Borrelia nietonii sp. nov.: Relapsing Fever Spirochetes Transmitted By the Tick Ornithodoros hermsi Designated Previously as Borrelia hermsii Genomic Group II.

Background: The taxonomic status of the relapsing fever spirochete Borrelia hermsii in western North America was established in 1942 and based solely on its specific association with the soft tick vector Ornithodoros hermsi. Multilocus sequence typing (MLST) of the 16S rRNA, flaB, gyrB, glpQ, and 16S-23S rRNA intergenic spacer of B. hermsii isolates collected over many years from various geographic locations and biological sources identified two distinct clades designated previously as B. hermsii Genomic Group I (GGI) and Genomic Group II (GGII). To better assess the taxonomic relationship of these two genomic groups to each other and other species of Borrelia, DNA sequences of the entire linear chromosome were determined. Materials and Methods: Genomic DNA samples were prepared from 11 spirochete isolates grown in Barbour-Stoenner-Kelly-H medium. From these preparations, DNA sequences of the entire linear chromosome of two isolates of B. hermsii belonging to each genomic group and seven additional species were determined. Results: Chromosomal sequences of four isolates of B. hermsii contained 919,212 to 922,307 base pairs. DNA sequence identities between the two genomic groups of B. hermsii were 95.86-95.99%, which were more divergent than chromosomal sequences comparing Borrelia parkeri and Borrelia turicatae (97.13%), Borrelia recurrentis and Borrelia duttonii (97.07%), and Borrelia crocidurae and B. duttonii (97.09%). The 3' end of the chromosome of the two GGII isolates also contained a unique intact oppA gene absent from all other species examined. Conclusion: Previous MLST and the chromosomal sequences presented herein support the division of the B. hermsii species complex into two species, B. hermsii sensu stricto ( = GGI) and Borrelia nietonii sp. nov. ( = GGII). We name this unique relapsing fever spirochete in honor of our late friend and colleague Dr. Nathan Nieto for his outstanding contributions to our understanding of tick-borne relapsing fever.

Genetically diverse mouse models of SARS-CoV-2 infection reproduce clinical variation in type I interferon and cytokine responses in COVID-19.

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.

Genetically diverse mouse models of SARS-CoV-2 infection reproduce clinical variation in type I interferon and cytokine responses in COVID-19.

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19) and is influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the genetically diverse Collaborative Cross (CC) founder strains crossed to human ACE2 transgenic mice (K18-hACE2) that confers susceptibility to SARS-CoV-2. Infection of CC x K18- hACE2 resulted in a spectrum of survival, viral replication kinetics, and immune profiles. Importantly, in contrast to the K18-hACE2 model, early type I interferon (IFN-I) and regulated proinflammatory responses were required for control of SARS-CoV-2 replication in PWK x K18-hACE2 mice that were highly resistant to disease. Thus, virus dynamics and inflammation observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding anti-coronavirus immunity.

Transcriptomic profiling of the digestive tract of the rat flea, Xenopsylla cheopis, following blood feeding and infection with Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a highly lethal pathogen transmitted by the bite of infected fleas. Once ingested by a flea, Y. pestis establish a replicative niche in the gut and produce a biofilm that promotes foregut colonization and transmission. The rat flea Xenopsylla cheopis is an important vector to several zoonotic bacterial pathogens including Y. pestis. Some fleas naturally clear themselves of infection; however, the physiological and immunological mechanisms by which this occurs are largely uncharacterized. To address this, RNA was extracted, sequenced, and distinct transcript profiles were assembled de novo from X. cheopis digestive tracts isolated from fleas that were either: 1) not fed for 5 days; 2) fed sterile blood; or 3) fed blood containing ~5x108 CFU/ml Y. pestis KIM6+. Analysis and comparison of the transcript profiles resulted in identification of 23 annotated (and 11 unknown or uncharacterized) digestive tract transcripts that comprise the early transcriptional response of the rat flea gut to infection with Y. pestis. The data indicate that production of antimicrobial peptides regulated by the immune-deficiency pathway (IMD) is the primary flea immune response to infection with Y. pestis. The remaining infection-responsive transcripts, not obviously associated with the immune response, were involved in at least one of 3 physiological themes: 1) alterations to chemosensation and gut peristalsis; 2) modification of digestion and metabolism; and 3) production of chitin-binding proteins (peritrophins). Despite producing several peritrophin transcripts shortly after feeding, including a subset that were infection-responsive, no thick peritrophic membrane was detectable by histochemistry or electron microscopy of rat flea guts for the first 24 hours following blood-feeding. Here we discuss the physiological implications of rat flea infection-responsive transcripts, the function of X. cheopis peritrophins, and the mechanisms by which Y. pestis may be cleared from the flea gut.

Molluscum Contagiosum Virus Transcriptome in Abortively Infected Cultured Cells and a Human Skin Lesion.

UNLABELLED: Molluscum contagiosum virus (MOCV), the only circulating human-specific poxvirus, has a worldwide distribution and causes benign skin lesions that may persist for months in young children and severe infections in immunosuppressed adults. Studies of MOCV are restricted by the lack of an efficient animal model or a cell culture replication system. We used next-generation sequencing to analyze and compare polyadenylated RNAs from abortive MOCV infections of several cell lines and a human skin lesion. Viral RNAs were detected for 14 days after MOCV infection of cultured cells; however, there was little change in the RNA species during this time and a similar pattern occurred in the presence of an inhibitor of protein synthesis, indicating a block preventing postreplicative gene expression. Moreover, a considerable number of MOCV RNAs mapped to homologs of orthopoxvirus early genes, but few did so to homologs of intermediate or late genes. The RNAs made during in vitro infections represent a subset of RNAs detected in human skin lesions which mapped to homologs of numerous postreplicative as well as early orthopoxvirus genes. Transfection experiments using fluorescent protein and luciferase reporters demonstrated that vaccinia virus recognized MOCV intermediate and late promoters, indicating similar gene regulation. The specific recognition of the intermediate promoter in MOCV-infected cells provided evidence for the synthesis of intermediate transcription factors, which are products of early genes, but not for late transcription factors. Transcriptome sequencing (RNA-seq) and reporter gene assays may be useful for testing engineered cell lines and conditions that ultimately could provide an in vitro replication system. IMPORTANCE: The inability to propagate molluscum contagiosum virus, which causes benign skin lesions in young children and more extensive infections in immunosuppressed adults, has constrained our understanding of the biology of this human-specific virus. In the present study, we characterized the RNAs synthesized in abortively infected cultured cells and a human skin lesion by next-generation sequencing. These studies provided an initial transcription map of the MOCV genome, suggested temporal regulation of gene expression, and indicated that the in vitro replication block occurs prior to intermediate and late gene expression. RNA-seq and reporter assays, as described here, may help to further evaluate MOCV gene expression and define conditions that could enable MOCV replication in vitro.

Genome sequencing of Giardia lamblia genotypes A2 and B isolates (DH and GS) and comparative analysis with the genomes of genotypes A1 and E (WB and Pig).

Giardia lamblia (syn G. intestinalis, G. duodenalis) is the most common pathogenic intestinal parasite of humans worldwide and is a frequent cause of endemic and epidemic diarrhea. G. lamblia is divided into eight genotypes (A-H) which infect a wide range of mammals and humans, but human infections are caused by Genotypes A and B. To unambiguously determine the relationship among genotypes, we sequenced GS and DH (Genotypes B and A2) to high depth coverage and compared the assemblies with the nearly completed WB genome and draft sequencing surveys of Genotypes E (P15; pig isolate) and B (GS; human isolate). Our results identified DH as the smallest Giardia genome sequenced to date, while GS is the largest. Our open reading frame analyses and phylogenetic analyses showed that GS was more distant from the other three genomes than any of the other three were from each other. Whole-genome comparisons of DH_A2 and GS_B with the optically mapped WB_A1 demonstrated substantial synteny across all five chromosomes but also included a number of rearrangements, inversions, and chromosomal translocations that were more common toward the chromosome ends. However, the WB_A1/GS_B alignment demonstrated only about 70% sequence identity across the syntenic regions. Our findings add to information presented in previous reports suggesting that GS is a different species of Giardia as supported by the degree of genomic diversity, coding capacity, heterozygosity, phylogenetic distance, and known biological differences from WB_A1 and other G. lamblia genotypes.

