Gotowa bibliografia na temat „Reassortment”

Swine have often been considered as a mixing vessel for different influenza strains. In order to assess their role in more detail, we undertook a retrospective sequencing study to detect and characterize the reassortants present in European swine and to estimate the rate of reassortment between H1N1, H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding segments. We analysed 69 newly obtained whole genome sequences of subtypes H1N1–H3N2 from swine influenza viruses sampled between 1982 and 2008, using Illumina and 454 platforms. Analyses of these genomes, together with previously published genomes, revealed a large monophyletic clade of Eurasian swine-lineage polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently examined reassortments between the haemagglutinin and neuraminidase segments and estimated the reassortment rates between lineages using a recently developed evolutionary analysis method. High rates of reassortment between H1N2 and H1N1 Eurasian swine lineages were detected in European strains, with an average of one reassortment every 2–3 years. This rapid reassortment results from co-circulating lineages in swine, and in consequence we should expect further reassortments between currently circulating swine strains and the recent swine-origin H1N1v pandemic strain.

When two influenza viruses co-infect the same cell, they can exchange genome segments in a process known as reassortment. Reassortment is an important source of genetic diversity and is known to have been involved in the emergence of most pandemic influenza strains. However, because of the difficulty in identifying reassortments events from viral sequence data, little is known about its role in the evolution of the seasonal influenza viruses. Here we introduce TreeKnit, a method that infers ancestral reassortment graphs (ARG) from two segment trees. It is based on topological differences between trees, and proceeds in a greedy fashion by finding regions that are compatible in the two trees. Using simulated genealogies with reassortments, we show that TreeKnit performs well in a wide range of settings and that it is as accurate as a more principled bayesian method, while being orders of magnitude faster. Finally, we show that it is possible to use the inferred ARG to better resolve segment trees and to construct more informative visualizations of reassortments.

Reassortment among the RNA segments of Influenza A virus caused the two most recent human influenza pandemics; recently, reassortment has generated viral genotypes associated with outbreaks of avian H5N1 influenza in Asia and Europe. A statistical analysis has been developed for the systematic identification and characterization of reassortant viruses. The analysis was applied to the genes of the replication complex of 152 avian influenza A viruses isolated between 1966 and 2004 from predominantly terrestrial and domestic aquatic avian species. The results indicated that reassortment among these genes was pervasive throughout this period and throughout both the Eurasian and North American lineages of the virus. Evidence is presented that the circulating genotypes of the replication complex are being replaced continually by novel genotypes created by reassortment. No constraints for coordinated reassortment among genes of the replication complex were evident; rather, reassortment almost always proceeded one segment at a time. A maximum-likelihood estimate of the rate of reassortment was derived. For significantly diverged Asian avian influenza A viruses from the period 1991–2004, it was estimated that the median duration between creation of a new genotype and its next segment reassortment was 3 years. Reassortments that introduced previously unobserved influenza genetic material were detected. These findings point to substantial potential for rapid generation of novel avian influenza A viruses, emphasizing the importance of intensive surveillance of these host species in preparation for a possible pandemic.

The influenza A virus is a negative-stranded RNA virus composed of eight segmented RNA molecules, including polymerases (PB2, PB1, PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (MP), and nonstructure gene (NS). The influenza A viruses are notorious for rapid mutations, frequent reassortments, and possible recombinations. Among these evolutionary events, reassortments refer to exchanges of discrete RNA segments between co-infected influenza viruses, and they have facilitated the generation of pandemic and epidemic strains. Thus, identification of reassortments will be critical for pandemic and epidemic prevention and control. This paper presents a reassortment identification method based on distance measurement using complete composition vector (CCV) and segment clustering using a minimum spanning tree (MST) algorithm. By applying this method, we identified 34 potential reassortment clusters among 2,641 PB2 segments of influenza A viruses. Among the 83 serotypes tested, at least 56 (67.46%) exchanged their fragments with another serotype of influenza A viruses. These identified reassortments involve 1,957 H2N1 and 1,968 H3N2 influenza pandemic strains as well as H5N1 avian influenza virus isolates, which have generated the potential for a future pandemic threat. More frequent reassortments were found to occur in wild birds, especially migratory birds. This MST clustering program is written in Java and will be available upon request.

ABSTRACTThe reassortment of gene segments between influenza viruses increases genomic diversity and plays an important role in viral evolution. We have shown previously that this process is highly efficient within a coinfected cell and, given synchronous coinfection at moderate or high doses, can give rise to ∼60 to 70% of progeny shed from an animal host. Conversely, reassortmentin vivocan be rendered undetectable by lowering viral doses or extending the time between infections. One might also predict that seeding of transmitted viruses into different sites within the target tissue could limit subsequent reassortment. Given the potential for stochastic factors to restrict reassortment during natural infection, we sought to determine its efficiency in a host coinfected through transmission. Two scenarios were tested in a guinea pig model, using influenza A/Panama/2007/99 (H3N2) virus (wt) and a silently mutated variant (var) thereof as parental virus strains. In the first, coinfection was achieved by exposing a naive guinea pig to two cagemates, one infected with wt and the other with var virus. When such exposure led to coinfection, robust reassortment was typically seen, with 50 to 100% of isolates carrying reassortant genomes at one or more time points. In the second scenario, naive guinea pigs were exposed to a cagemate that had been coinoculated with wt and var viruses. Here, reassortment occurred in the coinoculated donor host, multiple variants were transmitted, and reassortants were prevalent in the recipient host. Together, these results demonstrate the immense potential for reassortment to generate viral diversity in nature.IMPORTANCEInfluenza viruses evolve rapidly under selection due to the generation of viral diversity through two mechanisms. The first is the introduction of random errors into the genome by the viral polymerase, which occurs with a frequency of approximately 10−5errors/nucleotide replicated. The second is reassortment, or the exchange of gene segments between viruses. Reassortment is known to occur readily under well-controlled laboratory conditions, but its frequency in nature is not clear. Here, we tested the hypothesis that reassortment efficiency following coinfection through transmission would be reduced compared to that seen with coinoculation. Contrary to this hypothesis, our results indicate that coinfection achieved through transmission supports high levels of reassortment. These results suggest that reassortment is not exquisitely sensitive to stochastic effects associated with transmission and likely occurs in nature whenever a host is infected productively with more than one influenza A virus.

