populations and designing cost-effective surveillance strategies for infectious diseases. In this study, we assessed the farming and trading practices in the Vietnamese swine familial farming sector, which accounts for most pigs in Vietnam, and for which disease surveillance is a major challenge. Farmers from two communes of a Red River Delta Province (northern Vietnam) were interviewed, along with traders involved in pig transactions. Major differences in the trade structure were observed between the two communes. One commune had mainly transversal trades, that is between farms of equivalent sizes, whereas the other had pyramidal trades, that is from larger to smaller farms. Companies and large familial farrow-to-finish farms were likely to act as major sources of disease spread through pig sales, demonstrating their importance for disease control. Familial fattening farms with high pig purchases were at greater risk of disease introduction and should be targeted for disease detection as part of a risk-based surveillance. In contrast, many other familial farms were isolated or weakly connected to the swine trade network limiting their relevance for surveillance activities. However, some of these farms used boar hiring for breeding, increasing the risk of disease spread. Most familial farms were slaughtering pigs at the farm or in small local slaughterhouses, making the surveillance at the slaughterhouse inefficient. In terms of spatial distribution of the trades, the results suggested that northern provinces were highly connected and showed some connection with central and southern provinces. These results are useful to develop risk-based surveillance protocols for disease detection in the swine familial sector and to make recommendations for disease control.

Syndromic surveillance of abortions in beef cattle based on the prospective analysis of spatio-temporal variations of calvings.

Our objective was to study the ability of a syndromic surveillance system to identify spatio-temporal clusters of drops in the number of calvings among beef cows during the Bluetongue epizootic of 2007 and 2008, based on calving seasons. France was partitioned into 300 iso-populated units, i.e. units with quite the same number of beef cattle. Only 1% of clusters were unlikely to be related to Bluetongue. Clusters were detected during the calving season of primary infection by Bluetongue in 28% (n = 23) of the units first infected in 2007, and in 87% (n = 184) of the units first infected in 2008. In units in which a first cluster was detected over their calving season of primary infection, Bluetongue was detected more rapidly after the start of the calving season and its prevalence was higher than in other units. We believe that this type of syndromic surveillance system could improve the surveillance of abortive events in French cattle. Besides, our approach should be used to develop syndromic surveillance systems for other diseases and purposes, and in other settings, to avoid "false" alarms due to isolated events and homogenize the ability to detect abnormal variations of indicator amongst iso-populated units.

When private actors matter: Information-sharing network and surveillance of Highly Pathogenic Avian Influenza in Vietnam.

The effectiveness of animal health surveillance systems depends on their capacity to gather sanitary information from the animal production sector. In order to assess this capacity we analyzed the flow of sanitary information regarding Highly Pathogenic Avian Influenza (HPAI) suspicions in poultry in Vietnam. Participatory methods were applied to assess the type of actors and likelihood of information sharing between actors in case of HPAI suspicion in poultry. While the reporting of HPAI suspicions is mandatory, private actors had more access to information than public actors. Actors of the upstream sector (medicine and feed sellers) played a key role in the diffusion of information. The central role of these actors and the influence of the information flow on the adoption by poultry production stakeholders of behaviors limiting (e.g. prevention measures) or promoting disease transmission (e.g. increased animal movements) should be accounted for in the design of surveillance and control programs.

Modelling the expected rate of laboratory biosafety breakdowns involving rinderpest virus in the post-eradication era.

Now that we are in the rinderpest post-eradication era, attention is focused on the risk of re-introduction. A semi-quantitative risk assessment identified accidental use of rinderpest virus in laboratories as the most likely cause of re-introduction. However there is little data available on the rates of laboratory biosafety breakdowns in general. In addition, any predictions based on past events are subject to various uncertainties. The aims of this study were therefore to investigate the potential usefulness of historical data for predicting the future risk of rinderpest release via laboratory biosafety breakdowns, and to investigate the impacts of the various uncertainties on these predictions. Data were collected using a worldwide online survey of laboratories, a structured search of ProMED reports and discussion with experts. A stochastic model was constructed to predict the number of laboratory biosafety breakdowns involving rinderpest that will occur over the next 10 years, based on: (1) the historical rate of biosafety breakdowns; and (2) the change in the number of laboratories that will have rinderpest virus in the next 10 years compared to historically. The search identified five breakdowns, all of which occurred during 1970-2000 and all of which were identified via discussions with experts. Assuming that our search for historical events had a sensitivity of over 60% and there has been at least a 40% reduction in the underlying risk (attributable to decreased laboratory activity post eradication) the most likely number of biosafety events worldwide was estimated to be zero over a 10 year period. However, the risk of at least one biosafety breakdown remains greater than 1 in 10,000 unless the sensitivity was at least 99% or the number of laboratories has decreased by at least 99% (based on 2000-2010 during which there were no biosafety breakdowns).