Pervasive initiation and 3'-end formation of poxvirus postreplicative RNAs.

Poxviruses are large DNA viruses that replicate within the cytoplasm and encode a complete transcription system, including a multisubunit RNA polymerase, stage-specific transcription factors, capping and methylating enzymes, and a poly(A) polymerase. Expression of the more than 200 open reading frames by vaccinia virus, the prototype poxvirus, is temporally regulated: early mRNAs are synthesized immediately after infection, whereas intermediate and late mRNAs are synthesized following genome replication. The postreplicative transcripts are heterogeneous in length and overlap the entire genome, which pose obstacles for high resolution mapping. We used tag-based methods in conjunction with high throughput cDNA sequencing to determine the precise 5'-capped and 3'-polyadenylated ends of postreplicative RNAs. Polymerase slippage during initiation of intermediate and late RNA synthesis results in a 5'-poly(A) leader that allowed the unambiguous identification of true transcription start sites. Ninety RNA start sites were located just upstream of intermediate and late open reading frames, but many more appeared anomalous, occurring within coding and non-coding regions, indicating pervasive transcription initiation. We confirmed the presence of functional promoter sequences upstream of representative anomalous start sites and demonstrated that alternative start sites within open reading frames could generate truncated isoforms of proteins. In an analogous manner, poly(A) sequences allowed accurate mapping of the numerous 3'-ends of postreplicative RNAs, which were preceded by a pyrimidine-rich sequence in the DNA coding strand. The distribution of postreplicative promoter sequences throughout the genome provides enormous transcriptional complexity, and the large number of previously unmapped RNAs may have novel functions.

Genome sequence of erythromelalgia-related poxvirus identifies it as an ectromelia virus strain.

Erythromelagia is a condition characterized by attacks of burning pain and inflammation in the extremeties. An epidemic form of this syndrome occurs in secondary students in rural China and a virus referred to as erythromelalgia-associated poxvirus (ERPV) was reported to have been recovered from throat swabs in 1987. Studies performed at the time suggested that ERPV belongs to the orthopoxvirus genus and has similarities with ectromelia virus, the causative agent of mousepox. We have determined the complete genome sequence of ERPV and demonstrated that it has 99.8% identity to the Naval strain of ectromelia virus and a slighly lower identity to the Moscow strain. Small DNA deletions in the Naval genome that are absent from ERPV may suggest that the sequenced strain of Naval was not the immediate progenitor of ERPV.

Expression profiling of the intermediate and late stages of poxvirus replication.

The double-stranded DNA genome of vaccinia virus (VACV), the prototype poxvirus, contains approximately 200 open reading frames (ORFs) that are transcribed at early, intermediate, and late stages of infection. Previous high-throughput deep RNA sequencing allowed us to map 118 VACV early genes that are expressed before viral DNA replication and 93 postreplicative genes. However, the intermediate- and late-stage postreplicative genes could not be differentiated. Here, we synchronized infections with a reversible inhibitor of DNA replication and used a VACV mutant that conditionally transcribes late genes to sequence the two classes of mRNAs. In addition, each postreplicative ORF was individually expressed under conditions that distinguished intermediate and late classes. We identified 38 VACV genes that belong to the late class and 53 that belong to the intermediate class, with some of the latter continuing to be expressed late. These data allowed us to prepare a genome-wide early, intermediate, and late transcription map. Inspection of sequences upstream of these ORFs revealed distinctive characteristics of intermediate and late promoters and suggested that some promoters have intermediate and late elements. The intermediate genes encoded many DNA binding/packaging and core-associated proteins in addition to late transcription factors; the late genes encoded many morphogenesis and mature virion membrane proteins, including those involved in entry, in addition to early transcription factors. The top-ranked antigens for CD4(+) T cells and B cells were mainly intermediate rather than late gene products. The differentiation of intermediate and late genes may enhance understanding of poxvirus replication and lead to improvements in expression vectors and recombinant vaccines.

Genome-wide analysis of the 5' and 3' ends of vaccinia virus early mRNAs delineates regulatory sequences of annotated and anomalous transcripts.

Poxviruses are large DNA viruses that encode a multisubunit RNA polymerase, stage-specific transcription factors, and enzymes that cap and polyadenylate mRNAs within the cytoplasm of infected animal cells. Genome-wide microarray and RNA-seq technologies have been used to profile the transcriptome of vaccinia virus (VACV), the prototype member of the family. Here, we adapted tag-based methods in conjunction with SOLiD and Illumina deep sequencing platforms to determine the precise 5' and 3' ends of VACV early mRNAs and map the putative transcription start sites (TSSs) and polyadenylation sites (PASs). Individual and clustered TSSs were found preceding 104 annotated open reading frames (ORFs), excluding pseudogenes. In the majority of cases, a 15-nucleotide consensus core motif was present upstream of the ORF. This motif, however, was also present at numerous other locations, indicating that it was insufficient for transcription initiation. Further analysis revealed a 10-nucleotide AT-rich spacer following functional core motifs that may facilitate DNA unwinding. Additional putative TSSs occurred in anomalous locations that may expand the functional repertoire of the VACV genome. However, many of the anomalous TSSs lacked an upstream core motif, raising the possibility that they arose by a processing mechanism as has been proposed for eukaryotic systems. Discrete and clustered PASs occurred about 40 nucleotides after an UUUUUNU termination signal. However, a large number of PASs were not preceded by this motif, suggesting alternative polyadenylation mechanisms. Pyrimidine-rich coding strand sequences were found immediately upstream of both types of PASs, signifying an additional feature of VACV 3'-end formation and polyadenylation.

Simultaneous high-resolution analysis of vaccinia virus and host cell transcriptomes by deep RNA sequencing.

Deep RNA sequencing was used to simultaneously analyze vaccinia virus (VACV) and HeLa cell transcriptomes at progressive times following infection. VACV, the prototypic member of the poxvirus family, replicates in the cytoplasm and contains a double-stranded DNA genome with approximately 200 closely spaced open reading frames (ORFs). The acquisition of a total of nearly 500 million short cDNA sequences allowed construction of temporal strand-specific maps of the entire VACV transcriptome at single-base resolution and analysis of over 14,000 host mRNAs. Before viral DNA replication, transcripts from 118 VACV ORFs were detected; after replication, transcripts from 93 additional ORFs were characterized. The high resolution permitted determination of the precise boundaries of many mRNAs including read-through transcripts and location of mRNA start sites and adjacent promoters. Temporal analysis revealed two clusters of early mRNAs that were synthesized in the presence of inhibitors of protein as well as DNA synthesis, indicating that they do not correspond to separate immediate- and delayed-early classes as defined for other DNA viruses. The proportion of viral RNAs reached 25-55% of the total at 4 h. This rapid change, resulting in a relative decrease of the vast majority of host mRNAs, can contribute to the profound shutdown of host protein synthesis and blunting of antiviral responses. At 2 h, however, a minority of cellular mRNAs was increased. The overrepresented functional categories of the up-regulated RNAs were NF-kappaB cascade, apoptosis, signal transduction, and ligand-mediated signaling, which likely represent the host response to invasion.