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1.  Infectious Bronchitis Virus Generates Spherules from Zippered Endoplasmic Reticulum Membranes 
mBio  2013;4(5):e00801-13.
ABSTRACT
Replication of positive-sense RNA viruses is associated with the rearrangement of cellular membranes. Previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian cell line but also in primary avian cells and in epithelial cells of ex vivo tracheal organ cultures. In all cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes.
IMPORTANCE All positive-sense single-stranded RNA viruses induce rearranged cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.
IMPORTANCE
All positive-sense single-stranded RNA viruses induce rearranged cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.
doi:10.1128/mBio.00801-13
PMCID: PMC3812713  PMID: 24149513
2.  A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq 
BMC Genomics  2014;15(1):828.
Background
Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template.
Results
The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately flanking the poly(C) region.
Conclusions
We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-828) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-828
PMCID: PMC4247156  PMID: 25269623
Next generation sequencing; Whole genome sequencing; Foot-and-mouth disease virus; Genome; RNA; virus; FMDV
3.  Visualizing the autophagy pathway in avian cells and its application to studying infectious bronchitis virus 
Autophagy  2013;9(4):496-509.
Autophagy is a highly conserved cellular response to starvation that leads to the degradation of organelles and long-lived proteins in lysosomes and is important for cellular homeostasis, tissue development and as a defense against aggregated proteins, damaged organelles and infectious agents. Although autophagy has been studied in many animal species, reagents to study autophagy in avian systems are lacking. Microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) is an important marker for autophagy and is used to follow autophagosome formation. Here we report the cloning of avian LC3 paralogs A, B and C from the domestic chicken, Gallus gallus domesticus, and the production of replication-deficient, recombinant adenovirus vectors expressing these avian LC3s tagged with EGFP and FLAG-mCherry. An additional recombinant adenovirus expressing EGFP-tagged LC3B containing a G120A mutation was also generated. These vectors can be used as tools to visualize autophagosome formation and fusion with endosomes/lysosomes in avian cells and provide a valuable resource for studying autophagy in avian cells. We have used them to study autophagy during replication of infectious bronchitis virus (IBV). IBV induced autophagic signaling in mammalian Vero cells but not primary avian chick kidney cells or the avian DF1 cell line. Furthermore, induction or inhibition of autophagy did not affect IBV replication, suggesting that classical autophagy may not be important for virus replication. However, expression of IBV nonstructural protein 6 alone did induce autophagic signaling in avian cells, as seen previously in mammalian cells. This may suggest that IBV can inhibit or control autophagy in avian cells, although IBV did not appear to inhibit autophagy induced by starvation or rapamycin treatment.
doi:10.4161/auto.23465
PMCID: PMC3627666  PMID: 23328491
chicken; avian; GFP-LC3; autophagy; primary cells; recombinant adenovirus; coronavirus; infectious bronchitis virus; LC3A; LC3B; LC3C
4.  Coronavirus nsp6 proteins generate autophagosomes from the endoplasmic reticulum via an omegasome intermediate 
Autophagy  2011;7(11):1335-1347.
Autophagy is a cellular response to starvation which generates autophagosomes to carry cellular organelles and long-lived proteins to lysosomes for degradation. Degradation through autophagy can provide an innate defense against virus infection, or conversely autophagosomes can promote infection by facilitating assembly of replicase proteins. We demonstrate that the avian coronavirus, infectious bronchitis virus (IBV), activates autophagy. A screen of individual IBV nonstructural proteins (nsps) showed that autophagy was activated by IBV nsp6. This property was shared with nsp6 of mammalian coronaviruses mouse hepatitis virus, and severe acute respiratory syndrome virus, and the equivalent nsp5–7 of the arterivirus porcine reproductive and respiratory syndrome virus. These multiple-spanning transmembrane proteins located to the endoplasmic reticulum (ER) where they generated Atg5 and LC3II -positive vesicles, and vesicle formation was dependent on Atg5 and class III PI3 kinase. The vesicles recruited double-FYVE-domain containing protein (DFCP) indicating localized concentration of phosphatidylinositol 3 phosphate, and therefore shared many features with omegasomes formed from the ER in response to starvation. Omegasomes induced by viral nsp6 matured into autophagosomes that delivered LC3 to lysosomes and therefore recruited and recycled the proteins needed for autophagosome nucleation, expansion, cellular trafficking and delivery of cargo to lysosomes. The coronavirus nsp6 proteins activated omegasome and autophagosome formation independently of starvation, but activation did not involve direct inhibition of mTOR signaling, activation of sirtuin 1 or induction of ER stress.