Genetic control of HgCl2-induced IgE and autoimmunity by a 117-kb interval on rat chromosome 9 through CD4 CD45RChigh T cells.

Gold or mercury salts trigger a dramatic IgE response and a CD4 T-cell-dependent nephropathy in Brown-Norway (BN), but not in Lewis (LEW) rats. We previously identified the 1.1-Mb Iresp3 (immunoglobin response QTL3) locus on chromosome 9 that controls these gold salt-triggered immune disorders. In the present work, we investigated the genetic control of HgCl(2)-induced immunological disorders and assessed the relative contribution of the CD45RC(high) and CD45RC(low) CD4 T-cell subpopulations in this control. By using interval-specific congenic lines, we narrowed down Iresp3 locus to 117-kb and showed that BN rats congenic for the LEW 117-kb were protected from HgCl(2)-triggered IgE response and nephropathy. This 117-kb interval also controls CD45RC expression by CD4 T cells and the ability of CD45RC(high) CD4 T cells to trigger the autoimmune disorders resulting from HgCl(2) administration. This 117-kb region contains four genes, including Vav1, a strong candidate gene according to its cellular function and exclusive expression in hematopoietic cells. Thus, this study highlights the role of the CD45RC(high) CD4 T-cell subpopulation in the opposite susceptibility of BN and LEW rats to HgCl(2)-triggered immune disorders and identifies a 117-kb interval on chromosome 9 that has a key role in their functions.

Impact of the implementation of rest days in live bird markets on the dynamics of H5N1 highly pathogenic avian influenza.

Live bird markets (LBMs) act as a network 'hub' and potential reservoir of infection for domestic poultry. They may therefore be responsible for sustaining H5N1 highly pathogenic avian influenza (HPAI) virus circulation within the poultry sector, and thus a suitable target for implementing control strategies. We developed a stochastic transmission model to understand how market functioning impacts on the transmission dynamics. We then investigated the potential for rest days-periods during which markets are emptied and disinfected-to modulate the dynamics of H5N1 HPAI within the poultry sector using a stochastic meta-population model. Our results suggest that under plausible parameter scenarios, HPAI H5N1 could be sustained silently within LBMs with the time spent by poultry in markets and the frequency of introduction of new susceptible birds' dominant factors determining sustained silent spread. Compared with interventions applied in farms (i.e. stamping out, vaccination), our model shows that frequent rest days are an effective means to reduce HPAI transmission. Furthermore, our model predicts that full market closure would be only slightly more effective than rest days to reduce transmission. Strategies applied within markets could thus help to control transmission of the disease.

An assessment of the feasibility of a poultry tracing scheme for smallholders in Vietnam.

Tracing movements could assist the implementation of bio-containment measures during a disease outbreak. To evaluate the potential for implementing a tracing system for a poultry supply chain in northern Vietnam, a four-month longitudinal study was conducted to identify marketing practices associated with poultry traceability. Poultry sold in batches were traced between farms and markets, and their traceability was assessed upon market arrival. A total of 315 batches were released from the farms; 37% arrived at a market, from which 57.3% were 'traceable'. The results of the multivariable analysis showed that traceability was associated with farms operating through no more than two traders (Odds ratio [OR] = 5.97, 95% CI 1.15-30.92) and batches brought to the market on the day of purchase (OR = 4.05, 95% CI 1.23-13.27). No specific incentives were provided to farmers or traders. Results suggest that there is potential for implementing a poultry traceability scheme, although the tracing methodology should be refined.

Induction of autoimmunity through bystander effects. Lessons from immunological disorders induced by heavy metals.

Autoreactive T cells exist in healthy individuals and represent a potential reservoir of pathogenic effectors which, when stimulated by microbial adjuvants, could trigger an autoimmune disease. Experimental studies have indicated that xenobiotics, well defined from a chemical point of view, could promote the differentiation of autoreactive T cells towards a pathogenic pathway. It is therefore theoretically possible that compounds present in vaccines such as thiomersal or aluminium hydroxyde can trigger autoimmune reactions through bystander effects. Mercury and gold in rodents can induce immunological disorders with autoimmune reactions. In vitro, both activate signal transduction pathways that result in the expression of cytokines, particularly of IL-4 and IFNgamma. In a suitable microenvironment heavy metals could therefore favour the activation of autoreactive T cells. In that respect, genetic background is of major importance. Genome-wide searches in the rat have shown that overlapping chromosomal regions control the immunological disorders induced by gold salt treatment, the development of experimental autoimmune encephalomyelitis and the CD45RC(high)/CD45RC(low)CD4(+)T cells balance. The identification and functional characterization of genes controlling these phenotypes may shed light on key regulatory mechanisms of immune responses. This should help to improve efficacy and safety of vaccines.

The balance between CD45RChigh and CD45RClow CD4 T cells in rats is intrinsic to bone marrow-derived cells and is genetically controlled.

The level of CD45RC expression differentiates rat CD4 T cells in two subpopulations, CD45RC(high) and CD45RC(low), that have different cytokine profiles and functions. Interestingly, Lewis (LEW) and Brown Norway (BN) rats, two strains that differ in their ability to mount type 1 and type 2 immune responses and in their susceptibility to autoimmune diseases, exhibit distinct CD45RC(high)/CD45RC(low) CD4 T cell ratios. The CD45RC(high) subpopulation predominates in LEW rats, and the CD45RC(low) subpopulation in BN rats. In this study, we found that the antiinflammatory cytokines, IL-4, IL-10, and IL-13, are exclusively produced by the CD45RC(low) CD4 T cells. Using bone marrow chimeras, we showed that the difference in the CD45RC(high)/CD45RC(low) CD4 T cell ratio between naive LEW and BN rats is intrinsic to hemopoietic cells. Furthermore, a genome-wide search for loci controlling the balance between T cell subpopulations was conducted in a (LEW x BN) F(2) intercross. Genome scanning identified one quantitative trait locus on chromosome 9 (approximately 17 centiMorgan (cM); log of the odds ratio (LOD) score 3.9). In addition, two regions on chromosomes 10 (approximately 28 cM; LOD score 3.1) and 20 (approximately 40 cM; LOD ratio score 3) that contain, respectively, a cytokine gene cluster and the MHC region were suggestive for linkage. Interestingly, overlapping regions on these chromosomes have been implicated in the susceptibility to various immune-mediated disorders. The identification and functional characterization of genes in these regions controlling the CD45RC(high)/CD45RC(low) Th cell subpopulations may shed light on key regulatory mechanisms of pathogenic immune responses.

Cellular and genetic factors involved in the difference between Brown Norway and Lewis rats to develop respectively type-2 and type-1 immune-mediated diseases.

The understanding of the mechanisms of immune tolerance and the unravelling of the pathophysiology of autoimmune diseases rely on animal models. In this respect, BN and LEW rats represent models of choice to study immune-mediated diseases from the cellular and genetic points of view. Indeed, BN and LEW rats are extremes with respect to their polarisation of the immune response as well as their susceptibility to autoimmune diseases. LEW rats are susceptible to Th1-mediated autoimmune diseases while BN rats are highly susceptible to Th2-mediated autoimmune disease. Comparison of the T cell compartment between LEW and BN rats revealed several important differences. 1) A MHC-dependent quantitative difference that is due to a defect in the CD8 T cell compartment in BN rats. 2) A qualitative MHC-independent difference that is related to a high frequency of CD45RClow CD4 and CD8 T cell subsets, producing IL-4, IL-13, IL-10 and TGF-beta in BN rats as compared to LEW rats. 3) Interestingly, the genetic studies showed that susceptibility to Th1-mediated experimental autoimmune encephalomyelitis, and to Th2-mediated disorders triggered by gold salts as well as the difference in the CD4SRChigh/CD45RClow ratio between LEW and BN rats are genetically determined by regions on chromosomes 9, 10 and 20.

Anti-SSA/Ro52 autoantibodies blocking the cardiac 5-HT4 serotoninergic receptor could explain neonatal lupus congenital heart block.