ABSTRACTSwine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression.IMPORTANCEInfluenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

Reassortment is an important source of genetic diversity in segmented viruses and is the main source of novel pathogenic influenza viruses. Despite this, studying the reassortment process has been constrained by the lack of a coherent, model-based inference framework. Here, we introduce a coalescent-based model that allows us to explicitly model the joint coalescent and reassortment process. In order to perform inference under this model, we present an efficient Markov chain Monte Carlo algorithm to sample rooted networks and the embedding of phylogenetic trees within networks. This algorithm provides the means to jointly infer coalescent and reassortment rates with the reassortment network and the embedding of segments in that network from full-genome sequence data. Studying reassortment patterns of different human influenza datasets, we find large differences in reassortment rates across different human influenza viruses. Additionally, we find that reassortment events predominantly occur on selectively fitter parts of reassortment networks showing that on a population level, reassortment positively contributes to the fitness of human influenza viruses.

Abstract Genomic reassortment is an important genetic event in the generation of emerging influenza viruses, which can cause numerous serious flu endemics and epidemics within hosts or even across different hosts. However, there is no dedicated and comprehensive repository for reassortment events among influenza viruses. Here, we present FluReassort, a database for understanding the genomic reassortment events in influenza viruses. Through manual curation of thousands of literature references, the database compiles 204 reassortment events among 56 subtypes of influenza A viruses isolated in 37 different countries. FluReassort provides an interface for the visualization and evolutionary analysis of reassortment events, allowing users to view the events through the phylogenetic analysis with varying parameters. The reassortment networks in FluReassort graphically summarize the correlation and causality between different subtypes of the influenza virus and facilitate the description and interpretation of the reassortment preference among subtypes. We believe FluReassort is a convenient and powerful platform for understanding the evolution of emerging influenza viruses. FluReassort is freely available at https://www.jianglab.tech/FluReassort.

Revealing the frequency and determinants of reassortment among RNA genome segments is fundamental to understanding basic aspects of the biology and evolution of the influenza virus. To estimate the extent of genomic reassortment in influenza viruses circulating in North American swine, we performed a phylogenetic analysis of 139 whole-genome viral sequences sampled during 1998–2011 and representing seven antigenically distinct viral lineages. The highest amounts of reassortment were detected between the H3 and the internal gene segments (PB2, PB1, PA, NP, M and NS), while the lowest reassortment frequencies were observed among the H1γ, H1pdm and neuraminidase segments, particularly N1. Less reassortment was observed among specific haemagglutinin–neuraminidase combinations that were more prevalent in swine, suggesting that some genome constellations may be evolutionarily more stable.

Bluetongue (BT) is a non-contagious viral disease of domestic and wild ruminants. Bluetongue virus (BTV), an Orbivirus that belongs to the family Reoviridae, is the causing agent of the disease. The virus consists of a ten segmented double stranded (ds) RNA genome and currently 27 serotypes have been identified worldwide. The virus is transmitted by Culicoide biting midges (Diptera: Ceratoponidae) and the occurrence of the disease depends on the presence and abundance of competent vectors. In South Africa most European ovine breeds are more susceptible to the disease than indigenous sheep, while cattle and goats in general prove to be sub-clinically infected. During recent outbreaks in Europe (2008 2011) cattle showed severe clinical symptoms and mortality. The role of cattle in the epidemiology of the disease in South Africa is however poorly understood. Bluetongue virus has the ability to reassort its genome segments in vertebrate hosts or vectors which have been infected with more than one strain at the same time. This phenomenon has been reported previously. In 1987, reassortment was investigated in cattle between BTV serotypes 11 and 17 where six reassortants with unique genetic profiles were described. In 2008, in Europe, segment 5 of BTV serotype 16 was identical to the South African vaccine strain of BTV serotype 2. In India in 2013 studies showed that in some isolates obtained from an outbreak segment 6 of BTV serotype 21 were 97.6% identical to segment 6 of BTV serotype 16. Bluetongue disease is controlled by annual vaccination. In South Africa the freeze dried polyvalent BTV vaccine is mainly used to vaccinate sheep, and the vaccine consists of three bottles, each bottle includes five serotypes and each bottle is vaccinated at a three week interval, between August to October of each year. The vaccine proves to be effective in producing immunity against the disease but there are multiple side effects. The main concern is that vaccine virus can be detected during the viraemic period in inoculated sheep. The titre levels are also sufficient to be transferred to non-vaccinated animal hosts via Culicoides midges. The possibility of reassortment between genome segments of vaccine and wild type strain viruses when simultaneously infected therefore exists. This might result in the emergence of new virus serotypes with different phenotypic characteristics i.e. reversion of the live attenuated vaccine strain to a virulent strain. The aim of the project was to investigate the potential generation of genetic reassortant viruses between field and vaccine serotypes of BTV within cattle. Six cattle between the ages of six and twelve months were used. Before the onset of the project cattle were tested for antibodies specific to BTV using a commercial available cELISA and for viral nucleic acid with RT-PCR. Only animals showing negative results by both the tests were used in the trial. The animals were housed in vector-free stables for the duration of the trial. The cattle were divided into two groups; the first group was infected with BTV serotypes from Bottle B of the Onderstepoort Biological Products (OBP) vaccine (BTV serotype 3, 8, 9, 10 and 11), while the second group was infected with the same vaccine serotypes and simultaneously infected with a wild type BTV serotype 4. Blood samples were collected daily from the animals from Day 1 to Day 39 post inoculation. Viraemia was detected between day 2 to day 39 and in one of the animals viraemia could be detected until 39 days post inoculation using virus isolation. Buffy coats as well as first cell culture passages of buffy coats were used to isolate the virus using the plaque forming unit method. The vaccine parental strains were obtained from Bottle two of the vaccine using the plaque forming unit assay and the isolated viruses were serotyped using a serum neutralization assay. Plaques were isolated and amplified on Vero cells. BTV serotype 4 was isolated in the Department of Veterinary Tropical Diseases from a field sample. RNA was extracted from the isolated plaques as well as the six parental strains and compared using polyacrylamide gel electrophoresis (PAGE).
Dissertation (MSc)--University of Pretoria, 2015.
tm2016
Veterinary Tropical Diseases
MSc

