Comparative Genomics, Infectivity and Cytopathogenicity of American Isolates of Zika Virus that Developed Persistent Infections in Human Embryonic Kidney (HEK293) Cells.

Zika virus (ZIKV) transmission can cause serious fetal neurological abnormalities. ZIKV persistence in various human cells and tissues can serve as infectious reservoirs and post serious threats to public health. The human embryonic kidney (HEK293) cell line with known neuronal developmental properties was readily infected by ZIKV in a strain-dependent fashion. Significant cytopathic effect in HEK293 cells infected by the prototype MR 766 strain of ZIKV resulted in complete loss of cells, while small numbers of HEK293 cells infected by contemporary ZIKV isolates (PRV or FLR strain) continued to survive and regrow to confluency in the culture around two months after initial infection. Most, if not all, of the cells in the two resulting persistently ZIKV-infected HEK293 cell lines tested positive for ZIKV antigen. Compared to HEK293 control cells, the persistently ZIKV-infected HEK293 cells had slower growth rates with some cells undergoing apoptosis in culture. The "persistent ZIKVs" produced constitutively by both PRV and FLR strains ZIKV-infected HEK293 cells had significantly attenuated cell infectivity and/or cytopathogenicity. Comparative genome sequence analyses between the persistent ZIKVs and the original inoculum ZIKVs showed no clonal selection with specific gene mutations in the prolonged process of establishing persistently PRV strain ZIKV-infected HEK293 cells; while selection of ZIKV subclones with mutations in the envelope, protein pr and multiple NS genes was evident in developing persistently FLR strain ZIKV-infected HEK293 cell line. Our study provides molecular insights into the complex interplays of ZIKV and human host cells in establishing ZIKV persistence.

Identification and characterization of EBV genomes in spontaneously immortalized human peripheral blood B lymphocytes by NGS technology.

BACKGROUND: We conducted genomic sequencing to identify Epstein Barr Virus (EBV) genomes in 2 human peripheral blood B lymphocytes that underwent spontaneous immortalization promoted by mycoplasma infections in culture, using the high-throughput sequencing (HTS) Illumina MiSeq platform. The purpose of this study was to examine if rapid detection and characterization of a viral agent could be effectively achieved by HTS using a platform that has become readily available in general biology laboratories. RESULTS: Raw read sequences, averaging 175 bps in length, were mapped with DNA databases of human, bacteria, fungi and virus genomes using the CLC Genomics Workbench bioinformatics tool. Overall 37,757 out of 49,520,834 total reads in one lymphocyte line (# K4413-Mi) and 28,178 out of 45,335,960 reads in the other lymphocyte line (# K4123-Mi) were identified as EBV sequences. The two EBV genomes with estimated 35.22-fold and 31.06-fold sequence coverage respectively, designated K4413-Mi EBV and K4123-Mi EBV (GenBank accession number KC440852 and KC440851 respectively), are characteristic of type-1 EBV. CONCLUSIONS: Sequence comparison and phylogenetic analysis among K4413-Mi EBV, K4123-Mi EBV and the EBV genomes previously reported to GenBank as well as the NA12878 EBV genome assembled from database of the 1000 Genome Project showed that these 2 EBVs are most closely related to B95-8, an EBV previously isolated from a patient with infectious mononucleosis and WT-EBV. They are less similar to EBVs associated with nasopharyngeal carcinoma (NPC) from Hong Kong and China as well as the Akata strain of a case of Burkitt's lymphoma from Japan. They are most different from type 2 EBV found in Western African Burkitt's lymphoma.

Detection of MLV-related virus gene sequences in blood of patients with chronic fatigue syndrome and healthy blood donors.

Chronic fatigue syndrome (CFS) is a serious systemic illness of unknown cause. A recent study identified DNA from a xenotropic murine leukemia virus-related virus (XMRV) in peripheral blood mononuclear cells (PBMCs) from 68 of 101 patients (67%) by nested PCR, as compared with 8 of 218 (3.7%) healthy controls. However, four subsequent reports failed to detect any murine leukemia virus (MLV)-related virus gene sequences in blood of CFS patients. We examined 41 PBMC-derived DNA samples from 37 patients meeting accepted diagnostic criteria for CFS and found MLV-like virus gag gene sequences in 32 of 37 (86.5%) compared with only 3 of 44 (6.8%) healthy volunteer blood donors. No evidence of mouse DNA contamination was detected in the PCR assay system or the clinical samples. Seven of 8 gag-positive patients tested again positive in a sample obtained nearly 15 y later. In contrast to the reported findings of near-genetic identity of all XMRVs, we identified a genetically diverse group of MLV-related viruses. The gag and env sequences from CFS patients were more closely related to those of polytropic mouse endogenous retroviruses than to those of XMRVs and were even less closely related to those of ecotropic MLVs. Further studies are needed to determine whether the same strong association with MLV-related viruses is found in other groups of patients with CFS, whether these viruses play a causative role in the development of CFS, and whether they represent a threat to the blood supply.

Identification of DNA signatures suitable for use in development of real-time PCR assays by whole-genome sequence approaches: use of Streptococcus pyogenes in a pilot study.

A stepwise computational approach using three layers of publicly available software was found to effectively identify DNA signatures for Streptococcus pyogenes. PCR testing validated that 9 out of 15 signature-derived primer sets could detect as low as 5 fg of target DNA with high specificity. The selected signature-derived primer sets were successfully evaluated against all 23 clinical isolates. The approach is readily applicable for designing molecular assays for rapid detection and characterization of various pathogenic bacteria.

Epstein-Barr Virus in Burkitt Lymphoma in Africa Reveals a Limited Set of Whole Genome and LMP-1 Sequence Patterns: Analysis of Archival Datasets and Field Samples From Uganda, Tanzania, and Kenya.

Epstein-Barr virus (EBV) is associated with endemic Burkitt lymphoma (eBL), but the contribution of EBV variants is ill-defined. Studies of EBV whole genome sequences (WGS) have identified phylogroups that appear to be distinct for Asian versus non-Asian EBV, but samples from BL or Africa, where EBV was first discovered, are under-represented. We conducted a phylogenetic analysis of EBV WGS and LMP-1 sequences obtained primarily from BL patients in Africa and representative non-African EBV from other conditions or regions using data from GenBank, Sequence Read Archive, or Genomic Data Commons for the Burkitt Lymphoma Genome Sequencing Project (BLGSP) to generate data to support the use of a simpler biomarker of geographic or phenotypic associations. We also investigated LMP-1 patterns in 414 eBL cases and 414 geographically matched controls in the Epidemiology of Burkitt Lymphoma in East African children and minors (EMBLEM) study using LMP-1 PCR and Sanger sequencing. Phylogenetic analysis revealed distinct genetic patterns of African versus Asian EBV sequences. We identified 281 single nucleotide variations (SNVs) in LMP-1 promoter and coding region, which formed 12 unique patterns (A to L). Nine patterns (A, AB, C, D, F, I, J, K and L) predominated in African EBV, of which four were found in 92% of BL samples (A, AB, D, and H). Predominant patterns were B and G in Asia and H in Europe. EBV positivity in peripheral blood was detected in 95.6% of EMBLEM eBL cases versus 79.2% of the healthy controls (odds ratio [OR] =3.83; 95% confidence interval 2.06-7.14). LMP-1 was successfully sequenced in 66.7% of the EBV DNA positive cases but in 29.6% of the controls (ORs ranging 5-11 for different patterns). Four LMP-1 patterns (A, AB, D, and K) were detected in 63.1% of the cases versus 27.1% controls (ORs ranges: 5.58-11.4). Dual strain EBV infections were identified in WGS and PCR-Sanger data. In conclusion, EBV from Africa is phylogenetically separate from EBV in Asia. Genetic diversity in LMP-1 formed 12 patterns, which showed promising geographic and phenotypic associations. Presence of multiple strain infection should be considered in efforts to refine or improve EBV markers of ancestry or phenotype. Lay Summary: Epstein-Barr virus (EBV) infection, a ubiquitous infection, contributes to the etiology of both Burkitt Lymphoma (BL) and nasopharyngeal carcinoma, yet their global distributions vary geographically with no overlap. Genomic variation in EBV is suspected to play a role in the geographical patterns of these EBV-associated cancers, but relatively few EBV samples from BL have been comprehensively studied. We sought to compare phylogenetic patterns of EBV genomes obtained from BL samples in Africa and from tumor and non-tumor samples from elsewhere. We concluded that EBV obtained from BL in Africa is genetically separate from EBV in Asia. Through comprehensive analysis of nucleotide variations in EBV's LMP-1 gene, we describe 12 LMP-1 patterns, two of which (B and G) were found mostly in Asia. Four LMP-1 patterns (A, AB, D, and F) accounted for 92% of EBVs sequenced from BL in Africa. Our results identified extensive diversity of EBV, but BL in Africa was associated with a limited number of variants identified, which were different from those identified in Asia. Further research is needed to optimize the use of PCR and sequencing to study LMP-1 diversity for classification of EBV variants and for use in epidemiologic studies to characterize geographic and/or phenotypic associations of EBV variants with EBV-associated malignancies, including eBL.

The TOR complex 1 is distributed in endosomes and in retrograde vesicles that form from the vacuole membrane and plays an important role in the vacuole import and degradation pathway.

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.

Draft Genome Sequences of Blastococcus sp. Clones TML/M2B and TML/C7B, with Different Motilities, Isolated in a Laboratory.

Two novel Blastococcus sp. clones, TML/M2B and TML/C7B, with 2 different stable growth phenotypes, were isolated from a laboratory tissue culture. The draft genome sequences generated through genomic sequencing of clones TML/M2B and TML/C7B contain 4 and 2 contigs, respectively. The respective genome sizes are 4.10 Mb and 4.11 Mb, with G+C contents of 74.17% and 74.14%, respectively.

Genomic Alterations of Staphylococcus aureus ATCC 25923 after Prolonged Passage in the Laboratory.

Staphylococcus aureus reference strain ATCC 25923 has been maintained for more than a decade in our laboratory. Genomic study revealed that the resulting strain AFIPCBER_B_8.4 has lost a 37-kb genomic fragment of the ATCC 25923 parental strain. The missing fragment showed sequence similarity to genes of bacteriophage proteins.

Comparative genomics, infectivity and cytopathogenicity of Zika viruses produced by acutely and persistently infected human hematopoietic cell lines.

Zika virus (ZIKV), an arthropod-borne virus, has emerged as a major human pathogen. Prolonged or persistent ZIKV infection of human cells and tissues may serve as a reservoir for the virus and present serious challenges to the safety of public health. Human hematopoietic cell lines with different developmental properties revealed differences in susceptibility and outcomes to ZIKV infection. In three separate studies involving the prototypic MR 766 ZIKV strain and the human monocytic leukemia U937 cell line, ZIKV initially developed only a low-grade infection at a slow rate. After continuous culture for several months, persistently ZIKV-infected cell lines were observed with most, if not all, cells testing positive for ZIKV antigen. The infected cultures produced ZIKV RNA (v-RNA) and infectious ZIKVs persistently ("persistent ZIKVs") with distinct infectivity and pathogenicity when tested using various kinds of host cells. When the genomes of ZIKVs from the three persistently infected cell lines were compared with the genome of the prototypic MR 766 ZIKV strain, distinct sets of mutations specific to each cell line were found. Significantly, all three "persistent ZIKVs" were capable of infecting fresh U937 cells with high efficiency at rapid rates, resulting in the development of a new set of persistently ZIKV-infected U937 cell lines. The genomes of ZIKVs from the new set of persistently ZIKV-infected U937 cell lines were further analyzed for their different mutations. The 2nd generation of persistent ZIKVs continued to possess most of the distinct sets of mutations specific to the respective 1st generation of persistent ZIKVs. We anticipate that the study will contribute to the understanding of the fundamental biology of adaptive mutations and selection during viral persistence. The persistently ZIKV-infected human cell lines that we developed will also be useful to investigate critical molecular pathways of ZIKV persistence and to study drugs or countermeasures against ZIKV infections and transmission.

N-terminal domain of yeast Hsp104 chaperone is dispensable for thermotolerance and prion propagation but necessary for curing prions by Hsp104 overexpression.

Hsp104 is a hexameric protein chaperone that resolubilizes stress-damaged proteins from aggregates. Hsp104 promotes [PSI(+)] prion propagation by breaking prion aggregates, which propagate as amyloid fibers, into more numerous prion "seeds." Inactivating Hsp104 cures cells of [PSI(+)] and other amyloid-like yeast prions. Overexpressing Hsp104 also eliminates [PSI(+)], presumably by completely resolubilizing prion aggregates. Inexplicably, however, excess Hsp104 does not cure the other prions. Here we identify missense mutations in Hsp104's amino-terminal domain (NTD), which is conserved among Hsp100 proteins but whose function is unknown, that improve [PSI(+)] propagation. Hsp104Delta147, engineered to lack the NTD, supported [PSI(+)] and functioned normally in thermotolerance and protein disaggregation. Hsp104Delta147 failed to cure [PSI(+)] when overexpressed, however, implying that excess Hsp104 does not eliminate [PSI(+)] by direct dissolution of prion aggregates. Curing of [PSI(+)] by overexpressing catalytically inactive Hsp104 (Hsp104KT), which interferes with endogenous Hsp104, did not require the NTD. We further found that Hsp104 mutants defective in threading peptides through the hexamer pore had reduced ability to support [PSI(+)] in proportion to protein resolubilization defects, suggesting that [PSI(+)] propagation depends on this threading and that Hsp104 "breaks" prion aggregates by extracting protein monomers from the amyloid fibers.

Intraspecies comparative genomics of three strains of Orientia tsutsugamushi with different antibiotic sensitivity.

We recently reported the genome of Orientia tsutsugamushi (OT) strain Karp (GenBank Accession #: NZ_LYMA00000000.2, https://www.ncbi.nlm.nih.gov/nuccore/NZ_LYMA00000000.2) with > 2 Mb in size through clone-based sequencing and high throughput genomic shotgun sequencing (HTS). The genomes of OT strains AFSC4 and AFSC7 were similarly sequenced by HTS Since strains AFSC4 (GenBank Accession #: NZ_LYMT00000000.1, https://www.ncbi.nlm.nih.gov/nuccore/1035784408) and AFSC7 (GenBank Accession #: NZ_LYMB00000000.1, https://www.ncbi.nlm.nih.gov/nuccore/1035854767) were more resistant to antibiotics than strain Karp, we conducted comparative analysis of the three draft genomes annotated by RAST server aimed to identify possible genetic bases of difference in microbial antibiotic sensitivity. Intraspecies comparative genomics analysis of the three OT strains revealed that two ORFs encoding hypothetical proteins in both strains AFSC4 and AFSC7 are absent in strain Karp.

Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species.

Culture-based identification methods have been the gold standard for the diagnosis of fungal infection. Currently, molecular technologies such as real-time PCR assays with short turnaround time can provide desirable alternatives for the rapid detection of Candida microbes. However, most of the published PCR primer sets are not Candida specific and likely to amplify DNA from common environmental contaminants, such as Aspergillus microbes. In this study, we designed pan-Candida primer sets based on the ribosomal DNA-coding regions conserved within Candida but distinct from those of Aspergillus and Penicillium. We demonstrate that the final two selected pan-Candida primer sets would not amplify Aspergillus DNA and could be used to differentiate eight medically important Candida pathogens in real-time PCR assays based on their melting profiles, with a sensitivity of detection as low as 10 fg of Candida genomic DNA. Moreover, we further evaluated and selected species-specific primer sets covering Candida albicans, Candida glabrata, Candida tropicalis, and Candida dubliniensis and show that they had high sensitivity and specificity. These real-time PCR primer sets could potentially be assembled into a single PCR array for the rapid detection of Candida species in various clinical settings, such as corneal transplantation.

Genomic Sequencing of Orientia tsutsugamushi Strain Karp, an Assembly Comparable to the Genome Size of the Strain Ikeda.

Orientia tsutsugamushi, an intracellular bacterium, belongs to the family Rickettsiaceae This study presents the draft genome sequence of strain Karp, with 2.0 Mb as the size of the completed genome. This nearly finished draft genome sequence was annotated with the RAST server and the contents compared to those of the other strains.

DNA Sequence Signatures for Rapid Detection of Six Target Bacterial Pathogens Using PCR Assays.

Using Streptococcus pyogenes as a model, we previously established a stepwise computational workflow to effectively identify species-specific DNA signatures that could be used as PCR primer sets to detect target bacteria with high specificity and sensitivity. In this study, we extended the workflow for the rapid development of PCR assays targeting Enterococcus faecalis, Enterococcus faecium, Clostridium perfringens, Clostridium difficile, Clostridium tetani, and Staphylococcus aureus, which are of safety concern for human tissue intended for transplantation. Twenty-one primer sets that had sensitivity of detecting 5-50 fg DNA from target bacteria with high specificity were selected. These selected primer sets can be used in a PCR array for detecting target bacteria with high sensitivity and specificity. The workflow could be widely applicable for the rapid development of PCR-based assays for a wide range of target bacteria, including those of biothreat agents.

Draft Genome Sequence of Pantoea sp. Strain MBLJ3, Isolated in a Laboratory Environmental Control Study.

This report describes the draft genome sequence of a newly isolated strain, Pantoea sp. MBLJ3. The genome is 4.8 Mb in size, with a G+C content of 54.27%, and it contains 4,522 protein-coding sequences, 69 tRNA genes, and 5 rRNA genes.

Mixed group of Rhizobiales microbes in lung and blood of a patient with fatal pulmonary illness.

We examined the microbial composition in the diseased lung and early-phase microbial cultures from the blood of a patient with a rapidly progressing fatal pulmonary illness. Although no microbes could be isolated from such cultures during the initial study, the HTS-microbiome study revealed the presence of a unique mixture of alphaproteobacteria, composed mainly of different families of Rhizobiales microbes. Microbial 16S rDNA sequences matching closely to Afipia cberi were identified mainly in the patient's diseased lung tissue, but only rarely in the early-phase blood cultures. Conversely, the high abundance of sequences found in early-phase blood cultures of different broth media matched closely with those of the families Methylobacteriaceae, Phyllobacteriaceae and Sphingomonadaceae. The two species that successfully adapted to grow in a laboratory culture system were A. cberi and Mesorhizobium hominis, which eventually were isolated from a previously cryopreserved blood culture of SP4 broth. Many other species, including members of the Bradyrhizobiaceae and Phyllobacteriaceae families, and all members of the Methylobacteriaceae and Sphingomonadaceae families identified by HTS remained non-cultivated. We developed specific PCR primers and FISH probes, which detected the target Rhizobiales microbes in former blood cultures and autopsy lung tissues. It is unclear what role these Rhizobiales microbes might have played in the patient's complex disease process. However, the above mentioned assays should help in rapidly detecting and identifying these previously unrecognized Rhizobiales microbes in patients.

Genome sequencing and annotation of Afipia septicemium strain OHSU_II.

We report the 5.1 Mb noncontiguous draft genome of Afipia septicemium strain OHSU_II, isolated from blood of a female patient. The genome consists of 5,087,893 bp circular chromosome with no identifiable autonomous plasmid with a G + C content of 61.09% and contains 4898 protein-coding genes and 49 RNA genes including 3 rRNA genes and 46 tRNA genes.

Isolation of novel Afipia septicemium and identification of previously unknown bacteria Bradyrhizobium sp. OHSU_III from blood of patients with poorly defined illnesses.

Cultures previously set up for isolation of mycoplasmal agents from blood of patients with poorly-defined illnesses, although not yielding positive results, were cryopreserved because of suspicion of having low numbers of unknown microbes living in an inactive state in the broth. We re-initiated a set of 3 cultures for analysis of the "uncultivable" or poorly-grown microbes using NGS technology. Broth of cultures from 3 blood samples, submitted from OHSU between 2000 and 2004, were inoculated into culture flasks containing fresh modified SP4 medium and kept at room temperature (RT), 30°C and 35°C. The cultures showing evidence of microbial growth were expanded and subjected to DNA analysis by genomic sequencing using Illumina MiSeq. Two of the 3 re-initiated blood cultures kept at RT after 7-8 weeks showed evidence of microbial growth that gradually reached into a cell density with detectable turbidity. The microbes in the broth when streaked on SP4 agar plates produced microscopic colonies in ∼ 2 weeks. Genomic studies revealed that the microbes isolated from the 2 blood cultures were a novel Afipia species, tentatively named Afipia septicemium. Microbes in the 3(rd) culture (OHSU_III) kept at RT had a limited level of growth and could not reach a plateau with high cell density. Genomic sequencing identified the microbe in the culture as a previously unknown species of Bradyrhizobium bacteria. This study reports on the isolation of novel Afipia and Bradyrhizobium species. Isolation of Bradyrhizobium species bacteria has never been reported in humans. The study also reveals a previously unrecognized nature of hematogenous infections by the 2 unique groups of Bradyrhizobiaceae. Our studies show that improvement of culture system plus effective use of NGS technology can facilitate findings of infections by unusual microbes in patients having poorly-defined, sometimes mysterious illnesses.

Isolation and characterization of two novel bacteria Afipia cberi and Mesorhizobium hominis from blood of a patient afflicted with fatal pulmonary illness.

We recently isolated and discovered new Bradyrhizobiaceae microbes from the cryopreserved culture broth of blood samples from 3 patients with poorly defined illnesses using modified SP4 media and culture conditions coupled with genomic sequencing. Using a similar protocol, we studied a previously cryopreserved culture broth of blood sample from a patient who had succumbed to an acute onset of fulminant pulmonary illness. We report that two phases of microbial growth were observed in the re-initiated culture. Biochemical and genomic characterization revealed microbes isolated from the first phase of growth were new Afipia species of Bradyrhizobiaceae, tentatively named A. cberi with a ~ 5 MB chromosome that was different from those of all previously known Afipia microbes including the newly discovered A. septicemium. The microbes isolated from the second phase of growth were prominent sugar assimilators, novel Phyllobacteriaceae, phylogenetically most closely related to Mesorhizobium and tentatively named M. hominis with a ~ 5.5 MB chromosome. All A. cberi isolates carry a circular ~ 140 KB plasmid. Some M. hominis isolates possess a circular ~ 412 KB plasmid that can be lost in prolonged culture or passage. No antibiotics resistant genes could be identified in both of the A. cberi and M. hominis plasmids. Antibiotic susceptibility studies using broth culture systems revealed isolates of A. cberi could be sensitive to some antibiotics, but all isolates of M. hominis were resistant to essentially all tested antibiotics. However, the cell-free antibiotics susceptibility test results may not be applicable to clinical treatment against the microbes that are known to be capable of intracellular growth. It remains to be determined if the 2 previously unknown Rhizobiales were indeed pathogenic and played a role in the pulmonary disease process in this patient. Specific probes and methods will be developed to re-examine the diseased lungs from patient's autopsy.