The 52-kDa SSA/Ro (Ro52) ribonucleoprotein is an antigenic target strongly associated with the autoimmune response in mothers whose children develop neonatal lupus and congenital heart block. When sera from patients with systemic lupus erythematosus were used as autoimmune controls in an enzyme immunoassay to screen for antibodies against the human serotoninergic 5-HT4-receptor, a high correlation was found between the presence of anti-Ro52 protein antibodies in such sera and antibodies reacting with a synthetic peptide, corresponding to the second extracellular loop of the human 5-HT4 receptor (amino acid residues 165-185). Homology scanning between the 5-HT4 peptide and the sequence of the Ro52 protein indicated two potential common epitopes located between residues 365 and 396 of the Ro52 protein. Cross-reactivity was found between the peptide derived from the 5-HT4 receptor, and a peptide corresponding to residues 365-382 of the Ro52 protein. Autoantibodies, affinity-purified on the 5-HT4 receptor peptide, specifically recognized both the Ro52 protein and the 5-HT4 receptor protein in immunoblots. The affinity-purified antibodies antagonized the serotonin-induced L-type Ca channel activation on human atrial cells. This effect could explain the electrophysiological abnormalities in neonatal lupus.

Rat chromosome 9 bears a major susceptibility locus for IgE response.

Injection of Brown Norway (BN) rats with gold salts provides a model to analyze the genetic control of the IgE response. A cohort of F2 progeny of susceptible BN and resistant LEW strains has been studied to carry out a genome-wide search for loci controlling the IgE response. Genome scanning identified two previously described loci, Atps1 and Atps2, and a new locus, Atps3. Atps1 linked to the MHC and Atps2 linked to the cytokine gene cluster that included the IL-4 region have been previously associated with serum IgE concentrations and with other Th2-dependent immune manifestations triggered by gold salts. The new interval, Atps3, identified on chromosome 9 (Lod score = 16), appears to play a major role in the control of the IgE response since it accounts for 31% of the genetic variance. Moreover, Atps3 is linked to anti-laminin antibody response and to glomerular immunoglobulin deposits. The identification and functional characterization of genes involved in these regions, particularly in Atps3, may shed light on the pathogenesis of atopic diseases in man.

A dominant role for the thymus and MHC genes in determining the peripheral CD4/CD8 T cell ratio in the rat.

During their development, immature CD4CD8 double positive thymocytes become committed to either the CD4 or CD8 lineage. The final size of the peripheral CD4 and CD8 T cell compartments depends on thymic output and on the differential survival and proliferation of the respective T cell subsets in the periphery. Our results reveal that the development of the distinct peripheral CD4/CD8 T cell ratio between Lewis and Brown Norway rats originates in the thymus and, as shown by the use of radiation bone marrow chimeras, is determined by selection on radio-resistant stromal cells. Furthermore, this difference is strictly correlated with the MHC haplotype and is the result of a reduction in the absolute number of CD8 T cells in Brown Norway rats. These data suggest that the distinct CD4/CD8 T cell ratio between these two rat strains is the consequence of differential interactions of the TCR/CD8 coreceptor complex with the respective MHC class I haplotypes during selection in the thymus.

Longitudinal determination of antiphospholipid antibodies in lupus patients without previous manifestations of antiphospholipid syndrome. A prospective study.

OBJECTIVE: To investigate whether serial anticardiolipin determination contributes to the clinical management of patients with systemic lupus erythematosus (SLE) with no previous sign of the antiphospholipid syndrome (APS). METHODS: In 90 patients with SLE with no previous clinical manifestations of the APS, repeated clinical evaluations were performed, and serial blood samples (obtained over a 30 month period, range 13-53 mo) were screened for antiphospholipid antibodies (aPL). Anticardiolipin antibodies (aCL) were detected using an ELISA and considered positive if the result was >25 GPL on 2 separate occasions. Patients were not required to be on a specific treatment regimen during the study. RESULTS: Thirty-four patients (37%) had at least one positive ELISA and 11 (11/90, 12%) 2 positive ELISA for IgG aCL during the study. Lupus anticoagulant (LAC) was found in 16 patients, and a false positive VDRL in 5. At study completion, the total number of clinical or laboratory events associated with APS was 30. In univariate analysis, aCL was significantly associated only with LAC (p<0.012). Presence of aCL also correlated with hemoglobin level, anti-DNA antibody, leukocyte count, and the SLE Disease Activity Index (SLEDAI). LAC and aCL were significantly associated with each other (OR 5.17; 95% CI 1.5-17.7), but LAC had a better positive predictive value than aCL for arterial thrombosis and neurological events. CONCLUSION: Among our patients with SLE without previous clinical manifestations of APS, positive aCL did not predict the occurrence of APS within the next 3 years, but was statistically related to the clinical disease activity (SLEDAI).