doi:10.4161/auto.7.11.16642
PMCID: PMC3242798  PMID: 21799305
autophagy; omegasome; endoplasmic reticulum; coronavirus; nonstructural proteins
5.  Integrating genetic and epidemiological data to determine transmission pathways of foot-and-mouth disease virus 
Estimating detailed transmission trees that reflect the relationships between infected individuals or populations during a disease outbreak often provides valuable insights into both the nature of disease transmission and the overall dynamics of the underlying epidemiological process. These trees may be based on epidemiological data that relate to the timing of infection and infectiousness, or genetic data that show the genetic relatedness of pathogens isolated from infected individuals. Genetic data are becoming increasingly important in the estimation of transmission trees of viral pathogens due to their inherently high mutation rate. Here, we propose a maximum-likelihood approach that allows epidemiological and genetic data to be combined within the same analysis to infer probable transmission trees. We apply this approach to data from 20 farms infected during the 2001 UK foot-and-mouth disease outbreak, using complete viral genome sequences from each infected farm and information on when farms were first estimated to have developed clinical disease and when livestock on these farms were culled. Incorporating known infection links due to animal movement prior to imposition of the national movement ban results in the reduction of the number of trees from 41 472 that are consistent with the genetic data to 1728, of which just 4 represent more than 95% of the total likelihood calculated using a model that accounts for the epidemiological data. These trees differ in several ways from those constructed prior to the availability of genetic data.
doi:10.1098/rspb.2007.1442
PMCID: PMC2599933  PMID: 18230598
foot-and-mouth disease virus; transmission trees; contact tracing; complete genome sequencing
6.  Transmission Pathways of Foot-and-Mouth Disease Virus in the United Kingdom in 2007 
PLoS Pathogens  2008;4(4):e1000050.
Foot-and-mouth disease (FMD) virus causes an acute vesicular disease of domesticated and wild ruminants and pigs. Identifying sources of FMD outbreaks is often confounded by incomplete epidemiological evidence and the numerous routes by which virus can spread (movements of infected animals or their products, contaminated persons, objects, and aerosols). Here, we show that the outbreaks of FMD in the United Kingdom in August 2007 were caused by a derivative of FMDV O1 BFS 1860, a virus strain handled at two FMD laboratories located on a single site at Pirbright in Surrey. Genetic analysis of complete viral genomes generated in real-time reveals a probable chain of transmission events, predicting undisclosed infected premises, and connecting the second cluster of outbreaks in September to those in August. Complete genome sequence analysis of FMD viruses conducted in real-time have identified the initial and intermediate sources of these outbreaks and demonstrate the value of such techniques in providing information useful to contemporary disease control programmes.
Author Summary
Foot-and-mouth disease (FMD) outbreaks in the United Kingdom during August and September 2007 have caused severe disruption to the farming sector and cost hundreds of millions of pounds. Investigating and determining the source of these outbreaks is imperative for their effective management and future prevention. Foot-and-mouth disease virus (FMDV) has a high mutation rate, resulting in rapid evolution. We show how complete genome sequences (acquired within 24–48 h of sample receipt) can be used to track FMDV movement from farm to farm in real time. This helped to determine the most likely source of the outbreak, assisted ongoing epidemiological investigations as to whether these field cases were linked to single or multiple releases from the source, and predicted the existence of undetected intermediate infected premises.
doi:10.1371/journal.ppat.1000050
PMCID: PMC2277462  PMID: 18421380
7.  Molecular Epidemiology of the Foot-and-Mouth Disease Virus Outbreak in the United Kingdom in 2001▿  
Journal of Virology  2006;80(22):11274-11282.
The objective of this study was to quantify the extent to which the genetic diversity of foot-and-mouth disease virus (FMDV) arising over the course of infection of an individual animal becomes fixed, is transmitted to other animals, and thereby accumulates over the course of an outbreak. Complete consensus sequences of 23 genomes (each of 8,200 nucleotides) of FMDV were recovered directly from epithelium tissue acquired from 21 farms infected over a nearly 7-month period during the 2001 FMDV outbreak in the United Kingdom. An analysis of these consensus sequences revealed very few apparently ambiguous sites but clear evidence of 197 nucleotide substitutions at 191 different sites. We estimated the rate of nucleotide substitution to be 2.26 × 10−5 per site per day (95% confidence interval [CI], 1.75 × 10−5 to 2.80 × 10−5) and nucleotide substitutions to accrue in the consensus sequence at an average rate of 1.5 substitutions per farm infection. This is a sufficiently high rate showing that detailed histories of the transmission pathways can be reliably reconstructed. Coalescent methods indicated that the date at which FMDV first infected livestock in the United Kingdom was 7 February 2001 (95% CI, 20 January to 19 February 2001), which was identical to estimates obtained on the basis of purely clinical evidence. Nucleotide changes appeared to have occurred evenly across the genome, and within the open reading frame, the ratio of nonsynonymous-to-synonymous change was 0.09. The ability to recover particular transmission pathways of acutely acting RNA pathogens from genetic data will help resolve uncertainties about how virus is spread and could help in the control of future epidemics.
doi:10.1128/JVI.01236-06
PMCID: PMC1642183  PMID: 16971422

Results 1-7 (7)