Replication of porcine circoviruses

Porcine circoviruses are circular single-stranded DNA viruses that infect swine and wild boars. Two species of porcine circoviruses exist. Porcine circovirus type 1 is non pathogenic contrary to porcine circovirus type 2 which is associated with the disease known as Post-weaning Multisystemic Wasting Syndrome. Porcine circovirus DNA has been shown to replicate by a rolling circle mechanism. Other studies have revealed similar mechanisms of rolling-circle replication in plasmids and single-stranded viruses such as Geminivirus. Three elements are important in rolling-circle replication: i) a gene encoding initiator protein, ii) a double strand origin, and iii) a single strand origin. However, differences exist between viruses and plasmids and between viruses. Porcine circovirus replication probably involves a "melting pot" rather than "cruciform" rolling-circle mechanism

Modification of PCV-2 virulence by substitution of the genogroup motif of the capsid protein

Porcine circovirus type 2 (PCV-2) is the causal agent of the post-weaning multisystemic wasting syndrome (PMWS). PCV-2 are small single-stranded circular DNA viruses clustered into two main genogroups: PCV-2a and PCV-2b. Each genogroup present a specific highly-conserved motif of six amino acids (between amino acids 86 and 91) in the PCV-2 capsid protein. The aim of this study was to verify whether the motif located in the capsid protein and specific to each PCV-2 genogroup contributes to virulence. Two parental DNA clones, PCV-2a and PCV-2b, were constructed as well as two mutants DNA clones, PCV-2a/motif 2b and PCV-2b/motif 2a by exchanging the capsid motif of each genogroup. The four DNA clones were characterized in vitro as well as in vivo. Cells transfected by the four DNA clones produced infectious viruses. In specific-pathogen-free piglets transfected by the four infectious DNA clones, PCV-2b/motif 2a virulence was not attenuated while the PCV-2a/motif 2b virulence was drastically reduced compared to their parent virulence. These results suggest that the amino acids between positions 86 and 91 of the capsid protein are determinant for the virulence of isolates. However, the environment of this motif seems also involved

Infectious Bronchitis Coronavirus: Genome Evolution in Vaccinated and Non-Vaccinated SPF Chickens

Infectious Bronchitis virus (IBV) continues to cause significant economic losses for the chicken industry despite the use of many live IBV vaccines around the world. Several authors have suggested that vaccine-induced partial protection may contribute to the emergence of new IBV strains. In order to study this hypothesis, three passages of a challenge IBV were made in SPF chickens sham inoculated or vaccinated at day of age using a live vaccine heterologous to the challenge virus. All birds that were challenged with vaccine heterologous virus were positive for viral RNA. NGS analysis of viral RNA in the unvaccinated group showed a rapid selection of seven genetic variants, finally modifying the consensus genome of the viral population. Among them, five were non-synonymous, modifying one position in NSP 8, one in NSP 13, and three in the Spike protein. In the vaccinated group, one genetic variant was selected over the three passages. This synonymous modification was absent from the unvaccinated group. Under these conditions, the genome population of an IBV challenge virus evolved rapidly in both heterologous vaccinated and non-vaccinated birds, while the genetic changes that were selected and the locations of these were very different between the two groups

Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog

The avibirnavirus infectious bursal disease virus (IBDV) is responsible for a highly contagious and sometimes lethal disease of chickens (Gallus gallus). IBDV genetic variation is well-described for both field and live-attenuated vaccine strains, however, the dynamics and selection pressures behind this genetic evolution remain poorly documented. Here, genetically homogeneous virus stocks were generated using reverse genetics for a very virulent strain, rvv, and a vaccine-related strain, rCu-1. These viruses were serially passaged at controlled multiplicities of infection in several biological systems, including primary chickens B cells, the main cell type targeted by IBDV in vivo. Passages were also performed in the absence or presence of a strong selective pressure using the antiviral nucleoside analog 7-deaza-2′-C-methyladenosine (7DMA). Next Generation Sequencing (NGS) of viral genomes after the last passage in each biological system revealed that (i) a higher viral diversity was generated in segment A than in segment B, regardless 7DMA treatment and viral strain, (ii) diversity in segment B was increased by 7DMA treatment in both viruses, (iii) passaging of IBDV in primary chicken B cells, regardless of 7DMA treatment, did not select cell-culture adapted variants of rvv, preserving its capsid protein (VP2) properties, (iv) mutations in coding and non-coding regions of rCu-1 segment A could potentially associate to higher viral fitness, and (v) a specific selection, upon 7DMA addition, of a Thr329Ala substitution occurred in the viral polymerase VP1. The latter change, together with Ala270Thr change in VP2, proved to be associated with viral attenuation in vivo. These results identify genome sequences that are important for IBDV evolution in response to selection pressures. Such information will help tailor better strategies for controlling IBDV infection in chickens

