Multiple novel non-canonically transcribed sub-genomic mRNAs produced by avian coronavirus infectious bronchitis virus.

Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified.

Attenuation of Infectious Bronchitis Virus in Eggs Results in Different Patterns of Genomic Variation across Multiple Replicates.

The gammacoronavirus infectious bronchitis virus (IBV) causes an acute, highly contagious respiratory disease of poultry. Live attenuated vaccines are traditionally generated by serial passage of a virulent strain in embryonated chicken eggs; however, the molecular mechanism of attenuation is unknown. M41-CK, a virulent lab-adapted strain of IBV, was egg passaged over 100 times in four parallel independent replicates. All four final egg-passaged viruses were attenuated in vivo and exhibited similar growth phenotypes in adult chicken kidney cells and ex vivo tracheal organ cultures. The virus populations were sequenced by 454 pyrosequencing at the end of passaging, and the results showed that overall sequence diversity in the IBV population increased but the four replicates only had between 11 and 17 consensus-level single nucleotide polymorphisms (SNPs). Although hot spots of variation were identified in spike and nucleocapsid structural proteins as well as the 3' untranslated region, each attenuated virus possessed a different pattern of genomic variation. Overall, only a small number of consensus-level SNPs were acquired during egg passage, leaving a potentially short route back to virulence. These results highlight the unpredictable nature of attenuation by serial egg passage and the need to develop mechanisms to rationally attenuate IBV for the next generation of effective vaccines.IMPORTANCE Infectious bronchitis remains a major problem in the global poultry industry, despite the existence of many different vaccines. IBV vaccines are currently developed by serial passage of a virulent strain on embryonated hen's eggs until attenuation; however, little is known about the evolution of the viral population during the process of attenuation. High-throughput sequencing of four replicates of a serially egg-passaged IBV revealed a different pattern of genomic variation in each attenuated replicate and few consensus-level SNPs. This raises concerns that only a small number of genomic mutations are required to revert to a virulent phenotype, which may result in vaccine breakdown in the field. The observed hot spots of variation in the attenuated viruses have the potential to be used in the rational attenuation of virulent IBV for next-generation vaccine design.

Mass Die-Off of Saiga Antelopes, Kazakhstan, 2015.

In 2015, a mass die-off of ≈200,000 saiga antelopes in central Kazakhstan was caused by hemorrhagic septicemia attributable to the bacterium Pasteurella multocida serotype B. Previous analyses have indicated that environmental triggers associated with weather conditions, specifically air moisture and temperature in the region of the saiga antelope calving during the 10-day period running up to the event, were critical to the proliferation of latent bacteria and were comparable to conditions accompanying historically similar die-offs in the same areas. We investigated whether additional viral or bacterial pathogens could be detected in samples from affected animals using 3 different high-throughput sequencing approaches. We did not identify pathogens associated with commensal bacterial opportunisms in blood, kidney, or lung samples and thus concluded that P. multocida serotype B was the primary cause of the disease.

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.

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.

African swine fever virus NAM P1/95 is a mixture of genotype I and genotype VIII viruses.

African swine fever virus causes a lethal hemorrhagic disease of domestic pigs. The NAM P1/1995 isolate was originally described as B646L genotype XVIII; however, full genome sequencing revealed that this assignment was incorrect.

Sequencing and Analysis of Lumpy Skin Disease Virus Whole Genomes Reveals a New Viral Subgroup in West and Central Africa.

Lumpy skin disease virus (LSDV) is a member of the capripoxvirus (CPPV) genus of the Poxviridae family. LSDV is a rapidly emerging, high-consequence pathogen of cattle, recently spreading from Africa and the Middle East into Europe and Asia. We have sequenced the whole genome of historical LSDV isolates from the Pirbright Institute virus archive, and field isolates from recent disease outbreaks in Sri Lanka, Mongolia, Nigeria and Ethiopia. These genome sequences were compared to published genomes and classified into different subgroups. Two subgroups contained vaccine or vaccine-like samples ("Neethling-like" clade 1.1 and "Kenya-like" subgroup, clade 1.2.2). One subgroup was associated with outbreaks of LSD in the Middle East/Europe (clade 1.2.1) and a previously unreported subgroup originated from cases of LSD in west and central Africa (clade 1.2.3). Isolates were also identified that contained a mix of genes from both wildtype and vaccine samples (vaccine-like recombinants, grouped in clade 2). Whole genome sequencing and analysis of LSDV strains isolated from different regions of Africa, Europe and Asia have provided new knowledge of the drivers of LSDV emergence, and will inform future disease control strategies.

Inactivation of Plasmodium falciparum in whole blood by riboflavin plus irradiation.

BACKGROUND: Malaria parasites are frequently transmitted by unscreened blood transfusions in Africa. Pathogen reduction methods in whole blood would thus greatly improve blood safety. We aimed to determine the efficacy of riboflavin plus irradiation for treatment of whole blood infected with Plasmodium falciparum. STUDY DESIGN AND METHODS: Blood was inoculated with 10(4) or 10(5) parasites/mL and riboflavin treated with or without ultraviolet (UV) irradiation (40-160 J/mL red blood cells [mL(RBCs)]). Parasite genome integrity was assessed by quantitative amplification inhibition assays, and P. falciparum viability was monitored in vitro. RESULTS: Riboflavin alone did not affect parasite genome integrity or parasite viability. Application of UV after riboflavin treatment disrupted parasite genome integrity, reducing polymerase-dependent amplification by up to 2 logs (99%). At 80 J/mL(RBCs), riboflavin plus irradiation prevented recovery of viable parasites in vitro for 2 weeks, whereas untreated controls typically recovered to approximately 2% parasitemia after 4 days of in vitro culture. Exposure of blood to 160 J/mL(RBCs) was not associated with significant hemolysis. CONCLUSIONS: Riboflavin plus irradiation treatment of whole blood damages parasite genomes and drastically reduces P. falciparum viability in vitro. In the absence of suitable malaria screening assays, parasite inactivation should be investigated for prevention of transfusion-transmitted malaria in highly endemic areas.

Whole-Genome Sequencing Protocols for IBV and Other Coronaviruses Using High-Throughput Sequencing.

This chapter reports the high-throughput sequencing protocol for sequencing Coronaviruses and other positive strand viruses to produce a dataset of significant depth of coverage. The protocol describes sequencing of infectious bronchitis virus propagated in embryonated eggs and harvested in the allantoic fluid. The protocol is composed of three main steps-enrichment of the allantoic fluid using ultracentrifugation, extraction of total RNA from allantoic fluid, and library preparation from total RNA to DNA sequencing libraries. The workflow will be suitable for all coronaviruses using high-throughput sequencing platforms.

Genome Sequencing of Foot-and-Mouth Disease Virus Type O Isolate GRE/23/94.

The complete genome of a foot-and-mouth disease type O virus originating from an epidemic in Greece in 1994 is reported. This virus belongs to the Middle East-South Asia topotype.

Investigating intra-host and intra-herd sequence diversity of foot-and-mouth disease virus.

Due to the poor-fidelity of the enzymes involved in RNA genome replication, foot-and-mouth disease (FMD) virus samples comprise of unique polymorphic populations. In this study, deep sequencing was utilised to characterise the diversity of FMD virus (FMDV) populations in 6 infected cattle present on a single farm during the series of outbreaks in the UK in 2007. A novel RT-PCR method was developed to amplify a 7.6kb nucleotide fragment encompassing the polyprotein coding region of the FMDV genome. Illumina sequencing of each sample identified the fine polymorphic structures at each nucleotide position, from consensus level changes to variants present at a 0.24% frequency. These data were used to investigate population dynamics of FMDV at both herd and host levels, evaluate the impact of host on the viral swarm structure and to identify transmission links with viruses recovered from other farms in the same series of outbreaks. In 7 samples, from 6 different animals, a total of 5 consensus level variants were identified, in addition to 104 sub-consensus variants of which 22 were shared between 2 or more animals. Further analysis revealed differences in swarm structures from samples derived from the same animal suggesting the presence of distinct viral populations evolving independently at different lesion sites within the same infected animal.

Rheumatoid arthritis is associated with IgG antibodies to human endogenous retrovirus gag matrix: a potential pathogenic mechanism of disease?

OBJECTIVE: Human endogenous retrovirus (HERV)-K10 has been implicated in the etiology and pathogenesis of rheumatoid arthritis (RA). A secondary immune response to this virus might suggest an antigen-driven response in patients. The Gag region of HERV-K10 could provide a key epitope important for immunological reactivity. We investigated the presence of IgG antibodies to this region and assessed its significance in RA. METHODS: We determined an antigenic peptide on the matrix segment of HERV-K10 and developed an ELISA system to detect IgG antibodies in patients with RA and controls. The presence of antibodies to the matrix peptide (denoted as MAG1: RIGKELKQAGRKGNI) was correlated with patient details. RESULTS: On screening patients' serum, we found a significantly higher mean IgG antibody response to MAG1 in 30 patients with RA as compared to 23 patients with inflammatory bowel disease (p = 0.003), 29 patients with osteoarthritis (p = 0.001), and 43 healthy individuals (p = 0.002). Reactivity was not observed to a control peptide possessing a nonhomologous amino acid sequence. On evaluating clinical details with serological activity, no correlation with disease duration (p = 0.128), sex (p = 0.768), or rheumatoid factor status (p = 0.576) was found. CONCLUSION: A significantly elevated IgG antibody response to an HERV-K10 Gag matrix peptide was observed in patients with RA, suggesting that the exposure of HERV-K10 may cause a secondary, antigenic driven immune response in RA.