La nature segmentée du génome des virus de la grippe A (IAV) permet une évolution rapide par réassortiment génétique. Bien que le nombre théorique de génotypes issus d'un réassortiment entre deux virus soit de 256 (28), la panoplie complète des différents génotypes n'a jamais été observée et certains gènes ont tendance à co-ségréger, suggérant que le réassortiment génétique est biaisé. Cependant, à ce jour, les contraintes qui façonnent le réassortiment génétique restent largement méconnues. L'objectif de mon projet est de progresser dans la compréhension des règles sous-jacentes au réassortiment génétique afin d'améliorer notre capacité à prédire le réassortiment entre les IAV circulant dans la nature.Nous avons dans un premier temps étudié l’incompatibilité entre sous-unités hétérologues de la polymérase virale (FluPol) réunies suite à un réassortiment génétique. En effet, nous avons observé qu'un virus réassortant dont le segment PB2 dérive du virus A/WSN/33 (WSN) dans un fond génétique A/PR/8/34 (PR8) était atténué, malgré un degré d’identité de 97% entre les protéines PR8-PB2 et WSN-PB2. Des passages en série indépendants ont conduit à la sélection de révertants phénotypiques portant des mutations secondaires distinctes sur les sous-unités PA, PB1 et PB2. L’impact de ces mutations a été étudié par génétique inverse et à l’aide de tests d’activité sur les polymérase virales. Pour chaque virus révertant, au moins une mutation a été localisée à l'interface de dimérisation de FluPol et s'est avérée réguler son taux de dimérisation. La mutation PA-E349K en particulier joue un rôle majeur dans la correction d'un défaut initial de réplication virale (ARNc -> ARNv). Nos résultats montrent que les sous-unités de la FluPol co-évoluent non seulement pour assurer des interactions optimales entre sous-unités, mais également des niveaux appropriés de dimérisation, indispensables à une réplication efficace. Ainsi, la dimérisation de la FluPol pourrait être l’un des facteurs limitant l’issue du réassortiment génétique.Parallèlement, afin d’étudier le réassortiment génétique de manière exhaustive et avec une puissance statistique suffisante, nous avons cherché à adapter un système déjà éprouvé de microfluidique en goutte pour un séquençage ciblé, à haut débit et massivement parallélisé, de > 105 IAV issus d’un réassortiment entre deux IAVs. Pour établir la faisabilité du système deux souches virales saisonnières circulantes ont été choisies et des amorces ciblant les huit segments d’ARNv de chaque virus ont été conçues, testées et optimisées. Une expérience contrôle préliminaire réalisée sur des cellules uniques infectées individuellement, montre que les informations sont correctement préservées au niveau de la cellule unique mais que la détection des segments et des souches était déséquilibrée. De nouvelles amorces ont été conçues et des stratégies d'amplification alternatives mises en œuvre et optimisées. Après analyse du réassortiment entre les deux souches saisonnières et validation des données par comparaison avec les données de surveillance, notre système sera appliqué au réassortiment génétique entre les virus saisonniers humains et les virus animaux d’intérêt zoonotique.À long terme, les données générées via notre plateforme devraient aider à la compréhension des mécanismes de réassortiment génétique entre virus influenza. Notre plateforme pourrait également devenir un outil prédictif s’ajoutant aux outils d'évaluation du risque de pandémie grippale ainsi qu’un outil de surveillance
The segmented nature of the genome of influenza A viruses (IAVs) allows rapid evolution by genetic reassortment. Although the theoretical number of genotypes that can emerge from reassortment between two viruses is 256 (28), the full panel of different genotypes was never observed and certain genes tend to co-segregate, suggesting that genetic reassortment is biased. However, to date, the constraints that shape genetic reassortment remain largely unknown. The objective of my project is to make progress in understanding the rules underlying genetic reassortment in order to improve our capacity to predict reassortment among co-circulating IAVs.First, we investigated the incompatibility between non-cognate subunits of the influenza polymerase complex (FluPol) brought together by genetic reassortment. Indeed, we observed that a 7:1 reassortant virus whose PB2 segment derives from the A/WSN/33 (WSN) virus in an otherwise A/PR/8/34 (PR8) backbone was attenuated, despite a 97% identity between the PR8- and WSN-PB2 proteins. Independent serial passages led to the selection of phenotypic revertants bearing distinct second-site mutations on PA, PB1 and PB2. The constellation of mutations present on the revertant viruses was studied using reverse genetics and cell-based reconstitution of the viral polymerase. For each revertant virus, at least one mutation was located at the FluPol dimerization interface and was found to regulate the levels of FluPol dimer. For one of them, PA-E349K, a major role in correcting an initial defect in viral replication (cRNA -> vRNA) was demonstrated. Hence, our results show that the FluPol subunits co-evolve not only to ensure optimal inter-subunit interactions but also proper levels of dimerization of the heterotrimer, essential for efficient viral RNA replication. Thus, we suggest that FluPol dimerization is one of the factors that can restrict the outcome of genetic reassortment.In parallel, in order to study the outcome of genetic reassortment comprehensively and achieve adequate statistical power, we aimed at adapting a proven droplet-based microfluidic single-cell RNA-seq system for customized high-throughput massively parallelized targeted sequencing of > 105 reassortant IAVs. For a proof-of-concept, two circulating seasonal viral strains were chosen and gene specific primers targeting their eight segments were designed, tested and optimized. From a preliminary compartimentalized control experiment, we found that single cell information was well preserved but that segment and strain detection were imbalanced. New primers were designed and alternative amplification strategies were implemented and optimized. A new control experiment will be performed prior to analysis of reassortment between the two seasonal strains and validation of the data by comparison with surveillance data. Once validated, our system will be applied to genetic reassortment between human seasonal viruses and animal viruses of zoonotic interest. In the long term, the data generated through our platform should help understanding the mechanism of IAV genetic reassortment and become a valuable predictive tool added to the Pandemic Influenza Risk Assessment Tools for pandemic preparedness

RNA viruses are among the most virulent microorganisms that threaten the health of humans and livestock. Among the most socio-economically important of the known RNA viruses are those found in the family Orthomyxovirus. In this era of rapid low-cost genome sequencing and advancements in computational biology techniques, many previously difficult research questions relating to the molecular epidemiology and evolutionary dynamics of these viruses can now be answered with ease. Using sequence data together with associated meta-data, in chapter two of this dissertation I tested the hypothesis that the Influenza A/H1N1 2009 pandemic virus was introduced multiple times into Africa, and subsequently dispersed heterogeneously across the continent. I further tested to what degree factors such as road distances and air travel distances impacted the observed pattern of spread of this virus in Africa using a generalised linear modelbased approach. The results suggested that their were multiple simultaneous introductions of 2009 pandemic A/H1N1 into Africa, and geographical distance and human mobility through air travel played an important role towards dissemination. In chapter three, I set out to test two hypotheses: (1) that there is no difference in the frequency of reassortments among the segments that constitute influenza virus genomes; and (2) that there is epochal temporal reassortment among influenza viruses and that all geographical regions are equally likely sources of epidemiologically important influenza virus reassortant lineages. The findings suggested that surface segments are more frequently exchanges than internal genes and that North America/Asia, Oceania, and Asia could be the most likely source locations for reassortant Influenza A, B and C virus lineages respectively. In chapter four of this thesis, I explored the formation of RNA secondary structures within the genomes of orthomyxoviruses belonging to five genera: Influenza A, B and C, Infectious Salmon Anaemia Virus and Thogotovirus using in silico RNA folding predictions and additional molecular evolution and phylogenetic tests to show that structured regions may be biologically functional. The presence of some conserved structures across the five genera is likely a reflection of the biological importance of these structures, warranting further investigation regarding their role in the evolution and possible development of antiviral resistance. The studies herein demonstrate that pathogen genomics-based analytical approaches are useful both for understanding the mechanisms that drive the evolution and spread of rapidly evolving viral pathogens such as orthomyxoviruses, and for illuminating how these approaches could be leveraged to improve the management of these pathogens.

Rokshana Parvin Molecular epidemiology and biological properties of avian influenza viruses of subtype H5N1 and H9N2 Institute of Virology Submitted in November 2014 Pages 106, Figures 7, Table 1, References 339, Publications 4 Keywords: Avian Influenza Virus, H5N1, H9N2, Reassortment, Mutation, Replication and Growth kinetics Introduction Avian influenza viruses (AIVs) are the major cause of significant disease outbreaks with high morbidity and mortality worldwide in domestic birds resulting in great economic losses. Especially the subtypes of highly pathogenic avian influenza viruses (HPAIV) H5N1 and low pathogenic avian influenza viruses (LPAIV) H9N2 became the most prevalent AIVs in poultry causing regular disease outbreaks in many countries of Asia, the Middle East and Europe and are still ongoing events. Therefore, continues monitoring, surveillance and characterization of the circulating viruses are of high priority. Objectives The current study was designed for three main objectives; i) Molecular epidemiology of the HPAIV H5N1 in migratory birds in Bangladesh, ii) Molecular characterization of the AIV subtype H9N2 and iii) Biological properties of the AIV subtype H9N2. Materials and methods In first the part of the investigations, two HPAIV H5N1 strains were confirmed from 205 pools of fecal surveillance samples in Bangladesh. The two isolated H5N1 viruses were characterized by genome amplification and sequence analysis of the all eight genome segments. In the second part of the investigations, a confirmed AIV H9N2 from a retrospective analysis derived from a poultry farm in Bangladesh was characterized. Furthermore, three AI-H9N2 viruses were isolated and characterized from a commercial broiler and broiler breeder flock with clinical respiratory manifestations in Egypt. Full length genome amplification, cloning, sequencing and comprehensive phylogenetic analyses were performed for all eight genome segments. In the final part of the study, four selected Eurasian lineage H9N2 viruses - three G1 sub-lineages H9N2 and one European wild bird H9N2 virus - were propagated in embryonated chicken eggs (ECE) and Madin-Darby canine kidney epithelial cell culture systems. The ECE-grown and cell culture-grown viruses were monitored for replication kinetics based on tissue culture infectious dose (TCID50), hemagglutination assay (HA) and quantitative real time RT-PCR (qRT-PCR). The cellular morphology after infections was analyzed by immunofluorescence assay and cellular ELISA was performed to screen the sensitivity of the viruses to amantadine. Results The two newly isolated HPAIV H5N1 strains from migratory birds belonged to clade 2.3.2.1 and clustered together with other recently isolated viruses in Bangladesh derived from ducks, chickens, quails and crow. The amino acid sequences were also genetically similar although, some unique amino acid substitutions were observed. These substitutions were not related to the known conserved region of the molecular determinants of the virus. The phylogenetic analyses of the isolated AIV H9N2 from Bangladesh and Egypt revealed their close relationship with their respective contemporary isolates and maintained ancestor relation with A/Quail/HK/G1/1997 confirming that all studied H9N2 belonged to G1 sub-lineage. All six internal gene segments of the Bangladeshi AIV H9N2 showed high sequence homology with the HPAIV subtype H7N3 from Pakistan. In addition, also the PB1 internal gene showed high nucleotide homologies with a recently circulating HPAI-H5N1 virus from Bangladesh. Thus, the Bangladeshi AIV H9N2 is genetically a unique strain which shares internal gene segments with different HPAI viruses and takes part in reassortment events. On the other hand, the internal gene segments of the Egyptian H9N2 viruses were similar to the other members of the G1 sub-lineage with no evidence of reassortment events. In this virus rather point mutations within their respective gene segments are observed. With regard to the biological characterization, the three G1-H9N2 viruses produced comparatively higher titer than the Eurasian wild type-AIV H9N2. Overall, the ECE-grown viruses yielded higher titers than cell culture-grown viruses. Following a single passage in cell culture, individual nucleotide substitutions were noticed in HA, NA and NS gene sequences but none of them are related to the conserved region that can alter virus pathogenesis or virulence. All of the studied H9N2 viruses were sensitive to amantadine. Conclusion The present study demonstrated for the first time the presence of HPAI H5N1 in the wild migratory bird population in Bangladesh and determine as one of the major cause to introduce the new clade of HPAIV H5N1 into the Bangladeshi poultry flocks. The Bangladeshi AIV H9N2 strain has exhibited two independent reassortment events with HPAIV of subtype H7N3 and H5N1.The Egyptian AIV H9N2 strains were limited to regular point mutations which is very common for AIVs. The G1-H9N2 viruses showed a higher replication profile when compared to European wild bird-AIV H9N2. Both the ECE and MDCK cell system allowed efficient replication but the ECE system is considered as the better cultivation system for H9N2 viruses in order to get maximum amounts of virus within a short time period. In this study new strains of AIV H9N2 and H5N1 with significant genetic constitutions were described. Thus, continuous monitoring of the field samples, rapid reporting soon after outbreaks, molecular characterization to confirm the emergence of new reassortant strains and the biological properties to know its impact on the virulence are recommended
Rokshana Parvin Molekulare Epidemiologie und biologische Charakterisierung von aviären Influenzaviren der Subtypen H5N1 und H9N2 Institut für Virologie Eingereicht im November 2014 Seiten 106, Abbildungen 7, Tabelle 1, Literaturangaben 339 , Publikationen 4 Schlüsselwörter: Aviäres Influenza Virus, H5N1, H9N2, Reassortment, Mutation, Replikation und Wachstumskinetik Einleitung Weltweit kommt es in der Geflügelproduktion durch Infektionen mit aviären Influenzaviren (AIV) zu hohen Morbiditäts- und Mortalitätsraten und damit verbunden zu hohen wirtschaftlichen Verlusten. Zu den bedeutenden AIV in der Geflügelwirtschaft werden die hoch pathogenen aviären Influenzaviren (HPAIV) des Subtyps H5N1 sowie AIV des Subtyps H9N2 gezählt. Letztere besitzen die Charakteristika von niedrigpathogenen aviären Influenzaviren. Durch diese Subtypen kommt es regelmäßig in vielen Ländern in Asien, im Nahen Osten und Europa zu wiederholten Krankheitsgeschehen. Dies bedingt die dringende Notwendigkeit von andauerndem Monitoring, Überwachung und Charakterisierung der zirkulierenden Viren. Ziele der Untersuchungen Die vorliegende Studie soll folgende drei Hauptfragestellungen beantworten: i) Molekulare Epidemiologie des HPAIV H5N1 bei Zugvögeln in Bangladesch, ii) Molekulare Charakterisierung von AIV des Subtyps H9N2 und iii) Biologische Eigenschaften von AIV des Subtyps H9N2. Materialien und Methoden Der erste Teil der Arbeit befasst sich mit zwei HPAIV Stämmen des Subtyps H5N1, welche im Monitoring Programm in Bangladesch von insgesamt 205 gepolten Kotproben, isoliert wurden. Die Charakterisierung der beiden Isolate erfolgte durch Vervielfältigung der acht Genomsegmente und nachfolgende phylogenetische Analysen. Der zweite Teil der Arbeit beschreibt die retrospektive Analyse eines AIV des Subtyps H9N2, welches von einer Geflügelproduktionsanlage in Bangladesch eingesandt wurde. Weiterhin wurden aus einer Geflügelmast- und Legehennenhaltung mit respiratorischer Symptomatik drei AIV des Subtyps H9N2 isoliert und charakterisiert. Auch hier wurde das gesamte Genom amplifiziert, kloniert und nachfolgend phylogenetisch analysiert. Im letzten Teil der Studie wurden vier europäische AIV H9N2 Isolate, von welchen 3 Isolate zur H9N2 Sublinie G1 gehören und ein Isolat von einem Wildvogel selektiert und in embryonierten Hühnereiern (EHE) und auf Madin-Darby canine kidney (MDCK) Zellen passagiert. Mittels 50% tissue culture infectious dose (TCID50), Hämagglutinationstest (HA) und RT-real-time-PCR (qRT-PCR) wurden von diesen so passagierten Viren die Vermehrungskinetik bestimmt. Die Morphologie der infizierten Zellen nach Infektion wurde mittels Immunfluoreszenztest analysiert. Eine Bestimmung der Amantadin Empfindlichkeit dieser Viren erfolgte mit einem ELISA. Ergebnisse Die beiden neuen HPAIV des Subtyps H5N1 von Zugvögeln können in die Clade 2.3.2.1 eingeordnet werden und clustern mit kürzlich aus Enten, Hühnern, Wachteln und Krähen isolierten AIV aus Bangladesch. Eine Verwandtschaft der Viren konnte auch auf Ebene der Aminosäure Sequenz gezeigt werden, obwohl einige einzigartige Aminosäure Austausche nachgewiesen wurden. Diese Austausche zeigen keine Verbindung mit bekannten konservierten Regionen der molekularen Determinanten der Viren. Die phylogenetische Analyse der AIV aus Bangladesch und Ägypten zeigt eine deutliche Verbindung mit den derzeit zirkulierenden AIV auf diesem geographischen Gebiet sowie die Verwandtschaft zu dem Isolat A/Quail/HK/G1/1997. Dies bestätigt, dass die in dieser Studie analysierten AIV zu der Subline G1 gehören. Alle sechs internen Gensegmente des AIV H9N2 aus Bangladesch zeigen eine hohe Sequenz Homologie mit einem HPAIV des Subtyps H7N3 aus Pakistan. Zusätzlich zeigt das interne Gene PB1 eine hohe Homologie auf Nukleinsäureebene zu einem derzeit in Bangladesch zirkulierenden HPAIV des Subtyps H5N1. Somit ist das AIV H9N2 aus Bangladesch als ein einzigartiges Isolat anzusehen, welches durch Reassortierung interne Gensegmente mit hochpathogenen AIV teilt. Im Gegensatz dazu, sind die internen Gene des AIV H9N2 aus Ägypten sehr ähnlich zu anderen Mitgliedern der Sublinie G1, welche keine Hinweise auf Reassorierung zeigen. Nur einzelne Punktmutationen konnten in den entsprechenden Gensegmenten nachgewiesen werden. In Hinblick auf die biologische Charakterisierung, konnte in den drei AIV H9N2 der Sublinie G1 vergleichsweise höhere Titer nachgewiesen werden als in einem europäischen AIV H9N2 Wildtypisolat. Insgesamt zeigten die in EHE passagierten Viren höhere Titer als die MDCK-Zell passagierten Viren. Schon nach einer Passage auf Zellkultur konnten einzelne Nukleotidaustausche in den HA, NA und NS kodierenden Gensegmenten nachgewiesen werden, wobei keine dieser Veränderungen einen Einfluss auf konservierte Regionen haben, die die Pathogenese oder Virulenz der Viren beeinflussen. Alle untersuchten H9N2 Viren sind sensitiv gegenüber Amantadin. Schlussfolgerungen Die vorliegende Studie zeigt erstmalig das Vorkommen von HPAIV H5N1 bei Zugvögeln in Bangladesch, welches als Haupteintragsquelle der neuen HPAIV H5N1 in der dortigen Geflügelhaltung angesehen wird. Das AIV H9N2 aus Bangladesch zeigt zwei unabhängige Reassortierungen mit HPAIV des Subtyps H7N3 und H5N1. Hingegen zeigt das ägyptische AIV H9N2 Punktmutationen, welche sehr typisch für diese Viren sind. Die hier untersuchten AIV H9N2 der Sublinie G1 zeigen im Vergleich zu einem europäischen AIV H9N2 eine höhere Replikationsrate. Eine Replikation der Viren konnte in EHE und MDCK-Zellen gezeigt werden, jedoch wird das EHE als das geeignetere System für die Kultivierung von H9N2 Viren betrachtet, da hier in einer kürzeren Zeitspanne mehr Virus produziert werden kann. Des Weiteren konnten in dieser Studie neue Isolate von AIV des Subtyps H9N2 und H5N1mit einem bedeutenden genetischen Aufbau beschrieben werden. Daher wird ein kontinuierliches Monitoring von Feldproben, unverzügliche Meldung von Ausbruchsgeschehen, die molekulare Charakterisierung zur Dokumentation eventuell auftretender neuer Reassortanten sowie Untersuchungen der biologischer Eigenschaften zur Virulenzbestimmung empfohlen

Influenza B viruses are a significant cause of disease and influenza B antigens are present in all human vaccines. Achieving suitable yields of seed viruses is often difficult for vaccine manufacturers. With influenza A viruses increases in yields have been achieved by the preparation of reassortants between a high-yielding donor strain and an epidemic strain. However, reassortment of influenza B viruses for the preparation of seeds has not been usually undertaken due to the lack suitable donor strains. Such an approach, which formed the basis of this thesis, could improve vaccine yields, lower costs and introduce a further element of predictability to vaccine manufacture. Potential donor strains were prepared from B/Lee/40 (B/Lee) by two approaches involving the selection of stable cold- and high- temperature mutants. Initial passaging was undertaken in specific-pathogen-free (SPF) chicken embryo kidney (CEK) cultures and later passage in SPF embryonated chicken eggs. Both approaches were successful, although a smaller number of viable progeny could be isolated from plaques obtained at 38„aC. Potential donor strains, isolated by selection at either 25 or 38„aC and plaque-purified in SPF CEK cultures, were tested for haemagglutinin and infectious titre, in comparison with the original parental strain by three methods, and for differences in antigenicity by cross-haemagglutination-inhibition tests. Potential donor strains selected at temperatures of 25„aC (C25) and 38„aC (H38) produced haemagglutination titres of 320 units/50ƒÝL and infectivities of 8.57 and 8.39 50% egg infectious doses, respectively, when grown in eggs at the permissive temperature (34„aC). Reassorting experiments using the B/Lee-derived potential donor strains C25 and H38 and the epidemic strain, B/Johannesburg/5/99 (B/Johannesburg), showed that the preparation of reassortant progeny with both epidemic strain HA and NA was difficult. Only 1/24 of the resulting reassortants possessed both the HA and NA of the epidemic strain. None of the reassortant progeny produced in reassorting experiments using C25 and H38 and the epidemic strain B/Panama/45/90 (B/Panama) possessed the desired 6:2 gene constellation (i.e. genes for the two surface antigens of the epidemic strain and the remainder from the donor strain). The infectious titre of selected progeny from the reassortment experiments were determined by three methods and compared with their respective epidemic parents. Yields of several influenza B epidemic strains and potential donor strains were measured after growth in Madin-Darby canine kidney (MDCK) cells prepared in serum-containing (SC) and animal- and human-derived protein-free (AHPF) media. Optimal multiplicities of infection were determined for B/Panama, B/Johannesburg and C25 in MDCK cultures grown in SC medium. A series of experiments were then undertaken to determine the maximum virus yields in MDCK cells grown in SC medium, followed by a further experiment using C25, B/Panama, B/Johannesburg, and selected reassortants after preparation in AHPF medium. Cell culture yields from 5/6 viruses grown in MDCK cells prepared in AHPF medium were higher than in cells prepared in SC medium and approached those obtained in eggs.

RNA viruses have the fastest evolutionary rates amongst protein-coding organisms on the planet. Ease of sequencing, advanced techniques of analysis and global health and economic concerns have all contributed to the recognition of RNA viruses as a robust research platform. Phylogenetic methods have been at the forefront of analytical techniques used to understand the dynamics of RNA viruses - during natural circulation in populations and in individual hosts, within epidemics, across species barriers and over billions of years that viruses have been around. Most of the work presented in this thesis employs phylogenetic incongruity arising from reassortment and recombination to gain insights into the genomes and populations of RNA viruses. Chapter 2 explores the selection regimes Ebola virus has experienced following a year of circulation in humans inWest Africa, as well as its recent history. Chapter 3 investigates the extent of recombination in MERS-CoV, a novel human pathogen with an obscure epidemiology, which is suggestive of frequent co-infection of some hosts. Chapter 4, on the other hand, documents a pattern of non-intuitive linkage between some segments of the human-endemic influenza B virus genome and explores its potential to speciate. Chapter 5 builds upon chapter 4 and attempts to describe small-scale reassortment between two segments of influenza B virus and the overall migration patterns of influenza B virus in Scotland. Chapter 6 exploits the independence of segments of influenza D virus, a recently described cattle pathogen, and coalescent theory to disentangle the origins of this virus. This thesis exemplifies the success of modern sequencing methods, which, together with the use of sophisticated analytical techniques, have uncovered a wealth of information hidden away in molecular sequences of RNA viruses. The work presented herein demonstrates how reticulate evolution can be exploited as a reliable, and sometimes indispensable, marker to improve inference of evolutionary forces in RNA viruses.

Influenza is a highly infectious, acute illness which has affected humans and animals since ancient times. Influenza viruses form the Orthomyxoviridae family and are grouped into types A, B, and C on the basis of the antigenic nature of the internal nucleocapsid or the matrix protein. Infl uenza A viruses infect a large variety of animal species, including humans, pigs, horses, sea mammals, and birds, occasionally producing devastating pandemics in humans, such as in 1918 when it has been estimated that between 50–100 million deaths occurred worldwide.There are two important viral surface glycoproteins, the haemagglutinin (HA) and neuraminidase (NA). The HA binds to sialic acid receptors on the membrane of host cells and is the primary antigen against which a host’s antibody response is targeted. The NA cleaves the sialic acid bond attaching new viral particles to the cell membrane of host cells allowing their release. The NA is also the target of the neuraminidase inhibitor class of antiviral agents that include oseltamivir and zanamivir and newer agents such as peramivir. Both these glycoproteins are important antigens for inducing protective immunity in the host and therefore show the greatest variation.Influenza A viruses are classified into 16 antigenically distinct HA (H1–16) and 9 NA subtypes (N1–9). Although viruses of relatively few subtype combinations have been isolated from mammalian species, all subtypes, in most combinations, have been isolated from birds. Each virus possesses one HA and one NA subtype.Last century, the sudden emergence of antigenically different strains in humans, termed antigenic shift, occurred on three occasions, 1918 (H1N1), 1957 (H2N2) and 1968 (H3N2), resulting in pandemics. The frequent epidemics that occur between the pandemics are as a result of gradual antigenic change in the prevalent virus, termed antigenic drift. Epidemics throughout the world occur in the human population due to infection with influenza A viruses, such as H1N1 and H3N2 subtypes, or with influenza B virus. Phylogenetic studies have led to the suggestion that aquatic birds that show no signs of disease could be the source of many influenza A viruses in other species. The 1918 H1N1 pandemic strain is thought to have arisen as a result of spontaneous mutations within an avian H1N1 virus. However, most pandemic strains, such as the 1957 H2N2, 1968 H3N2 and 2009 pandemic H1N1, are considered to have emerged by genetic re-assortment of the segmented RNA genome of the virus, with the avian and human influenza A viruses infecting the same host.Influenza viruses do not pass readily between humans and birds but transmission between humans and other animals has been demonstrated. This has led to the suggestion that the proposed reassortment of human and avian influenza viruses takes place in an intermediate animal with subsequent infection of the human population. Pigs have been considered the leading contender for the role of intermediary because they may serve as hosts for productive infections of both avian and human viruses, and there is good evidence that they have been involved in interspecies transmission of influenza viruses; particularly the spread of H1N1 viruses to humans. Apart from public health measures related to the rapid identification of cases and isolation. The main control measures for influenza virus infections in human populations involves immunization and antiviral prophylaxis or treatment.

Is an infection that is already widespread in humans likely to cause a pandemic? As discussed previously, that really gets down to a matter of terminology. The influenza A viruses that emerge as a consequence of genetic reassortment, perhaps between viruses circulating in us...

Different molecular aspects of avian viruses are presented such as the role and importance of capsid proteins and viral glycoproteins in viral pathogenesis, in development of diagnostic methods and the creation of recombinant vaccines is discussed. Mechanisms of viral evolution such as mutation, recombination and reassortment are included, and their role in the origin of diverse influenza, infectious bursal disease, avian reovirus is commented.

Most animal pathogens that infect humans employ an intermediate host. The original hosts of most coronaviruses are various species of bat. The original host of all flu viruses is the duck. But we tend to catch coronaviruses from animals that have been infected by bats. Influenza is more likely to be passed on by chickens or pigs. By eating animals, we engineer many opportunities for species, and their pathogens, to mix and mingle. We turn animals into intermediate hosts of harmful pathogens by inserting them into a particular point on a food chain that leads, ultimately, to us. These ideas are explained via SARS-CoV-1&2, the Spanish flu, the H5N1 influenza virus, and Nipah virus, among others. The role played by animal agriculture in virus mutation and reassortment is explained. By no longer eating animals, we would largely eliminate the threat of zoonotic diseases.

Influenza is caused by a virus with a segmented RNA genome. The virus can infect humans, pigs and other mammals and birds. The viral envelope contains two key proteins, haemagglutinin (HA) and neuraminidase (NA). There are sixteen known variants of the HA and nine of the NA. During an infection, HA binds to receptors on airway epithelial cells. Human influenza viruses and bird influenza viruses have different receptors. Some animals, such as pigs have both types of receptors. NA removes sialic acid residues from viral glycoproteins and plays a role in the infection process. The RNA polymerase of influenza is error-prone and this results in mutations in HA and NA (antigenic drift). Some of these mutants are more infectious and cause seasonal influenza. If an avian influenza virus and a human influenza virus infect an animal with both virus receptors, such as a pig, reassortment of the eight RNA segments from each virus can occur. If any of the 256 possible new combinations (antigenic shift) has increased virulence then it can cause a pandemic. Seasonal vaccination of those most at risk is the best preventative strategy but occasionally the virus changes in unexpected ways and the vaccines in use have reduced effectiveness.

In September 2006 an outbreak of 'Bluetongue like' disease struck the cattle herds in Israel. Over 100 dairy and beef cattle herds were affected. Epizootic hemorrhagic disease virus (EHDV) (an Orbivirusclosely related to bluetongue virus (BTV)), was isolated from samples collected from several herds during the outbreaks. Following are the aims of the study and summary of the results: which up until now were published in 6 articles in peer-reviewed journals. Three more articles are still under preparation: 1. To identify the origin of the virus: The virus identified was fully sequenced and compared with the sequences available in the GenBank. It appeared that while gene segment L2 was clustered with EHDV-7 isolated in Australia, most of the other segments were clustered with EHDV-6 isolates from South-Africa and Bahrain. This may suggest that the strain which affected Israel on 2006 may have been related to similar outbreaks which occurred in north-Africa at the same year and could also be a result of reassortment with an Australian strain (Wilson et al. article in preparation). Analysis of the serological results from Israel demonstrated that cows and calves were similarly positive as opposed to BTV for which seropositivity in cows was significantly higher than in calves. This finding also supports the hypothesis that the 2006 EHD outbreak in Israel was an incursive event and the virus was not present in Israel before this outbreak (Kedmi et al. Veterinary Journal, 2011) 2. To identify the vectors of this virus: In the US, Culicoides sonorensis was found as an efficient vector of EHDV as the virus was transmitted by midges fed on infected white tailed deer (WTD; Odocoileusvirginianus) to susceptible WTD (Ruder et al. Parasites and Vectors, 2012). We also examined the effect of temperature on replication of EHDV-7 in C. sonorensis and demonstrated that the time to detection of potentially competent midges decreased with increasing temperature (Ruder et al. in preparation). Although multiple attempts were made, we failed to evaluate wild-caught Culicoidesinsignisas a potential vector for EHDV-7; however, our finding that C. sonorensis is a competent vector is far more significant because this species is widespread in the U.S. As for Israeli Culicoides spp. the main species caught near farms affected during the outbreaks were C. imicolaand C. oxystoma. The vector competence studies performed in Israel were in a smaller scale than in the US due to lack of a laboratory colony of these species and due to lack of facilities to infect animals with vector borne diseases. However, we found both species to be susceptible for infection by EHDV. For C. oxystoma, 1/3 of the Culicoidesinfected were positive 11 days post feeding. 3. To identify the host and environmental factors influencing the level of exposure to EHDV, its spread and its associated morbidity: Analysis of the cattle morbidity in Israel showed that the disease resulted in an average loss of over 200 kg milk per cow in herds affected during September 2006 and 1.42% excess mortality in heavily infected herds (Kedmi et al. Journal of Dairy Science, 2010). Outbreak investigation showed that winds played a significant role in virus spread during the 2006 outbreak (Kedmi et al. Preventive Veterinary Medicine, 2010). Further studies showed that both sheep (Kedmi et al. Veterinary Microbiology, 2011) and wild ruminants did not play a significant role in virus spread in Israel (Kedmi et al. article in preparation). Clinical studies in WTD showed that this species is highly susceptibile to EHDV-7 infection and disease (Ruder et al. Journal of Wildlife Diseases, 2012). Experimental infection of Holstein cattle (cows and calves) yielded subclinical viremia (Ruder et al. in preparation). The findings of this study, which resulted in 6 articles, published in peer reviewed journals and 4 more articles which are in preparation, contributed to the dairy industry in Israel by defining the main factors associated with disease spread and assessment of disease impact. In the US, we demonstrated that sufficient conditions exist for potential virus establishment if EHDV-7 were introduced. The significant knowledge gained through this study will enable better decision making regarding prevention and control measures for EHDV and similar viruses, such as BTV.