Circovirus porcin de type 2: pouvoir pathogène et transmission

This report presents a summary of my research activities carried out as part of my research function on emerging viruses in swine at ANSES laboratory of Ploufragan. The research focused on the study of porcine circovirus type 2 (PCV-2), associated with several diseases in swine such as post-weaning multisystemic wasting syndrome. This work is divided into four axes: (i) the study of viral virulence factors that started with the construction of an infectious clone which allowed to study point mutants of the viral capsid; (ii) in vitro and in vivo host-pathogen interactions performed by transcriptomic analysis; (iii) the study of vertical and horizontal transmission of the virus and finally (iv) the study of diseases associated with PCV-2. All these research studies on a virus that causes a disease that emerged in the late 1990s in pigs allowed setting up a structure to be mobilized during emergence by identification of the associated agent, and carrying out research programs on the characteristic of the viral agent in order to implement adequate control measures.Ce mémoire présente une synthèse de mes activités de recherche réalisées dans le cadre de ma fonction de chargée de recherche sur les virus émergents dans l’espèce porcine à l’Anses laboratoire de Ploufragan. Les travaux de recherche présentés ont porté sur l’étude du circovirus porcin de type 2 (PCV-2), associé à de nombreuses maladies chez le porc dont la maladie d’amaigrissement du porcelet. Ces travaux se déclinent en quatre axes : (i) l’étude des facteurs de virulence de virus qui a débuté par la construction d’un clone infectieux et qui a permis d’étudier des mutants ponctuels dans la capside virale ; (ii) l’étude in vitro et in vivo des interactions hôte-pathogène par analyse transcriptomique ; (iii) l’étude de la transmission verticale et horizontale du virus et enfin (iv) l’étude des maladies associées au PCV-2. L’ensemble de ces travaux de recherche sur un virus responsable d’une maladie qui a émergé à la fin des années 1990 chez le porc a permis de mettre en place une structure destinée à être mobilisée en situation de suspicion d’émergence virale par l’identification de l’agent associé et de réaliser des projets de recherche sur la caractéristique de l’agent viral afin de pouvoir mettre en place des mesures de contrôle adéquates

Etude du rôle des interactions énergétiques et des communications intercellulaires dans la formation de biofilms bactériens en milieu marin

En milieu marin, la majorité des bactéries croît au sein de biofilms. Notre étude s'est portée sur deux étapes de leur formation : la phase initiale d'adhésion impliquant des interactions énergétiques et la maturation au cours de laquelle le quorum sensing, à l'aide molécules de type acyl homosérine lactones (AHSL), serait responsable d'adaptations phénotypiques des cellules. Tout d'abord, des AHSL et des gènes du quorum sensing ont été recherchés chez Vibrio tapetis. La production de C6-HSL et de dipeptides cycliques ont été montrés. Puis, les propriétés physicochimiques de surface de bactéries marines pionnières d'adhésion ont été analysées. Ces souches présentent une hétérogénéité de ces propriétés malgré une majorité de bactérie avec une surface hydrophile. Ceci suggère le rôle de structures membranaires dans cette étape initiale comme l'antigène O des LPS. Enfin, l'influence de C4-HSL dans la formation de biofilm par ces bactérie a été montrée comme dépendant de la souche.LORIENT-BU (561212106) / SudocSudocFranceF

Impact of porcine circovirus type 2 (PCV2) infection on hepatitis E virus (HEV) infection and transmission under experimental conditions

Hepatitis E virus is a zoonotic pathogen for which pigs have been identified as the main reservoir in industrialised countries. HEV infection dynamics in pig herds and pigs are influenced by several factors, including herd practices and possibly co-infection with immunomodulating viruses. This study therefore investigates the impact of porcine circovirus type 2 (PCV2) on HEV infection and transmission through experimental HEV/PCV2 co-infection of specific-pathogen-free pigs. No statistical difference between HEV-only and HEV/PCV2-infected animals was found for either the infectious period or the quantity of HEV shed in faeces. The HEV latency period was shorter for HEV/PCV2 co-infected pigs than for HEV-only infected pigs (11.6 versus 12.3 days). Its direct transmission rate was three times higher in cases of HEV/PCV2 co-infection than in cases of HEV-only infection (0.12 versus 0.04). On the other hand, the HEV transmission rate through environmental accumulation was lower in cases of HEV/PCV2 co-infection (4.3.10(-6) versus 1.5.10(-5) g/RNA copies/day for HEV-only infected pigs). The time prior to HEV seroconversion was 1.9 times longer in HEV/PCV2 co-infected pigs (49.4 versus 25.6 days for HEV-only infected pigs). In conclusion, our study shows that PCV2 affects HEV infection and transmission in pigs under experimental conditions

Infectious bursal disease virus: predicting viral pathotype using machine learning models focused on early changes in total blood cell counts

Abstract Infectious bursal disease (IBD) is an avian viral disease caused in chickens by infectious bursal disease virus (IBDV). IBDV strains (Avibirnavirus genus, Birnaviridae family) exhibit different pathotypes, for which no molecular marker is available yet. The different pathotypes, ranging from sub-clinical to inducing immunosuppression and high mortality, are currently determined through a 10-day-long animal experiment designed to compare mortality and clinical score of the uncharacterized strain with references strains. Limits of this protocol lie within standardization and the extensive use of animal experimentation. The aim of this study was to establish a predictive model of viral pathotype based on a minimum number of early parameters measured during infection, allowing faster pathotyping of IBDV strains with improved ethics. We thus measured, at 2 and 4 days post-infection (dpi), the blood concentrations of various immune and coagulation related cells, the uricemia and the infectious viral load in the bursa of Fabricius of chicken infected under standardized conditions with a panel of viruses encompassing the different pathotypes of IBDV. Machine learning algorithms allowed establishing a predictive model of the pathotype based on early changes of the blood cell formula, whose accuracy reached 84.1%. Its accuracy to predict the attenuated and strictly immunosuppressive pathotypes was above 90%. The key parameters for this model were the blood concentrations of B cells, T cells, monocytes, granulocytes, thrombocytes and erythrocytes of infected chickens at 4 dpi. This predictive model could be a second option to traditional IBDV pathotyping that is faster, and more ethical

Quantification of porcine circovirus type 2 (PCV-2) within- and between-pen transmission in pigs

PCV-2 within- and between-pen transmission was quantified by estimating the daily transmission rate $\beta$ and the basic reproduction ratio ($R_0$) using a stochastic SEIR (Susceptible, Exposed, Infectious, Removed) model fitted on experimental data. Within-pen transmission was quantified by using four groups of eight SPF (specific pathogen-free) pigs (four infected and four susceptible pigs having direct contact). Between-pen transmission was studied in two groups of 16 SPF pigs (eight infected and eight susceptible pigs having indirect contact (10 cm distance)). Pigs were monitored twice a week (blood samples) and were tested for PCV-2 antibodies (ELISA test) and viral genome load in sera (real-time PCR). Transmission parameters $\beta_{\rm within}$ and $\beta_{\rm between}$ were estimated using a maximum likelihood method and the duration of infectiousness, to compute $R_0$, was estimated with a parametric survival model. Different assumptions were made to determine the end of infectiousness (seroconversion, seroconversion and decline in viral genome load, permanent infectiousness). $R_{0[\rm within]}$ (8.9 (5.1-15.4)) was greater when the end of infectiousness was assumed to be related to both seroconversion and a decline of PCV-2 genome load in sera (average duration of infectiousness = 32 days) compared with only seroconversion as the indicator of recovery ($R_{0[\rm within]}$ = 5.5 (3.3-9.0)). Whatever the assumption, between-pen $R_0$ (0.58 (0.23-1.47)) was always significantly lower than within-pen $R_0$. Only $\beta_{\rm within}$ was sensitive to the assumption on end of infectiousness and decreased with increasing duration of infectiousness. These results showed that PCV-2 transmission is influenced by contact structure that appears worth being taken into account in an epidemic model