Simultaneous rapid sequencing of multiple RNA virus genomes

AbstractComparing sequences of archived viruses collected over many years to the present allows the study of viral evolution and contributes to the design of new vaccines. However, the difficulty, time and expense of generating full-length sequences individually from each archived sample have hampered these studies. Next generation sequencing technologies have been utilized for analysis of clinical and environmental samples to identify viral pathogens that may be present. This has led to the discovery of many new, uncharacterized viruses from a number of viral families. Use of these sequencing technologies would be advantageous in examining viral evolution. In this study, a sequencing procedure was used to sequence simultaneously and rapidly multiple archived samples using a single standard protocol. This procedure utilized primers composed of 20 bases of known sequence with 8 random bases at the 3′-end that also served as an identifying barcode that allowed the differentiation each viral library following pooling and sequencing. This conferred sequence independence by random priming both first and second strand cDNA synthesis. Viral stocks were treated with a nuclease cocktail to reduce the presence of host nucleic acids. Viral RNA was extracted, followed by single tube random-primed double-stranded cDNA synthesis. The resultant cDNAs were amplified by primer-specific PCR, pooled, size fractionated and sequenced on the Ion Torrent PGM platform. The individual virus genomes were readily assembled by both de novo and template-assisted assembly methods. This procedure consistently resulted in near full length, if not full-length, genomic sequences and was used to sequence multiple bovine pestivirus and coronavirus isolates simultaneously

Draft Genome Sequence of a Mycobacterium avium Complex Isolate from a Broadbill Bird

Mycobacterium avium complex (MAC) organisms cause opportunistic infections in humans, yet their epidemiology remains poorly understood. They are slowly growing environmental and animal-associated mycobacteria that have little notoriety except for the strains that cause disseminated infections in HIV- infected humans (1). Most MAC organisms are classified taxonomically as a single species, M. avium, which is divided into at least four subspecies, M. avium subsp. avium, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum (2). The only other species in this group is M. intracellulare. Genotyping of this diverse bacterial group has been achieved using intergenic spacers (3) and rpoB sequence analysis (4, 5)

Estimation of viral richness from shotgun metagenomes using a frequency count approach

BACKGROUND: Viruses are important drivers of ecosystem functions, yet little is known about the vast majority of viruses. Viral shotgun metagenomics enables the investigation of broad ecological questions in phage communities. One ecological characteristic is species richness, which is the number of different species in a community. Viruses do not have a phylogenetic marker analogous to the bacterial 16S rRNA gene with which to estimate richness, and so contig spectra are employed to measure the number of virus taxa in a given community. A contig spectrum is generated from a viral shotgun metagenome by assembling the random sequence reads into groups of sequences that overlap (contigs) and counting the number of sequences that group within each contig. Current tools available to analyze contig spectra to estimate phage richness are limited by relying on rank-abundance data. RESULTS: We present statistical estimates of virus richness from contig spectra. The program CatchAll (http://www.northeastern.edu/catchall/) was used to analyze contig spectra in terms of frequency count data rather than rank-abundance, thus enabling formal statistical analyses. Also, the influence of potentially spurious low-frequency counts on richness estimates was minimized by two methods, empirical and statistical. The results show greater estimates of viral richness than previous calculations in nearly all environments analyzed, including swine feces and reclaimed fresh water. CONCLUSIONS: CatchAll yielded consistent estimates of richness across viral metagenomes from the same or similar environments. Additionally, analysis of pooled viral metagenomes from different environments via mixed contig spectra resulted in greater richness estimates than those of the component metagenomes. Using CatchAll to analyze contig spectra will improve estimations of richness from viral shotgun metagenomes, particularly from large datasets, by providing statistical measures of richness

Early antibody response against Mycobacterium avium subspecies paratuberculosis antigens in subclinical cattle

<p>Abstract</p> <p>Background</p> <p>Our laboratories have previously reported on the experimental infection of cattle with <it>Mycobacterium avium </it>subsp <it>paratuberculosis </it>(<it>M. paratuberculosis</it>) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 <it>M. paratuberculosis </it>coding sequences. These combined tools have enabled a unique look at the temporal analysis of <it>M. paratuberculosis </it>antigens within the native host. The primary objective of this study was to identify <it>M. paratuberculosis </it>antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection.</p> <p>Results</p> <p>Sera from two experimentally infected cattle, taken pre-inoculation and at day 70, 194 and 321 post infection, identified dynamic antibody reactivity among antigens with some showing an increased response over time and others showing declining levels of reactivity over the same time period. A <it>M. paratuberculosis </it>specific protein, encoded by MAP0862, was strongly detected initially, but the antibody response became weaker with time. The most reactive protein was a putative surface antigen encoded by MAP1087. A second protein, MAP1204, implicated in virulence, was also strongly detected by day 70 in both cattle. Subsequent experiments showed that these two proteins were detected with sera from 5 of 9 naturally infected cattle in the subclinical stage of Johne's disease.</p> <p>Conclusion</p> <p>Collectively these results demonstrate that <it>M. paratuberculosis </it>proteins are detected by sera from experimentally infected cattle as early as 70 days after exposure. These data further suggest at least two antigens may be useful in the early diagnosis of <it>M. paratuberculosis </it>infections. Finally, the construction and use of a protein array in this pilot study has led to a novel approach for discovery of <it>M. paratuberculosis </it>antigens.</p

Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association

<p>Abstract</p> <p>Background</p> <p>The genome of <it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>(<it>MAP</it>) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map.</p> <p>Results</p> <p>Our analysis of one of the isolates, <it>MAP </it>S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the <it>MAP </it>S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human <it>M. avium </it>subsp. <it>hominissuis </it>strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of <it>MAP </it>(JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level.</p> <p>Conclusions</p> <p>Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the <it>M. avium </it>complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of <it>MAP</it>. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for <it>M. avium </it>complex strains based on these genome sequences.</p

Isolation and characterization of saprophytic and pathogenic strains of Leptospira from water sources in the Midwestern United States

The genus Leptospira is a diverse and unique group of bacteria comprising multiple saprophytic and pathogenic species, which survive and persist in suitable moist environments. Pathogenic species cause human and animal leptospirosis, a global and neglected zoonotic disease. Disease transmission occurs by exposure to contaminated water and moist soil environments or by contact with domestic animals and wildlife acting as reservoir hosts that shed Leptospira via urine. Here, we describe the unexpected diversity of saprophytic and pathogenic species of Leptospira isolated from water in the Midwestern United States. Samples were collected by volunteers in 11 counties in Iowa from water sources, including puddles, sewage, creeks, ponds, lakes, and rivers, during the summer of 2021. One hundred and five water samples were tested by culture for the presence of saprophytic and pathogenic species and by lipL32 qPCR specific for the detection of pathogens; 82 (78.1%) were culture positive and five (4.8%) were positive by lipL32 qPCR. Whole genome sequencing of isolates cultured from water samples identified 10 species of saprophytes, namely L. montravelensis, L. kemamanensis, L. bandrabouensis, L. bourretii, L. bouyouniensis, L. chreensis, L. ellinghausenii, L. terpstrae, L. yanagawae, and L. abararensis, as well as three novel saprophytic species. Whole genome sequencing also identified two novel pathogenic species. The remaining cultures comprised mixed populations of saprophytic species and six comprised a mixture of saprophytic and pathogenic species. One of these mixed cultures was enriched to select for a clonal isolate of pathogenic Leptospira, strain WS101.C1, which was classified as L. interrogans serogroup Djasiman serovar Djasiman. Cumulatively, 9.5% (10/105) of water samples were positive for pathogenic Leptospira. This study emphasizes the diversity of Leptospira present in water sources in the Midwestern United States and provides unique opportunities to explore the geographic diversity and evolution of this genus. The identification of known and novel pathogenic species circulating in local water sources highlights their potential usefulness as diagnostic antigens, as well as the role of water in the transmission of infection to human and animal populations. Integrating knowledge on human, animal, and environmental health is essential to control and predict risk for zoonoses

Identification of equine mares as reservoir hosts for pathogenic species of Leptospira

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US

Emerging infectious disease implications of invasive mammalian species : the greater white-toothed shrew (Crocidura russula) is associated with a novel serovar of pathogenic Leptospira in Ireland

The greater white-toothed shrew (Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of approximately 7,600 km2 on the island. C. russula is normally distributed in Northern Africa and Western Europe, and was previously absent from the British Isles. Whilst invasive species can have dramatic and rapid impacts on faunal and floral communities, they may also be carriers of pathogens facilitating disease transmission in potentially naive populations. Pathogenic leptospires are endemic in Ireland and a significant cause of human and animal disease. From 18 trapped C. russula, 3 isolates of Leptospira were cultured. However, typing of these isolates by standard serological reference methods was negative, and suggested an, as yet, unidentified serovar. Sequence analysis of 16S ribosomal RNA and secY indicated that these novel isolates belong to Leptospira alstonii, a unique pathogenic species of which only 7 isolates have been described to date. Earlier isolations were limited geographically to China, Japan and Malaysia, and this leptospiral species had not previously been cultured from mammals. Restriction enzyme analysis (REA) further confirms the novelty of these strains since no similar patterns were observed with a reference database of leptospires. As with other pathogenic Leptospira species, these isolates contain lipL32 and do not grow in the presence of 8-azagunaine; however no evidence of disease was apparent after experimental infection of hamsters. These isolates are genetically related to L. alstonii but have a novel REA pattern; they represent a new serovar which we designate as serovar Room22. This study demonstrates that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira