Novel coronavirus and astrovirus in Delaware Bay shorebirds.

BACKGROUND: Wild birds are an important but to some extent under-studied reservoir for emerging pathogens. We used unbiased sequencing methods for virus discovery in shorebird samples from the Delaware Bay, USA; an important feeding ground for thousands of migratory birds. FINDINGS: Analysis of shorebird fecal samples indicated the presence of a novel astrovirus and coronavirus. A sanderling sample yielded sequences with distant homology to avian nephritis virus 1, an astrovirus associated with acute nephritis in poultry. A ruddy turnstone sample yielded sequences with homology to deltacoronaviruses. CONCLUSIONS: Our findings highlight shorebirds as a virus reservoir and the need to closely monitor wild bird populations for the emergence of novel virus variants.

Upolu virus and Aransas Bay virus, two presumptive bunyaviruses, are novel members of the family Orthomyxoviridae.

UNLABELLED: Emerging and zoonotic pathogens pose continuing threats to human health and ongoing challenges to diagnostics. As nucleic acid tests are playing increasingly prominent roles in diagnostics, the genetic characterization of molecularly uncharacterized agents is expected to significantly enhance detection and surveillance capabilities. We report the identification of two previously unrecognized members of the family Orthomyxoviridae, which includes the influenza viruses and the tick-transmitted Thogoto and Dhori viruses. We provide morphological, serologic, and genetic evidence that Upolu virus (UPOV) from Australia and Aransas Bay virus (ABV) from North America, both previously considered potential bunyaviruses based on electron microscopy and physicochemical features, are orthomyxoviruses instead. Their genomes show up to 68% nucleotide sequence identity to Thogoto virus (segment 2; ∼74% at the amino acid level) and a more distant relationship to Dhori virus, the two prototype viruses of the recognized species of the genus Thogotovirus. Despite sequence similarity, the coding potentials of UPOV and ABV differed from that of Thogoto virus, instead being like that of Dhori virus. Our findings suggest that the tick-transmitted viruses UPOV and ABV represent geographically distinct viruses in the genus Thogotovirus of the family Orthomyxoviridae that do not fit in the two currently recognized species of this genus. IMPORTANCE: Upolu virus (UPOV) and Aransas Bay virus (ABV) are shown to be orthomyxoviruses instead of bunyaviruses, as previously thought. Genetic characterization and adequate classification of agents are paramount in this molecular age to devise appropriate surveillance and diagnostics. Although more closely related to Thogoto virus by sequence, UPOV and ABV differ in their coding potentials by lacking a proposed pathogenicity factor. In this respect, they are similar to Dhori virus, which, despite the lack of a pathogenicity factor, can cause disease. These findings enable further studies into the evolution and pathogenicity of orthomyxoviruses.

Global mRNA decay analysis at single nucleotide resolution reveals segmental and positional degradation patterns in a Gram-positive bacterium.

BACKGROUND: Recent years have shown a marked increase in the use of next-generation sequencing technologies for quantification of gene expression (RNA sequencing, RNA-Seq). The expression level of a gene is a function of both its rate of transcription and RNA decay, and the influence of mRNA decay rates on gene expression in genome-wide studies of Gram-positive bacteria is under-investigated. RESULTS: In this work, we employed RNA-Seq in a genome-wide determination of mRNA half-lives in the Gram-positive bacterium Bacillus cereus. By utilizing a newly developed normalization protocol, RNA-Seq was used successfully to determine global mRNA decay rates at the single nucleotide level. The analysis revealed positional degradation patterns, with mRNAs being degraded from both ends of the molecule, indicating that both 5' to 3' and 3' to 5' directions of RNA decay are present in B. cereus. Other operons showed segmental degradation patterns where specific ORFs within polycistrons were degraded at variable rates, underlining the importance of RNA processing in gene regulation. We determined the half-lives for more than 2,700 ORFs in B. cereus ATCC 10987, ranging from less than one minute to more than fifteen minutes, and showed that mRNA decay rate correlates globally with mRNA expression level, GC content, and functional class of the ORF. CONCLUSIONS: To our knowledge, this study presents the first global analysis of mRNA decay in a bacterium at single nucleotide resolution. We provide a proof of principle for using RNA-Seq in bacterial mRNA decay analysis, revealing RNA processing patterns at the single nucleotide level.

Genetic characterization of the Wyeomyia group of orthobunyaviruses and their phylogenetic relationships.

Phylogenetic analyses can give new insights into the evolutionary history of viruses, especially of viruses with segmented genomes. However, sequence information for many viral families or genera is still limited and phylogenies based on single or short genome fragments can be misleading. We report the first genetic analysis of all three genome segments of Wyeomyia group viruses Wyeomyia, Taiassui, Macaua, Sororoca, Anhembi and Cachoeira Porteira (BeAr328208) in the genus Orthobunyavirus of the family Bunyaviridae. In addition, Tucunduba and Iaco viruses were identified as members of the Wyeomyia group. Features of Wyeomyia group members that distinguish them from other viruses in the Bunyamwera serogroup and from other orthobunyaviruses, including truncated NSs sequences that may not counteract the host's interferon response, were characterized. Our findings also suggest genome reassortment within the Wyeomyia group, identifying Macaua and Tucunduba viruses as M-segment reassortants that, in the case of Tucunduba virus, may have altered pathogenicity, stressing the need for whole-genome sequence information to facilitate characterization of orthobunyaviruses and their phylogenetic relationships.

Discovery of an ebolavirus-like filovirus in europe.

Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain.

Genetic characterization of K13965, a strain of Oak Vale virus from Western Australia.

K13965, an uncharacterized virus, was isolated in 1993 from Anopheles annulipes mosquitoes collected in the Kimberley region of northern Western Australia. Here, we report its genomic sequence, identify it as a rhabdovirus, and characterize its phylogenetic relationships. The genome comprises a P' (C) and SH protein similar to the recently characterized Tupaia and Durham viruses, and shows overlap between G and L genes. Comparison of K13965 genome sequence to other rhabdoviruses identified K13965 as a strain of the unclassified Australian Oak Vale rhabdovirus, whose complete genome sequence we also determined. Phylogenetic analysis of N and L sequences indicated genetic relationship to a recently proposed Sandjima virus clade, although the Oak Vale virus sequences form a branch separate from the African members of that group.

Novel picornavirus in Turkey poults with hepatitis, California, USA.

To identify a candidate etiologic agent for turkey viral hepatitis, we analyzed samples from diseased turkey poults from 8 commercial flocks in California, USA, that were collected during 2008-2010. High-throughput pyrosequencing of RNA from livers of poults with turkey viral hepatitis (TVH) revealed picornavirus sequences. Subsequent cloning of the ≈9-kb genome showed an organization similar to that of picornaviruses with conservation of motifs within the P1, P2, and P3 genome regions, but also unique features, including a 1.2-kb sequence of unknown function at the junction of P1 and P2 regions. Real-time PCR confirmed viral RNA in liver, bile, intestine, serum, and cloacal swab specimens from diseased poults. Analysis of liver by in situ hybridization with viral probes and immunohistochemical testing of serum demonstrated viral nucleic acid and protein in livers of diseased poults. Molecular, anatomic, and immunologic evidence suggests that TVH is caused by a novel picornavirus, tentatively named turkey hepatitis virus.

Identification of a severe acute respiratory syndrome coronavirus-like virus in a leaf-nosed bat in Nigeria.

Bats are reservoirs for emerging zoonotic viruses that can have a profound impact on human and animal health, including lyssaviruses, filoviruses, paramyxoviruses, and severe acute respiratory syndrome coronaviruses (SARS-CoVs). In the course of a project focused on pathogen discovery in contexts where human-bat contact might facilitate more efficient interspecies transmission of viruses, we surveyed gastrointestinal tissue obtained from bats collected in caves in Nigeria that are frequented by humans. Coronavirus consensus PCR and unbiased high-throughput pyrosequencing revealed the presence of coronavirus sequences related to those of SARS-CoV in a Commerson's leaf-nosed bat (Hipposideros commersoni). Additional genomic sequencing indicated that this virus, unlike subgroup 2b CoVs, which includes SARS-CoV, is unique, comprising three overlapping open reading frames between the M and N genes and two conserved stem-loop II motifs. Phylogenetic analyses in conjunction with these features suggest that this virus represents a new subgroup within group 2 CoVs.

Granada virus: a natural phlebovirus reassortant of the sandfly fever Naples serocomplex with low seroprevalence in humans.

A new member of the phlebovirus genus, tentatively named Granada virus, was detected in sandflies collected in Spain. By showing the presence of specific neutralizing antibodies in human serum collected in Granada, we show that Granada virus infects humans. The analysis of the complete genome of Granada virus revealed that this agent is likely to be a natural reassortant of the recently described Massilia virus (donor of the long and short segments) with a yet unidentified phlebovirus (donor of the medium segment).

Identification of GBV-D, a novel GB-like flavivirus from old world frugivorous bats (Pteropus giganteus) in Bangladesh.

Bats are reservoirs for a wide range of zoonotic agents including lyssa-, henipah-, SARS-like corona-, Marburg-, Ebola-, and astroviruses. In an effort to survey for the presence of other infectious agents, known and unknown, we screened sera from 16 Pteropus giganteus bats from Faridpur, Bangladesh, using high-throughput pyrosequencing. Sequence analyses indicated the presence of a previously undescribed virus that has approximately 50% identity at the amino acid level to GB virus A and C (GBV-A and -C). Viral nucleic acid was present in 5 of 98 sera (5%) from a single colony of free-ranging bats. Infection was not associated with evidence of hepatitis or hepatic dysfunction. Phylogenetic analysis indicates that this first GBV-like flavivirus reported in bats constitutes a distinct species within the Flaviviridae family and is ancestral to the GBV-A and -C virus clades.

Astrovirus encephalitis in boy with X-linked agammaglobulinemia.

Encephalitis is a major cause of death worldwide. Although >100 pathogens have been identified as causative agents, the pathogen is not determined for up to 75% of cases. This diagnostic failure impedes effective treatment and underscores the need for better tools and new approaches for detecting novel pathogens or determining new manifestations of known pathogens. Although astroviruses are commonly associated with gastroenteritis, they have not been associated with central nervous system disease. Using unbiased pyrosequencing, we detected an astrovirus as the causative agent for encephalitis in a 15-year-old boy with agammaglobulinemia; several laboratories had failed to identify the agent. Our findings expand the spectrum of causative agents associated with encephalitis and highlight unbiased molecular technology as a valuable tool for differential diagnosis of unexplained disease.

Moussa virus: a new member of the Rhabdoviridae family isolated from Culex decens mosquitoes in Côte d'Ivoire.

Characterization of arboviruses at the interface of pristine habitats and anthropogenic landscapes is crucial to comprehensive emergent disease surveillance and forecasting efforts. In context of a surveillance campaign in and around a West African rainforest, particles morphologically consistent with rhabdoviruses were identified in cell cultures infected with homogenates of trapped mosquitoes. RNA recovered from these cultures was used to derive the first complete genome sequence of a rhabdovirus isolated from Culex decens mosquitoes in Côte d'Ivoire, tentatively named Moussa virus (MOUV). MOUV shows the classical genome organization of rhabdoviruses, with five open reading frames (ORF) in a linear order. However, sequences show only limited conservation (12-33% identity at amino acid level), and ORF2 and ORF3 have no significant similarity to sequences deposited in GenBank. Phylogenetic analysis indicates a potential new species with distant relationship to Tupaia and Tibrogargan virus.

Transcriptome sequencing of the Microarray Quality Control (MAQC) RNA reference samples using next generation sequencing.

BACKGROUND: Transcriptome sequencing using next-generation sequencing platforms will soon be competing with DNA microarray technologies for global gene expression analysis. As a preliminary evaluation of these promising technologies, we performed deep sequencing of cDNA synthesized from the Microarray Quality Control (MAQC) reference RNA samples using Roche's 454 Genome Sequencer FLX. RESULTS: We generated more that 3.6 million sequence reads of average length 250 bp for the MAQC A and B samples and introduced a data analysis pipeline for translating cDNA read counts into gene expression levels. Using BLAST, 90% of the reads mapped to the human genome and 64% of the reads mapped to the RefSeq database of well annotated genes with e-values

Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa.

Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever-associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus.

Novel borna virus in psittacine birds with proventricular dilatation disease.

Pyrosequencing of cDNA from brains of parrots with proventricular dilatation disease (PDD), an unexplained fatal inflammatory central, autonomic, and peripheral nervous system disease, showed 2 strains of a novel Borna virus. Real-time PCR confirmed virus presence in brain, proventriculus, and adrenal gland of 3 birds with PDD but not in 4 unaffected birds.

A metagenomic survey of microbes in honey bee colony collapse disorder.

In colony collapse disorder (CCD), honey bee colonies inexplicably lose their workers. CCD has resulted in a loss of 50 to 90% of colonies in beekeeping operations across the United States. The observation that irradiated combs from affected colonies can be repopulated with naive bees suggests that infection may contribute to CCD. We used an unbiased metagenomic approach to survey microflora in CCD hives, normal hives, and imported royal jelly. Candidate pathogens were screened for significance of association with CCD by the examination of samples collected from several sites over a period of 3 years. One organism, Israeli acute paralysis virus of bees, was strongly correlated with CCD.

Wasp gene expression supports an evolutionary link between maternal behavior and eusociality.

The presence of workers that forgo reproduction and care for their siblings is a defining feature of eusociality and a major challenge for evolutionary theory. It has been proposed that worker behavior evolved from maternal care behavior. We explored this idea by studying gene expression in the primitively eusocial wasp Polistes metricus. Because little genomic information existed for this species, we used 454 sequencing to generate 391,157 brain complementary DNA reads, resulting in robust hits to 3017 genes from the honey bee genome, from which we identified and assayed orthologs of 32 honey bee behaviorally related genes. Wasp brain gene expression in workers was more similar to that in foundresses, which show maternal care, than to that in queens and gynes, which do not. Insulin-related genes were among the differentially regulated genes, suggesting that the evolution of eusociality involved major nutritional and reproductive pathways.

Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing.

The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies.