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Genetic Diversity and Evolution
Characterization, Mapping, and Distribution of the Two XMRV Parental Proviruses
Oya Cingöz, Tobias Paprotka, Krista A. Delviks-Frankenberry, Sheryl Wildt, Wei-Shau Hu, Vinay K. Pathak, John M. Coffin
Oya Cingöz
Department of Molecular Biology and Microbiology, Genetics Program, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, USA
Tobias Paprotka
Viral Mutation Section, HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland, USA
Krista A. Delviks-Frankenberry
Sheryl Wildt
Harlan Laboratories, Indianapolis, Indiana, USA
Wei-Shau Hu
Viral Recombination Section, HIV Drug Resistance Program, National Cancer Institute, Frederick, Maryland, USA
Vinay K. Pathak
John M. Coffin
DOI: 10.1128/JVI.06022-11
Xenotropic murine leukemia virus-related virus (XMRV) was previously reported to be associated with human prostate cancer and chronic fatigue syndrome. Our groups recently showed that XMRV was created through recombination between two endogenous murine retroviruses, PreXMRV-1 and PreXMRV-2, during the passaging of a prostate tumor xenograft in nude mice. Here, multiple approaches that led to the identification of PreXMRV-2, as well as the distribution of both parental proviruses among different mouse species, are described. The chromosomal loci of both proviruses were determined in the mouse genome, and integration site information was used to analyze the distribution of both proviruses in 48 laboratory mouse strains and 46 wild-derived strains. The strain distributions of PreXMRV-1 and PreXMRV-2 are quite different, the former being found predominantly in Asian mice and the latter in European mice, making it unlikely that the two XMRV ancestors could have recombined independently in the wild to generate an infectious virus. XMRV was not present in any of the mouse strains tested, and among the wild-derived mouse strains analyzed, not a single mouse carried both parental proviruses. Interestingly, PreXMRV-1 and PreXMRV-2 were found together in three laboratory strains, Hsd nude, NU/NU, and C57BR/cd, consistent with previous data that the recombination event that led to the generation of XMRV could have occurred only in the laboratory. The three laboratory strains carried the Xpr1n receptor variant nonpermissive to XMRV and xenotropic murine leukemia virus (X-MLV) infection, suggesting that the xenografted human tumor cells were required for the resulting XMRV recombinant to infect and propagate.
Gammaretroviruses have a broad host range, infecting organisms as diverse as mammals, birds, and reptiles (9). Perhaps the best-studied retroviruses within the gammaretrovirus genus are murine leukemia viruses (MLVs), which have both endogenous and exogenous counterparts. Mouse genomes contain a large number of endogenous MLVs, and different Mus subspecies are highly variable in the number of MLV insertions that they harbor.
Endogenous proviruses are carried as part of the genome of the host species and are subject to the same forces of evolution as their host, resulting in the slow accumulation of mutations over time that can eventually render them inactive. However, in some species there are intact endogenous proviruses that can be activated to produce infectious virus, through external stimuli or through recombination with other endogenous or exogenous viruses (31, 33).
XMRV (xenotropic murine leukemia virus-related virus) was first identified as a possible human pathogen in 2006 in a prostate cancer (PC) patient cohort (58). It was later reported in a high percentage of chronic fatigue syndrome (CFS) patients, as well as some healthy subjects, although no causal link between XMRV and any human disease has ever been established (35). In contrast, there are a large number of studies from many laboratories in which either no virus or no association with disease was found in a variety of human populations, despite the use of multiple highly sensitive detection methods (2, 10, 12–14, 17, 18, 21, 22, 26, 27, 44, 45, 47, 53, 59, 60). Strikingly, two recent replication studies by Knox et al. and Shin et al. showed that patients who were previously reported as XMRV positive were, in fact, XMRV negative (29, 49). Furthermore, viral sequences detected in human samples do not show the extent of diversity that would be expected from a human retrovirus that replicates within a population, suggesting that the observed clinical isolates could be explained by laboratory contamination from a single source (23). Finally, XMRV is highly susceptible to the antiviral effects of human cellular restriction factors, making the virus an unlikely candidate to overcome the blocks against its replication in the context of human infection (7, 8, 19, 40, 52). Taken together, these findings challenge the notion that XMRV is a genuine human virus.
The 22Rv1 cell line was derived from a human prostate tumor (CWR22) passaged repeatedly as xenografts in nude mice (50). 22Rv1 cells contain multiple XMRV insertions (28) and produce the virus at very high titers (∼109 to 1010 RNA copies/ml; data not shown). The virus produced by these cells differs by only 1 nucleotide (nt) from the consensus XMRV sequence reported in patient samples (28). In a recent study, analysis of genetic material from early and late xenograft samples of the tumor line that was used to generate 22Rv1 cells revealed the absence of XMRV from early samples but its presence in the late samples (39). Moreover, the discovery of two endogenous MLV proviruses, PreXMRV-1 and PreXMRV-2, present in the mouse tissues used to passage the xenografts was reported. A heterozygous progeny virion derived from these proviruses most likely generated XMRV by a unique recombination event (39). Here we describe multiple screening approaches that led to the identification of PreXMRV-2, the integration sites of the parental proviruses in the mouse genome, and their distribution among 94 different mouse strains.
DNA samples.The xenograft samples from the CWR22 prostate tumor were obtained as previously described (39). Some of the mouse DNA samples were kindly provided by the following individuals: Mus cervicolor popaeus (J53), Mus caroli (J136), Mus cookii (J135), and Mus spicilegus (Halbturn) (J131) from Christine Kozak (NIAID, Bethesda, MD); Mus macedonicus (XBS), Mus cervicolor cervicolor (CRV), Mus musculus bactrianus (BIR), Mus famulus (FAM), Mus platythrix (PTX), Mus spicilegus (ZRU), and Mus musculus musculus (MPB) from François Bonhomme (ISE, Montpellier, France); and C57BR/cd, LPT/Le NZW/Lac, PWK/Ph, and WMP/Pas from Greg Towers (UCL, London, United Kingdom); Harlan Sprague Dawley nude (Hsd nude) and NIH Swiss nude mouse DNAs were obtained from Harlan Laboratories, Indianapolis, IN. Mus dunni DNA was prepared from tail fibroblast (MDTF) cells. NCRNU-M DNA was purchased from Taconic Farms. NU/NU DNAs were prepared from spleens purchased from Charles River Laboratories (Wilmington, MA). All other mouse genomic DNA samples were purchased from The Jackson Laboratory (Bar Harbor, ME).
PCR.All PCR analyses were performed using either a Bio-Rad C1000 Thermal cycler or an MJ Research Peltier Thermal Cycler PTC200. For screening experiments and host-virus junction DNA amplification, 50 ng of mouse genomic DNA was used per 25-μl reaction mixture. GAGr (5′-TCCCCCAACAAAGCCACTCCA-3′) is specific for the XMRV gag leader deletion, and XmU3f (5′-GTCCTAGCCCTATAAAAAAGGGG-3′) is specific for the 2-nt insertion in the U3 region of the XMRV long terminal repeat (LTR), while XF6 (5′-GTACCCGCGCTTTTTGCTCC-3′), just upstream of XmU3f in the U3 region, would amplify many endogenous MLVs (Fig. 1A). To amplify PreXMRV-2 host-virus junctions, flanking primers for chromosome 12 (upstream, C12-1F, 5′-TGCTGGACAGAATCTCTGGTCTCT-3′; downstream, C12-4R, 5′-GATACTCAAGTGGTTCCCACCC-3′) were used in combination with internal provirus primers 129-1R (5′-GCGGTTTCGGCGTAAAACCGAAAGCA-3′) and envOUT1 (5′-CTGACCCAACAGTATCACCAACTC-3′), respectively. To amplify PreXMRV-1 host-virus junctions, flanking primers for chromosome 3 upstream, C3-2F (5′-GTAGCCATCAATGAGTTGTGAC-3′), and downstream, C3-4R (5′-GGATCTTCCAGTAGAACTATGTCC-3′), were used with XgR2 (5′-AGAGACAAAGACAAAACGATCGCCGGCC-3′) and XF6 (see above), respectively. The preintegration site of PreXMRV-1 was detected using the flanking primers for Chr.3 upstream, C3-1F (5′-CCACCACATATACGTACACCTTC-3′), and downstream, C3-6R (5′-GCAGTTTCTGGATGGTCATTCC-3′). β-Actin was amplified using the BA-F (5′-AGCCATGTACGTAGCCATCC-3′) and BA-R (5′-CTCTCAGCTGTGGTGGTGAA-3′) primer set, which amplifies both mouse and human β-actin. Intracisternal A-type particles (IAP) were amplified as described previously (38, 43).
Screening of mouse DNA for XMRV and XMRV-like elements by PCR. (A) Schematic representation of primers used (upper panel); their exact locations are indicated by arrows (lower panel). Positions are based on the VP62 XMRV genome (EF185282). MLV gag leader regions can be divided into three subgroups based on the length polymorphism. Unique restriction sites BsaAI and Eco53KI, which distinguish proviruses in the first group (0 bp; XMRV-like) from the rest (15 or 24 bp), are shown in boxes. (B) Sensitivity and specificity of the PCR assay. Serial dilutions of genomic DNA from 22Rv1 cells were amplified with the XMRV-specific primer set (GAGr and XmU3f) in the presence or absence of 50 ng of C57BL/6 genomic DNA. (C) The absence of XMRV (top row) and presence of an XMRV-like provirus (middle row) in a panel of representative mouse strains. The primer sets used for each screening assay are indicated on the right. Note that the NU/NU and Hsd nude mice are outbred strains; only one representative of each is shown. Analysis of larger numbers of individual mice from these outbred strains is shown in Fig. 5. β-Actin served as amplification control. NTC, no-template control.
For single-genome amplification, 3-fold serial dilutions of mouse genomic DNA were amplified with primer set 303F (5′-GCTAACTAGATCTGTATCTGGCGG-3′) and 1018R (5′-CTTTATAGAGGGGGTAAGGGCAG-3′) in the first round and then with 419shF (5′-ATCAGTTAACCTACCCGAGTCG-3′) and 628R (5′-GGTAGTTACGGTCTGTCCCAT-3′) in the second round, 10 reactions per dilution. The dilution at which 3/10 reactions are positive was selected, nested PCR was performed in a 96-well format, and reactions were analyzed by electrophoresis on precast 2% E-gels (Invitrogen). Positive reaction products were digested with BsaAI (NEB); fragments that remained undigested were isolated using a gel purification kit (Qiagen) and sequenced to confirm the presence of the gag leader deletion.
For Xpr1 cloning and sequencing, exons 10, 11, 12, and 13, comprising portions of the ECL3 and ECL4 loops, were amplified using the primers as previously cited (64).
Detection of provirus fragments in unblots.Unblot assays were performed as described previously, with slight modifications (55). The probe gL1 for detecting the gag leader deletion was 5′-TCGGACTTTTTGGAGTGGCTTTGTTGGGGG-3′. The probe was end labeled with [γ-32P]ATP using polynucleotide kinase (NEB) at 37°C for 1 h, and the labeled probe was cleaned using Micro Bio-Spin chromatography columns (Bio-Rad). Mouse genomic DNA (15 μg) either was directly digested with BsmI or Eco53KI (NEB) or was first obtained by whole-genome amplification from much smaller quantities using the REPLI-g midikit (Qiagen) and phenol extraction. Digested genomic DNA samples were electrophoresed on a 0.9% agarose gel for 20 to 30 h, the gel was dried under vacuum, and DNA was visualized by ethidium bromide (EtBr) staining. The dried gel was denatured for 15 min in 0.5 M NaOH, 1.5 M NaCl; neutralized for 15 min in 1 M Tris-HCl, 1.5 M NaCl, pH 8.0; and hybridized overnight with the radiolabeled probe (7.5 × 106 cpm) in hybridization buffer (5× SSPE [1× SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA (pH 7.7)] 0.1% SDS, pH 7.4). The hybridized gel was washed (2× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate], 0.1% SDS) and subjected to autoradiography.
Integration site mapping.The integration sites for PreXMRV-1 and PreXMRV-2 in the mouse genome were identified using the GenomeWalker kit (Clontech) according to the manufacturer's recommendations, with some modifications. To clone the PreXMRV-2 integration site, DBA/2J genomic DNA was digested with six restriction enzymes separately (NEB). Fragments with overhangs were blunted with the Quick Blunting kit (NEB). Adaptors supplied with the kit were ligated to fragment ends to construct genomic DNA libraries, which were serially diluted to avoid generation of recombinants during PCR. Nested PCR was performed with two sets of adaptor-specific primers (AP1 and AP2) provided with the kit and provirus-specific primers, 129-1R (see above) and 129-2R (5′-GGTCTGTCCCATGATCTCGAGAACAC-3′), on diluted libraries. Fragments from the first round of PCR were also subjected to BsaAI digestion (NEB) to prevent most proviruses without the gag leader deletion from being amplified in the second round. A single band was observed after the second PCR, which was cloned into pCR4-TOPO (Invitrogen). Clones were screened by PCR for the presence of a >1.2-kb insert as well as the absence of the restriction site by BsaAI digestion; those that passed both tests were sequenced. Five such clones revealed identical fragments containing the gag leader deletion, a complete 5′ LTR, and flanking cellular DNA, which mapped to mouse chromosome 12.
To clone the integration site of PreXMRV-1, C57L/J DNA was subjected to the same process as described for PreXMRV-2. Two of the restriction enzymes chosen were ApaLI and Hpy166II, both of which would digest most C57BL/6 MLVs in the LTR upstream of the provirus primer binding site and prevent their amplification but would leave PreXMRV-1 intact to be amplified. Nested PCR was performed with AP1 and AP2 supplied with the kit and provirus-specific primers XgR1 (5′-GCGACTCAGTCTATCGGATGACTGG-3′) and XgR2 (see above). Fragments were cloned, sequenced, and screened as described for PreXMRV-2. The correct clones with PreXMRV-1 LTR and flanking sequence mapped the provirus to mouse chromosome 3.
Primers were then designed for the flanking region based on the cloned sequence and combined with virus-specific primers to screen for and sequence both proviruses in different strains. Sequencing was performed by the Tufts University Core Facility.
Nucleotide sequence accession numbers.The complete provirus sequences of PreXMRV-1 and PreXMRV-2 plus the flanking regions in the mouse genome have been deposited in GenBank under the accession numbers HE599400 and HE599401, respectively.
Searching mouse genomes for XMRV-like elements.Four different strategies were employed to search for XMRV-like sequences in mouse genomes. In all cases, proviruses that contain stretches of identity to XMRV were detected; however, no XMRV provirus was present in any of the strains tested, as previously reported (39).
XMRV-specific PCR assay.The XMRV genome shows up to ∼95% identity with some MLVs, found as both endogenous and exogenous viruses in mice (58). To detect XMRV sequences in mouse DNA, a highly specific PCR assay was designed that selectively amplified the 5′ region of the XMRV genome but not any of the endogenous MLVs in C57BL/6 under the conditions used (25, 39). Due to the sequence similarity between XMRV and endogenous MLVs, primer selection for PCR was restricted to regions of the provirus that were sufficiently divergent. One of the features that distinguish the XMRV genome from most other MLVs is a 24-bp deletion in its gag leader region (Fig. 1A). This deletion was initially thought to be specific for XMRV but has recently been detected in the whole-genome shotgun (WGS) sequence of an inbred strain, 129X1/SvJ (GenBank ID, AAHY01591888.1) (11), and later in a few other strains using deep sequencing (23). A reverse primer (GAGr) spanning the gag leader deletion was used in combination with a forward primer (XmU3f) that includes a 2-bp insertion unique to the XMRV U3 region (Fig. 1A). In addition, the sensitivity and specificity of the assay were assessed (Fig. 1B).
In the initial screen with the XmU3f-GAGr primer set, XMRV-specific sequences were not detected in any of the 46 wild-derived or 48 laboratory mouse strains tested (39), while the 22Rv1 control cell line was always positive, implying that no endogenous provirus identical to XMRV was present in these strains (Fig. 1C).
The partial U3 region included in the 129X1/SvJ sequence revealed that the XMRV-specific 2-bp insertion was not present in the 129X1/SvJ provirus (Fig. 1A). Moreover, there was a 6-bp deletion in the XmU3f primer binding site, which prevented the detection of this provirus. A different primer was designed (XF6) to amplify the 129X1/SvJ provirus, which yielded a clear band in several strains when used with the GAGr primer (Fig. 1C). The PCR products from all strains were sequenced and confirmed to be identical to each other, differing from the 129X1/SvJ sequence at two positions (157 and 263 of the GenBank entry) where the deposited sequence has an extra C and T nucleotide, respectively.
SGS.To obtain more accurate sequence information and to rule out potential PCR recombination between different proviruses, a single-genome sequencing (SGS) approach was developed. MLV genomes were classified into three groups based on their gag leader profiles: those that contain (i) 0 bp (XMRV-like gag leader deletion), (ii) 15 bp, or (iii) 24 bp (Fig. 1A). The first group includes all XMRV isolates, as well as the uncharacterized provirus fragments identified by deep sequencing (23), while the second and third groups include all C57BL/6 proviruses and some exogenous MLVs (25). A diagnostic BsaAI restriction site was identified at the junction of the deletion, which would selectively digest a large fraction of the sequences in the last two groups (Fig. 1A). This selection method eliminated most "unwanted" proviruses and focused on those with the gag leader deletion, plus a few with a polymorphism at the restriction site.
Serial dilutions of genomic DNA were subjected to nested PCR with well-conserved MLV primers upstream and downstream of the gag leader deletion (see Materials and Methods). Positive reactions were analyzed by BsaAI digestion. Fragments that remained undigested (i.e., those with the gag leader deletion) were analyzed further by sequencing, and the presence of the deletion (or a polymorphism in the restriction site) was confirmed. Using this approach, a total of seven mouse strains were analyzed, three of which contained XMRV-like gag leader regions (Fig. 2B), providing further evidence that such elements are present as endogenous viruses in the genomes of several inbred mouse strains.
Detection of XMRV-like elements. (A) Outline of the SGS approach. BsaAI digestion profiling (Fig. 1A) distinguishes fragments with the deletion from the rest. (B) Alignment of sequences from three strains positive for the gag leader deletion (NU/J, NCRNU, and DBA/2J), the whole-genome shotgun (WGS) sequences from the 129X1/SvJ and DBA/2J genomes in GenBank, and other MLV sequences as a reference. Numbers reflect corresponding positions in the VP62 genome (EF185822). (C) Schematic representation of the Eco53KI sites and the gL1 probe binding site in MLV genomes. (D) Unblots of various mouse genomic DNA samples digested with Eco53KI and hybridized with a radiolabeled probe. A distinct provirus fragment corresponding to PreXMRV-2 was detected in six strains (arrow).
Detection of provirus fragments in unblots.To investigate the distribution of XMRV-like sequences in mouse genomes and to provide further support for the PCR screening data, genomic hybridization blot assays were performed using the unblotting technique (55). Briefly, genomic DNAs from various mouse strains were digested with a restriction enzyme, run on an agarose gel that was then dried down, and directly hybridized with a gag leader-specific radiolabeled probe that crossed the deletion site (see Materials and Methods). The enzyme Eco53KI was chosen since it would selectively digest proviruses lacking the gag leader deletion at the probe binding site while leaving proviruses with the deletion intact up to the next downstream restriction site (Fig. 1A and 2C). A single high-intensity band was detected in six out of 10 strains analyzed, in agreement with the genome screening results by PCR (Fig. 2D).
GenBank database searches.To investigate whether other mouse strains besides the reported 129X1/SvJ strain also carried the gag leader deletion (11, 23), an extensive search of the NCBI Trace Archives was performed by querying the gag leader region of XMRV. This search returned an additional three hits in the DBA/2J genome and one unassigned record with exactly the same sequence (GenBank IDs, ti:1098845661, 1096851092, 1096808503, and 1039132836). None of these sequence files extended beyond the LTR into the flanking region, providing no information about the integration site. Nevertheless, the presence of this XMRV-like sequence in multiple reads further confirmed our results and those of others (23) that XMRV-like endogenous sequences are indeed present in the genomes of several strains of mice.
Mapping the integration site of PreXMRV-1 and PreXMRV-2.Although multiple independent lines of evidence implied the presence of XMRV-like elements in various mouse genomes, further analysis required determination of their integration sites. A ligation-mediated PCR approach, described in Materials and Methods, was used to amplify and clone fragments of DBA/2J genomic DNA that, like PreXMRV-2, contained the specific gag leader deletion and included the complete 5′ LTR and flanking sequence. Knowledge of the flanking sequence, which mapped to chromosome 12 on the C57BL/6J genome sequence, allowed the design of primer pairs to amplify host-virus junctions from mouse genomic DNA (Fig. 3A).
Detection of PreXMRV-2 in mouse strains and CWR22 xenografts. (A) PCR approach to amplify host-virus junctions from genomic DNA. Regions of identity between XMRV and PreXMRV-2 are depicted as white bars; regions of difference are shown as black bars (not drawn to scale). (B) Genomic DNAs from representative mouse strains (left lanes) and early-passage samples of the CWR22 prostate cancer xenograft (right lanes) were analyzed for the presence of PreXMRV-2 (Table 1 has a complete list of mouse strains screened). In the 22Rv1 and CWR-R1 cell lines, the fragments detected by internal provirus primers are XMRV, as neither contains PreXMRV-2. NTC, no-template control.
The integration site of the provirus was confirmed by amplifying both 5′ and 3′ junctions from the DBA/2J genomic DNA, using flanking chromosomal primers and internal provirus primers (Fig. 3A and B). Both junction fragments contained a full-length LTR and the expected flanking region, as confirmed by sequencing, with a four-base "GGAA" target site duplication (TSD). The gag leader region included in the 5′ junction fragment was also sequenced again, confirming the presence of the 24-bp deletion. It is important to note that the provirus integration site is found within a rather complex, repetitive, and poorly sequenced region, making it impossible to use PCR to distinguish the preintegration site from the occupied integration site. We previously named this provirus PreXMRV-2 (GenBank ID, FR871850.1), to reflect its role in the generation of XMRV (39).
The integration site of PreXMRV-1 was cloned from C57L/J genomic DNA, using the same approach as that used to clone the integration site of PreXMRV-2, with slight modifications (see Materials and Methods). Sequencing the upstream flanking sequence, which was cloned together with the PreXMRV-1 LTR, revealed that the PreXMRV-1 insertion is found on mouse chromosome 3. Amplification of host-virus junctions from both ends followed by sequencing confirmed that the location indeed corresponded to PreXMRV-1, with a TSD of "GCAG" (Fig. 4A and B).
Detection of PreXMRV-1 in mouse strains and CWR22 xenografts. (A) PCR approach to amplify host-virus junctions from genomic DNA. Regions of identity between XMRV and PreXMRV-1 are depicted as white bars; regions of difference are shown as black bars (not drawn to scale). (B) Genomic DNAs from representative mouse strains (left lanes) and early-passage samples of the CWR22 prostate cancer xenograft (right lanes) were analyzed for the presence of PreXMRV-1 or its unoccupied integration site (Table 1 contains a complete list of mouse strains screened). In the 22Rv1 cell line, the fragment detected by internal provirus primers is XMRV. In the CWR-R1 cell line, which is known to contain a small number of cells derived from the mouse host (39), the provirus fragment is from both XMRV and PreXMRV-1, while the detected junction fragments are from PreXMRV-1 alone. NTC, no-template control.
Strain distribution of PreXMRV-1 and PreXMRV-2 and absence of XMRV.The discovery and characterization of PreXMRV-1 were previously described (39). To determine the distribution of the two XMRV precursors, a panel of mouse DNA samples from 48 laboratory and 46 wild-derived strains were screened for the presence of PreXMRV-1 and PreXMRV-2. As shown in Fig. 1C, no provirus identical to XMRV was present in any of the mouse strains tested (39). In the present analysis, 23 inbred, 2 outbred, and 2 wild-derived strains were positive for PreXMRV-2 (Table 1). Its strain distribution agreed perfectly with the earlier mouse genome screening results with XMRV-like gag leader-specific primers (Fig. 3B), consistent with PreXMRV-2 being the only provirus in these strains with the characteristic deletion. PreXMRV-1 was present in 4 inbred, 2 outbred, and 8 wild-derived strains (Table 1). Only one inbred strain (C57BR/cd) and two outbred strains (Hsd nude and NU/NU) contained both proviruses.
Mouse strains tested for the presence of XMRV,a PreXMRV-1, and PreXMRV-2
PreXMRV-1 seems to be quite rare among inbred strains; to date, it has been detected only in C57L/J, C58/J, C57BR/cd, and NZW/Lac mice. Although PreXMRV-2 is absent from the sequenced C57BL/6 mouse genome, it was present in nearly half of the inbred strains tested, suggesting that it is a common provirus insertion. The two wild-derived strains that harbor PreXMRV-2 belong to M. m. domesticus subspecies, SF/CamEi and SK/CamEi (Table 1). M. m. domesticus is native to the Near East, Europe, and Africa and later colonized the Americas and Australia as human travel between these continents became common (20). PreXMRV-1 was detected in several strains of Asian mice, M. m. castaneus and M. m. molossinus, the former being an ancestral species that gave rise to the latter, and also in the CALB/Rk strain thought to be a hybrid between M. m. domesticus and M. m. castaneus (24).
Interestingly, the only strains that harbor both PreXMRV-1 and PreXMRV-2 are one inbred (C57BR6/cd) and two outbred strains: the Harlan Sprague Dawley (Hsd) nude strain maintained by Harlan Laboratories and the NU/NU strain maintained by Charles River Laboratories (39). Both outbred strains are likely to have been used for passaging the prostate tumor xenografts that later gave rise to the 22Rv1 cell line (39). Because both are outbred strains, the numbers and distributions of specific proviruses may differ among individual mice. Therefore, the distribution of XMRV, PreXMRV-1, and PreXMRV-2 was analyzed in a large panel of Hsd nude mice (n = 49) and NIH Swiss mice (n = 10) (Fig. 5; Table 2). The NIH Swiss strain was included because it might have contributed to the outbred nude mouse strains (16). Among the Hsd nude mice analyzed, 27% were positive for PreXMRV-1 only, 10% were positive for PreXMRV-2 only, and 53% contained both proviruses, consistent with the null allele frequencies of 0.45 and 0.61, respectively (Fig. 5A and Table 2). Thus, about half of the mice within this outbred colony carried both XMRV parental proviruses (Fig. 5A). This result is consistent with the observation that some, but not all, of the mouse tissues associated with the early tumor xenografts contained both PreXMRV-1 and PreXMRV-2 (Fig. 3B and 4B) (39). NIH Swiss mice were more homogenous; all tested samples contained PreXMRV-2, while none contained PreXMRV-1 (Fig. 5B). The variability of the NU/NU outbred mice (n = 5) for the presence of PreXMRV-1 and PreXMRV-2 was shown previously; 2/5 mice contained both PreXMRV-1 and PreXMRV-2 (39). Most importantly, XMRV was not present in any of the 64 outbred samples tested, confirming previous data on its absence from various inbred and wild-derived strains (Fig. 5A and 5B) (39).
Variability of PreXMRV-1 and PreXMRV-2 and absence of XMRV in outbred mouse strains. Hsd nude mice (n = 49) (A) and NIH Swiss mice (n = 10) (B) were analyzed for the presence of XMRV, PreXMRV-1, and PreXMRV-2 by PCR as described for Fig. 3 and 4. The primers used for the detection of PreXMRV-1 in this figure also detect XMRV (primer set 8r-1bf [Fig. 4B]). However, the absence of XMRV was confirmed with XMRV-specific primers (top panels), and the presence of PreXMRV-1 was verified by primers flanking the integration site, with identical results (data not shown). Mouse IAP elements served as amplification controls. The Hsd nude sample used in Fig. 1C, 3B, and 4B is labeled as "Hsd #0." Null allele frequencies calculated from these results are shown in Table 2. The presence of PreXMRV-2 but not PreXMRV-1 in NIH Swiss mice is consistent with the detection of the former, but not the latter, in NIH 3T3 cells (36).
Frequency of PreXMRV-1 and PreXMRV-2 in outbred mice
The Xpr1 receptor in Hsd nude, NU/NU, C57BR/cd, and CWR22 xenograft mouse DNA is nonpermissive to XMRV entry.Xenotropic MLV (X-MLV) and polytropic MLV (P-MLV) use different variants of the XPR1 receptor (Fig. 6A) to gain entry into cells (63, 64). Many laboratory strains contain the Xpr1n allele, which does not support X-MLV entry, while P-MLV can use the protein encoded by this variant to infect cells. We determined whether XMRV, which has a xenotropic env gene, would have been able to infect the cells of the mice used for passaging the tumor, as well as the three mouse strains found to harbor both parental proviruses. Exon 13 of the Xpr1 gene, corresponding to the ECL4 region, was amplified and sequenced from Hsd nude (n = 21) and NU/NU (n = 5) mice (Fig. 6B). Every individual mouse tested among these two strains had the Xpr1n allele, which is nonpermissive to X-MLV entry, suggesting that the XMRV recombinant would not have been able to infect these mice. Exons 10, 11, and 12 of the Xpr1 gene, corresponding to the ECL3 region, were also sequenced from Hsd nude (n = 4) and NU/NU (n = 3) mice, and all carried the Xpr1n allele (Fig. 6B). All early xenografts, which contained some host mouse DNA from the original transplant, also carried the nonpermissive Xpr1n allele (Fig. 6C). The absence of a functional receptor to support X-MLV entry shows that XMRV could not have infected the cells of the host mouse in which it was created and provides further support for the requirement of the transplanted human cells for infection. Lastly, the C57BR/cd mouse, which is positive for both PreXMRV-1 and PreXMRV-2, contained the Xpr1n allele as well (Fig. 6B). This result is consistent with a previous report by Baliji et al. that showed that C57/BR, a strain related to C57BR/cd, also contains the Xpr1n allele (5).
Presence of the Xpr1n receptor allele in xenografts, outbred Hsd nude and NU/NU strains, and inbred C57BR/cd strains. (A) Known variants of the XPR1 receptor are shown, with the amino acid residues in the ECL3 and ECL4 loops responsible for the inability of the XPR1n allele to bind xenotropic MLV Env highlighted in bold (figure modified from reference 64). (B) ECL3 and ECL4 loops of the XPR1 receptor for Hsd nude, NU/NU, and C57BR/cd carry the Xpr1n allele, which does not support entry by a xenotropic MLV Env (30). (C) Early xenografts containing either PreXMRV-1 alone or the two proviruses together (39) carry the Xpr1n allele in the mouse tissues found associated with the xenografts.
Recent work identified two endogenous MLV proviruses from the xenografts of the CWR22 human prostate tumor passaged in nude mice (39), which led to the widely used 22Rv1 cell line, now known to produce XMRV (28, 50). Early xenograft passages of the tumor did not contain XMRV, whereas the later passages did, implying that XMRV infected the tumor after initial passaging and prior to generation of the 22Rv1 cell line (39). Moreover, the host mouse DNA found associated with the tumor contained both XMRV parental proviruses, strengthening the argument that recombination between these two endogenous MLVs led to the generation of XMRV. Here we described the experimental approaches leading to the identification of PreXMRV-2, the genomic locations of both proviruses, and their distribution among wild-derived and laboratory mouse strains.
The distribution of PreXMRV-1 and PreXMRV-2.As previously reported, XMRV is not present as a naturally occurring single provirus in any mouse genome tested, including ∼170 individual mice from 48 laboratory strains and 46 wild-derived strains (Table 1; Fig. 5A and B) (39). The fact that neither provirus is fixed in the subspecies where it occurs suggests that both are of fairly recent origin, having integrated after the origin of these subspecies less than 1 million years ago and prior to the radiation of M. m. domesticus in the Americas or the hybridization of M. m castaneus and M. m. musculus in Asia that gave rise to M. m. molossinus (20, 66).
The distribution of PreXMRV-1 and PreXMRV-2 is consistent with previous findings on Xmv and Pmv env distribution in wild mice (32). The only mice that we found to harbor both proviruses are the two outbred strains, Hsd nude and NU/NU (39), and one inbred strain, C57BR/cd (Table 1). Inbred mouse strains are largely derived from M. m. domesticus, with minor contributions from M. m. musculus and M. m. castaneus. Interestingly, among inbred mouse strains, the C57BR/cd genome contains some of the greatest M. m. musculus-derived content (65). It is particularly noteworthy that the two proviruses found their way into laboratory mice from wild subspecies found on two different continents, Asia (M. m. molossinus and M. m. castaneus) and Europe (M. m. domesticus), and are, therefore, very unlikely to have been present in the same mouse until their recent crossbreeding to create the fancy and laboratory mouse strains (61). In fact, not a single wild mouse strain was found to carry both PreXMRV-1 and PreXMRV-2, making it extremely unlikely that the two parents would be present in the same mouse in the wild, recombine, and cross species to infect humans.
Of the three strains that do carry both PreXMRV proviruses, two (Hsd nude and NU/NU) were most likely to have been used for the passaging of the CWR22 xenograft at the time that it became infected, although precise records were unavailable (37). We had previously shown the presence of both proviruses in xenografts obtained prior to 1996, at frequencies attributable to a low level of mouse DNA derived from the nude mouse host. In the case of PreXMRV-1, the flanking sequence was unknown, and the prior identification relied on internal provirus primers, some of which also recognized XMRV. Here, we confirmed the prior conclusion, using flanking sequence primers, finding that all the early xenograft samples were positive for the PreXMRV-1 provirus and that some, but not all, were positive for PreXMRV-2 (Fig. 3B and 4B). Furthermore, 3/7 of the samples were heterozygous for PreXMRV-1, as revealed by positive PCR signal with flanking primers alone (Fig. 4B). These results are consistent with the conclusion that either of the outbred nude mouse strains, Hsd nude and NU/NU, both of which are polymorphic at the two loci (Fig. 5A) (39), was a host for the CWR22 xenografts. Whatever the host strain, they clearly show that multiple passages off the tumor were through mice that contained both proviruses, providing ample opportunity for recombination to create XMRV.
The presence, in a number of mouse strains, of a provirus carrying the characteristic 24-bp gag leader deletion has been previously noted (11). Since the strain distribution of that provirus by PCR using gag leader-specific primers is in perfect agreement with that of PreXMRV-2, we conclude that this is the only provirus in these strains with the characteristic deletion. Proviruses with the gag leader deletion have also been detected in four inbred strains by deep sequencing (23). Although those fragments most likely correspond to PreXMRV-2, the short length of the deep sequence reads (167 bp) precludes a firm conclusion.
Recombination between PreXMRV-1 and PreXMRV-2.The generation of recombinant viruses with similar crossover patterns can occur multiple times, reflecting selection for advantageous properties such as receptor usage, expression in target cells, evasion of host restriction, and repair of defects in one or the other parental virus, resulting in a selective advantage to the recombinant virus. In the present case, PreXMRV-1 has obvious defects in gag and pol that were repaired by the corresponding regions in PreXMRV-2. It is worth emphasizing that the probability of generating the same XMRV recombinant with the same crossovers independently more than once is extremely small (39). In a hypothetical situation where by an extremely remote chance such an event did occur, the recombinant (XMRV) would be unable to reinfect the mouse target cells and spread, since the Hsd nude, the NU/NU, and the individual mice used to passage the xenografts carry the Xpr1n receptor variant (Fig. 6B and C), which is nonpermissive to XMRV infection (63, 64). The presence of the grafted human tumor tissue therefore gave the recombinant the opportunity to infect and propagate. The fact that multiple passages of the CWR22 xenograft took place in mice carrying both proviruses before the recombinant appeared suggests that the event must have been quite rare, possibly reflecting low levels of expression of the parental proviruses.
The biological properties of the parental proviruses are under investigation. As noted, PreXMRV-1 cannot be infectious due to mutations in gag and pol (39). Preliminary experiments using the cloned full-length PreXMRV-2 provirus to transfect XMRV-susceptible cells suggest that it is also not infectious (data not shown). It is not known whether PreXMRV-1 and PreXMRV-2 are expressed in mouse cells that harbor both proviruses. Further experiments are required to elucidate the infectious properties of potential recombinants that may arise between PreXMRV-1 and PreXMRV-2.
Detection of MLV-like sequences.A previous study testing human samples for the presence of XMRV reported the detection of Pmv/Mpmv sequences from the blood of CFS patients, as well as some healthy controls, by PCR (34). However, several instances of contamination of human tissues or laboratory reagents with very similar sequences have also been reported (29, 38, 43, 46, 49, 57). These results raise concerns about the potential for contamination by mouse DNA, since the only detection method relied on PCR and some of the detected provirus fragments were identical to known Pmvs found in mouse genomes.
It is important to point out that most published "XMRV-specific" PCR assays will in fact detect sequences that are identical between either PreXMRV-1 or -2 and XMRV and would yield a false-positive result if confronted with mouse DNA containing one or the other of these proviruses. Only PCR assays that rely on primers flanking one of the crossover points can be considered to be truly XMRV specific (39). In fact, of all the "XMRV-specific" PCR assays published to date, almost none are truly specific, since the primer sets would detect either PreXMRV-1 or PreXMRV-2 (4, 13, 17, 34, 35, 48, 53, 58). To our knowledge, the only exception is the primer set used by Hohn et al., where the first round of nested PCR spans a crossover site between PreXMRV-1 and PreXMRV-2 in the gag leader region (21).
PreXMRV-1 and PreXMRV-2 represent yet another addition to the growing list of endogenous MLV proviruses of mice. Their significance stems from the recombination between the two that ultimately led to the generation of XMRV during the passaging of a human prostate tumor as xenografts in mice. To our knowledge, no other MLV, recombinant or otherwise, acquired by human tissues upon transplantation into mice has ever received this much attention, even though such retroviral acquisitions by heterologous cells occur frequently during passage of human cells in mice or other species and have been known for more than 30 years (1, 6, 15, 56, 62, 67). Even more frequent is unintentional infection of cell lines with retroviruses in the laboratory, often going unnoticed for many years (3, 23, 28, 41, 42, 51, 54). Extreme measures are therefore necessary to avoid false positives, including routine screening of cell lines used in the laboratory, use of highly sensitive assays for detecting mouse DNA contamination as well as potential cross-contamination of assays with viruses released from cell lines, and finally, employing multiple independent assays to support findings, especially when made by PCR alone.
We thank Robert Silverman for the VP62 XMRV construct; Vineet KewalRamani for 22Rv1 cells; Alan Rein for 22Rv1 DNA; Greg Towers, Christine Kozak, and François Bonhomme for some of the mouse DNA samples; Julia Wildschutte for technical help; Wayne Frankel for helpful discussions; and the Tufts University Core Facility for sequencing.
This work was supported by grant R37 CA 089441 to J.M.C. and in part by an intramural Bench-to-Bedside Award to V.K.P. J.M.C. was a Research Professor of the American Cancer Society, with support from the F. M. Kirby Foundation. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
Received 15 August 2011.
Accepted 26 September 2011.
Accepted manuscript posted online 26 October 2011.
Address correspondence to John M. Coffin, john.coffin{at}tufts.edu.
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Journal of Virology Dec 2011, 86 (1) 328-338; DOI: 10.1128/JVI.06022-11
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Mast, M.A., and Turk, J.T., 1999, Environmental characteristics and water quality of Hydrologic Benchmark Network stations in the Midwestern United States, 1963-95: U.S. Geological Survey Circular 1173-B, 130 p.
The North Fork Whitewater River HBN Basin is located in the Wisconsin Driftless Section of the Central Lowland physiographic province in the southeastern corner of Minnesota (Figure 16. Map showing study area in the North Fork Whitewater River Basin and photograph of agricultural land). The basin drains an area of 262 km² and ranges in elevation from 235 to 400 m. The gently rolling landscape in the upper part of the basin is formed by a plateau of erosion- resistant rock. In the lower part of the basin, the plateau surface is strongly dissected by several steep-sided stream channels. The USGS gaging station is located 3.9 km west of Elba, Minn., at latitude 44×05'30'' and longitude 92×03'57''. The North Fork Whitewater River drains east into the Whitewater River, a tributary of the Mississippi River, and has a channel length of 47 km upstream from the gaging station and an average stream gradient of 1.9 m/km. Mean monthly discharge varies over a narrow range from 0.86 m³/s in January to 2.5 m³/s in March, and average annual runoff from the basin was 17 cm from 1933 through 1995 (Mitton and others, 1996). The climate is characterized by moderately warm summers and cold winters. Average daily air temperatures range from -10.7×C in January to 22.7×C in July (Lueth, 1994). Precipitation averages 84 cm annually and more than 70 percent falls from April through September. The average seasonal snowfall is 120 cm and, on average, 95 days of the year have at least 2 cm of snow cover (Lueth, 1994).
The North Fork Whitewater River Basin is in the Eastern Broadleaf Forest (Continental) ecoregion (Bailey and others, 1994), but only 16 percent of the basin is forested. Most forested areas are localized along the steep-sided slopes of the drainages in the lower part of the basin. Oak, shagbark hickory, bitternut hickory, aspen, and paper birch are the most common species. Large areas of the uplands at one time were covered by white oak, red oak, elm, basswood, maple, wild cherry, and pine that since have been cleared for farming. Soils in the basin are classified as Alfisols and mapped in the Seaton-NewGlarus-Palsgrove and Lacrescent-Lamoille soil associations (Lueth, 1994). The Seaton-NewGlarus-Palsgrove soils are well-drained silt loams developed in loess on the upland areas of the basin. A typical soil profile has a surface layer of grayish-brown silt loam that is 20 cm thick overlying a subsoil of dark yellowish-brown silt loam that extends to a depth of 150 cm. The soils generally have pH values between 5.1 and 7.3 and an organic-matter content of less than 3 percent in the upper 20 cm (McCleary and others, 1989). Soils in the Lacrescent-Lamoille series develop in loess and colluvium on steep-sided slopes along the drainages and consist of well-drained silt loams that contain between 20 and 60 percent cobbles and gravel.
Bedrock in the basin consists of gently dipping marine sedimentary rocks of Paleozoic age. The uplands plateau surface is capped by the resistant limestones of the Galena Formation and dolomite beds belonging to the Prairie du Chien Group of Paleozoic age (Mossler and Book, 1984). The softer shales and sandstones of the Decorah Shale, Platteville Formation, Glenwood Formation, St. Peter Sandstone, and Jordan Sandstone of Paleozoic age primarily crop out along the walls of the stream valleys (Mossler and Book, 1984). The Prairie du Chien Group and the Jordan Sandstone form the Prairie du Chien aquifer, which is the major water supply in the southeastern corner of Minnesota (Smith and Nemetz, 1996). The bedrock, particularly in the upland areas of the basin, is covered by till deposits of Pleistocene age. The till consists of a mixture of sand, silt, and clay that is calcareous where unweathered. A thin layer of loess mantles the till and the bedrock and ranges in thickness to as much as 3 m (Hobbs, 1988). The loess consists of a brownish silt loam that is leached of carbonate to a depth of 2 to 3 m. The steep walls along the stream channels are mantled by unsorted colluvium consisting of clasts of bedrock in a silt matrix derived from loess (Hobbs, 1984).
The North Fork Whitewater River Basin drains parts of Wabasha, Olmsted, and Winona Counties of Minnesota. Land ownership is 94 percent private and 6 percent State (W. Lorenzen, U.S. Department of Agriculture Natural Resources Conservation Service, written commun., 1996). Most State-owned land is part of the Whitewater State Forest and is located along a deeply incised section of the main channel of the North Fork Whitewater River in the lower part of the basin. More than 100 km of State and county roads traverse most areas of the basin. The incised section of the main river channel is accessible by hiking trails on State forest land; access to most tributaries is limited to rights-of-way at public road crossings. Many roads in the basin are plowed and heavily salted in winter.
Land cover in the basin is 64 percent cropland, 8 percent pasture land, 16 percent forest, 6 percent wetland, and 6 percent residential and urban land (W. Lorenzen, written commun., 1996). Feed grain and forage for dairy cattle and other livestock are the major farming enterprises in the watershed. Recent trends in the farm economy have shown a shift from dairy operations to cash crop production, including cannery crops (U.S. Department of Agriculture, 1996). The average farm is 254 acres in size, and approximately one-third of farms are dairies. Other human-related features in the basin include the townships of Viola (population 604), Elgin (population 761), and part of Plainview (population 2,768); a wastewater-treatment plant at Plainview; a State park campground; and a railroad line that parallels the upper reach of the main river channel. Controlling erosion from croplands is the principal land- management concern in the region (Argabright and others, 1996). Implementation of conservation practices, such as terracing, strip cropping, and reducing tillage, caused a reduction in soil erosion rates in the region of nearly 50 percent between 1930 and 1992 (Argabright and others, 1996). The U.S. Department of Agriculture instituted the CRP in 1985 as an additional means of reducing soil erosion from highly erodible croplands. Under the program, farmers were paid in exchange for planting permanent vegetation types, such as sod-forming grasses and legumes. About 25 percent of cropland in the basin was planted in grasses under the CRP program; however, as much as 50 percent of that grass cover has come back into production since 1996 (W. Lorenzen, written commun., 1996). In 1996, a long-term watershed implementation plan was adopted for the drainage basin of the Whitewater River, of which the North Fork Whitewater River is one of four major tributaries (U.S. Department of Agriculture, 1996). The goal of the plan is to improve water quality, reduce sedimentation and flooding, and improve wildlife habitat in the Whitewater River Basin by developing a partnership among watershed citizens, governmental agencies, environmental groups, and businesses.
The data set for the North Fork Whitewater River HBN Station analyzed for this report includes 218 water-quality samples that were collected from August 1967 through September 1993. Sampling frequency ranged from 8 to 12 samples per year during the period 1968 through 1982 and bimonthly from 1983 through 1993. Water-quality samples in the early part of the period of record were probably analyzed at a USGS laboratory in Columbus, Ohio, that operated until 1973 (Durum, 1978). After establishment of the central laboratory system, samples were analyzed at a laboratory in Salt Lake City, Utah, from 1973 through 1975 and at the NWQL in Arvada, Colo., from 1976 through 1995. Daily discharge records are available for the North Fork Whitewater River (station 05376000) from May 1939 through September 1941 and from July 1967 through September 1993.
not included in the calculation of the ion balance. The average nitrite-plus-nitrate concentration at this station was 220 meq/L, which is slightly less than 2 percent of the total ionic charge in solution. Time-series plots of the major dissolved constituents were inspected for evidence of method-related effects (fig. 17). No unusual patterns were apparent, except perhaps for nitrite plus nitrate, which had a pattern of elevated concentrations between 1980 and 1986 and again in 1992. The period of elevated nitrite-plus-nitrate concentrations in the 1980's seems to coincide with a change in the protocol for field preservation of nutrient samples. Before 1980, samples were preserved by chilling to 4×C; from 1980 through 1985, samples were preserved with mercuric chloride tablets; and beginning in 1986, the tablets were replaced by ampoules of mercuric chloride solution (Schertz and others, 1994). Schertz and others observed elevated organic plus ammonium nitrogen and ammonia nitrogen concentrations in several streams in Texas between 1980 and 1986, which were attributed to a positive bias in the data caused by the addition of mercuric chloride tablets. In 1986, when the tablets were replaced by the ampoules, the concentrations returned to normal levels of variation. Although Schertz and others (1994) only observed a bias in ammonium species, the pattern in the North Fork Whitewater River indicates that, perhaps, the mercuric chloride tablets may have biased the nitrite-plus-nitrate concentrations as well.
Median concentrations and ranges of major constituents in stream water at the North Fork Whitewater River Station and VWM concentrations in wet-only deposition measured at the Wildcat Mountain NADP station are listed in table 30. Precipitation chemistry at the NADP station, which is about 120 km east of the HBN station, was dilute and slightly acidic with a VWM pH of 4.8 for 6 years of record. The predominant cations were ammonium, which contributed 45 percent of the total cation charge, and calcium and hydrogen, which accounted for 26 and 20 percent, respectively. Sulfate accounted for 56 percent of the total anions and nitrate contributed 39 percent. The predominance of strong acid anions at this site indicates that precipitation may be affected by anthropogenic emissions of sulfur and nitrogen compounds, which cause acid rain. In addition, the relatively high ammonium concentrations at this NADP station may be linked to the high density of livestock in this region of the country.
a Data are volume-weighted mean concentrations for 1989-95.
Stream water in the North Fork Whitewater River is concentrated and strongly buffered; specific conductance ranged from 200 to 690 mS/cm, and alkalinity was between 1,240 and 5,700 meq/L (table 22). Calcium and magnesium were the predominant cations, and bicarbonate was the predominant anion. The predominance of these solutes in stream water is attributed to the presence of limestone bedrock and carbonate minerals in the overlying till and loess deposits. The median concentration of chloride in stream water was 240 meq/L, which is almost 100 times greater than the VWM concentration of 2.5 meq/L in precipitation. This large difference in concentration indicates that most stream-water chloride is derived from sources other than precipitation. Most stream-water chloride probably is derived from a combination of natural and human-related sources; however, road salt, sewage effluent, and fertilizer are probably the largest contributors. Sulfate concentrations also were much higher in stream water than in precipitation. The main source of sulfate, other than atmospheric deposition, probably is calcium sulfate minerals present in variable amounts in the limestone and shale bedrock. The median concentrations of nitrate and ammonium in stream water were 210 and 2.1 meq/L compared to 23 and 27 meq/L in precipitation, indicating that a substantial amount of inorganic nitrogen is exported from the basin. The most likely sources of stream-water nitrate in the basin include fertilizers, animal wastes, and wastewater effluent. The median stream-water nitrate concentration was similar to the concentration of 210 meq/L (3 mg/L), which is considered indicative of human-related pollution of ground water in southeastern Minnesota (Smith and Nemetz, 1995).
The solute composition of stream water was further evaluated by analyzing correlations between solutes and stream discharge (table 31). Discharge was poorly correlated with the major dissolved constituents, except for a weak positive correlation with nitrite plus nitrate (rho = 0.573). There also were few strong correlations among the solutes, except for a positive correlation between calcium and nitrate (rho = 0.764). The lack of strong correlations among these solutes probably is related to hydrologic factors. Streamflow is supplied primarily by ground-water discharge from a carbonate aquifer, which maintains a relatively constant flow rate and stream composition throughout most of the year. The correlations between nitrate and discharge and nitrate and calcium probably result from leaching of inorganic nitrogen from agricultural land and livestock yards during high-flow periods when discharge from shallow flow paths may be an important component of streamflow.
Results of the seasonal Kendall test for trends in discharge and major dissolved constituents are listed in table 32. Statistically significant trends were observed in discharge and most of the major dissolved constituents at the 0.01 probability level. Trends in calcium, magnesium, sodium, alkalinity, chloride, nitrite plus nitrate, and silica were similar for unadjusted and flow-adjusted concentrations, indicating that trends were not caused by the trend in stream discharge. The LOWESS curves in figure 17 show several different trend patterns at this station. The largest relative increases occurred in sodium, chloride, and nitrite-plus-nitrate concentrations, which at least doubled during the period of record. Most of the increase in sodium and chloride concentrations occurred before 1985; however, the magnitude of the trend in flow-adjusted chloride [10 (meq/L)/yr] was twice that of flow-adjusted sodium [5 (meq/L)/yr]. The upward trend in nitrite plus nitrate was more erratic than the trends in sodium and chloride, perhaps owing, in part, to analytical bias. By contrast, the increases in calcium, magnesium, potassium, alkalinity, sulfate, and silica concentrations were small (4 to 18 percent). Upward trends in sodium and chloride most likely are caused by changes in land-use activities in the basin, such as increased use of deicing salts, discharge from septic systems and the wastewater-treatment facility, and application of fertilizers to croplands. The difference in magnitude between the sodium and chloride trends was somewhat unexpected and may indicate part of the sodium from human-related sources is replacing calcium and magnesium on cation-exchange sites in the soil (Rittmaster and Shanley, 1995). The upward trend in nitrite-plus-nitrate concentrations probably also reflects changes in land-use activities because stream-water nitrogen is primarily derived from fertilizer and animal wastes. The source of trends in the remaining constituents is less clear. Landscape disturbance associated with croplands and residential areas has the potential to increase rates of base cation leaching and may result in higher stream-water alkalinity (Stoddard and Murdoch, 1991). Because sulfate and silica also are derived from geologic sources, landscape disturbance may affect stream-water concentrations of these solutes as well.
a Trend calculated for 1971-93.
Results of the surface-water synoptic sampling in the North Fork Whitewater River Basin on August 20, 1992, are listed in table 33, and the locations of the sampling sites are shown in figure 16. During the sampling period, discharge at the gaging station was about 1.1 m³/s compared to the median discharge of 0.9 m³/s for November (Lawrence, 1987), indicating that the basin was sampled during normal flow conditions for that time of year. The concentrations of most solutes measured at site 1 during the synoptic sampling were between the first- and third-quartile concentrations reported for the HBN station during the entire period of record (table 30). The tributary streams were similar in composition to stream water collected at the gaging station (site 1); calcium and magnesium were the predominant cations, and bicarbonate was the predominant anion. Ion balances were slightly negative for all samples and ranged from -0.4 to -2.1 percent.
The concentrations of the weathering-derived constituents did not vary markedly among the sampled tributary sites (table 33). For example, alkalinity ranged from a minimum of 4,980 meq/L at site 3 to a maximum of 5,800 meq/L in Dry Creek upstream from Elgin (site 7). Calcium concentrations ranged from 3,550 to 4,650 meq/L, and magnesium ranged from 2,000 to 2,880 meq/L. The widespread presence and rapid weathering rates of carbonate minerals in the bedrock and surfical deposits are the most likely reasons for the lack of substantial spatial variation in the concentrations of these solutes. Differences in the concentration of the remaining solutes seem to be related largely to human activities in the basin. Several solutes were elevated downstream from the wastewater-treatment plant at Plainview (site 9) compared to other sampling sites in the basin. For example, sodium and chloride concentrations were 6,090 and 5,630 meq/L, respectively, at site 9 compared to the average sodium and chloride concentrations of 310 and 700 meq/L, respectively, at the other sampling sites in the basin. The wastewater- treatment plant also apparently had a slight effect on stream-water potassium and sulfate concentrations based on the elevated concentrations of these solutes at site 9. Despite the high concentrations at site 9, the wastewater-treatment plant did not seem to substantially affect the surface-water chemistry at the gaging station (site 1), probably because of the low discharge at site 9 compared to the discharge at the gage. Nitrate concentrations at the sampling sites ranged from 240 meq/L at site 1 to 860 meq/L at site 2. All sites had nitrate concentrations greater than the background concentration of 210 meq/L (3 mg/L) for ground water in southeastern Minnesota (Smith and Nemetz, 1996), and two of the tributaries (sites 2 and 4) had concentrations at or greater than the drinking-water standard of 710 meq/L (10 mg/L), indicating widespread nitrate contamination of stream water in the basin.
Argabright, M.S., Cronshey, R.G., Helms, J.D., Pavelis, G.A., and Sinclair, H.R., Jr., 1996, Historical changes in soil erosion, 1930-1992, the northern Mississippi Valley Loess Hills: U.S. Department of Agriculture Natural Resources Conservation Service, Resource Economics and Social Sciences Division Historical Notes 5, 92 p.
Bailey, R.G., Avers, P.E., King, T., and McNab, W.H., eds., 1994, Ecoregions and subregions of the United States with supplementary table of map unit descriptions: Washington, D.C., U.S. Department of Agriculture Forest Service, scale 1:7,500,000(also at URL http://www.fs.fed.us/land/ecosysmgmt/ecoreg1_home.html, accessed 1998).
Hobbs, H.C., 1984, Surficial geology, in Geologic atlas of Winona County, Minnesota: Minnesota Geological Survey County Atlas Series, Atlas C-2, plate 3, scale 1:100,000.
Hobbs, H.C., 1988, Surficial geology, in Geologic atlas of Olmsted County, Minnesota: Minnesota Geological Survey County Atlas Series, Atlas C-3, plate 3, scale 1:100,000.
Lueth, R.A., 1994, Soil survey of Winona County, Minnesota: U.S. Department of Agriculture Soil Conservation Service, 278 p., 100 folded plates.
McCleary, F.E., Feusner, M.M., and Hamilton, S.J., 1989, Soil survey of Scioto County, Ohio: U.S. Department of Agriculture Soil Conservation Service, 207 p., 78 folded plates.
Mitton, G.B., Wakeman, E.S., and Guttormson, K.G., 1996, Water resources data, Minnesota, water year 1995: U.S. Geological Survey Water-Data Report MN-95-1, 389 p.
Mossler, J.H., and Book, P.R., 1984, Bedrock geology, in Geologic atlas of Winona County, Minnesota: Minnesota Geological Survey County Atlas Series, Atlas C-2, scale 1:100,000.
Rittmaster, R.L., and Shanley, J.B., 1995, Factors affecting water quality and net flux of solutes in two stream basins in the Quabbin Reservoir drainage basin, central Massachusetts, 1983-85: U.S. Geological Survey Water-Resources Investigations Report 94-4003, 66 p.
Schertz, T.L., Wells, F.C., and Ohe, D.J., 1994, Sources of trends in water-quality data for selected streams in Texas, 1975-89 water years: U.S. Geological Survey Water-Resources Investigations Report 94-4213, 49 p.
Smith, E.S., and Nemetz, D.A., 1996, Water quality along selected flowpaths in the Prairie du Chien-Jordon aquifer, southeastern Minnesota: U.S. Geological Survey Water-Resources Investigations Report 95-4115, 76 p.
Stoddard, J.L., and Murdoch, P.A., 1991, Catskill Mountains, in Charles, D.F., Acidic deposition and aquatic ecosystems-Regional case studies: New York, Springer-Verlag, p. 237-272.
U.S. Department of Agriculture, 1996, Watershed Plan and Environmental Assessment-Whitewater River Watershed, Olmsted, Wabasha, and Winona Counties, Minnesota: St. Paul, Minn., Natural Resources Conservation Service (also at URL http://www.mn.nrcs.usda.gov/watersheds/watershed.html).
Balaban, N.H., ed., 1988, Geologic atlas of Olmstead County, Minnesota: St. Paul, Minnesota Geological Survey, 9 plates containing 1. base map, 2. bedrock geology, 3. surficial geology, 4. depth to bedrock and bedrock topography, 5. sensitivity of the ground-water system to pollution, 6. sinkholes and sinkhole probability, 7. geology and well construction, 8. geology and well construction, and 9. geologic resources, scale 1:100,000.
Lueth, R.A., 1994, Soil survey of Winona County, Minnesota: U.S. Department of Agriculture Soil Conservation Service, 278 p.


FUGITIVE: Thomas Beihl [aka You Cant Hack For Crap Hahahaha !!!!] is wanted by Hope Bonding Co / Ann Marie�s Bsil Bonds as listed on the Fugitive Recovery Network's Most Wanted.
ALIASES: You Cant Hack For Crap Hahahaha !!!!
Crimes: DOR, FTA, Felony Bond Skipping and Interstate Flight to avoid prosecution . Just a wanna be computer guy, he is a real stupid person, he has admitted to being out of country, thats called "Interstate Flight to Avoid Prosecution" not to mention "Felony Bond skipping charges, the funniest part is his parents, they said plainly on the phone " haha I am not giving any information" so we are moving forward against them as well, thats our HAHAHA, its called "Aiding" a fugitive LOL!
Do not attempt to apprehend these individuals. Call the listed Agency with information.
Rewards may be available at the discretion of the Bail Bond/Apprehension company.


text:
Doctor urged psychiatric hospital for Germanwings pilot before
PARIS - Reuters
A file photo shows a French rescue worker who inspects the debris of the Germanwings Airbus A320 at the site of the crash. REUTERS Photo
A private doctor recommended that the German pilot who crashed a Germanwings jet into the Alps last year should be treated in a psychiatric hospital two weeks before the disaster, French investigators said March 13.
Prosecutors believe co-pilot Andreas Lubitz, who had a history of severe depression, barricaded himself into the cockpit and deliberately propelled his Airbus jet into a mountainside on March 24, killing all 150 people on board.
France's BEA air accident investigation office said in its final report that Lubitz had begun to show symptoms that could be consistent with a psychotic depressive episode in December 2014 and consulted several doctors over the following months, none of whom alerted aviation authorities or his employer.
Prosecutors have found evidence that the co-pilot, who also had eyesight problems and may have feared losing his job, had researched suicide methods and concealed his illness from his employer, sparking a debate on supervision and medical secrecy.
The BEA said his mental state had not generated any concerns reported by the pilots who flew with him.
However it cited a "lack of clear guidelines in German regulations" on when a threat to public safety outweighs the requirements of medical confidentiality.
Lubitz had been flying on a medical certificate that contained a waiver because of a severe depressive episode from August 2008 to July 2009. The waiver stated that the certificate would become invalid if there was a relapse into depression.
The BEA urged European authorities to carry out more research on the incapacitation of pilots, particularly where psychiatric issues are involved, and to tighten the rules for follow-up checks when pilots with a history of psychiatric problems are declared fit to fly.
UK leader's office apologizes for party before royal funeral
'Mega relocation' of Turkish Airlines to be aired on National Geographic
Family faces jail time after deadly exorcism ritual
Over 12,000 'dangerous dogs' registered across Turkey
Discovery of Turkish scientist to play key role in treatment of diabetes
Turkey eyes $35 billion in tourism revenues in 2022
UK PM fights for survival after lockdown party hangover
Biden tries to save voting rights bill in evening talks
Prince Andrew gives up military titles, patronages
Boris Johnson's office apologized to the royal family on Jan. 14 for holding staff parties in Downing Street on the eve of Prince Philip's funeral last year - the latest in a catalogue of allegedly lockdown-breaching gatherings that are threatening to topple the British prime minister.
Prices will stabilize by this summer: Minister
Rampant headline inflation rate in Turkey, which hit 36 percent last month, will not accelerate after January and should remain largely flat until a seasonal improvement in the cost of food leads to a general slowdown in the summer, Treasury and Finance Minister has said.
New coaches of Fenerbahçe, Galatasaray set for first tests
Two major Istanbul clubs, Fenerbahçe and Galatasaray, hope to put their Turkish Super Lig campaign back on track under new coaches.

redpajama_set_name: RedPajamaCommonCrawl


Gregory Kane | 6/20/2013, 10:48 p.m.
About six inches. That's how close the car came to hitting a boy at the intersection of Northern Parkway and Wabash Avenue.
I was on my way to, well, wherever the heck it was I was on my way to. I was stopped at the red light at Wabash Avenue and Northern Parkway, in the far left lane so I could make a left turn on to Northern Parkway.
There were three kids with buckets going from car to car soliciting donations. What were the donations for?
I have no idea. But everybody and his mama, at least in these parts, seems to think that soliciting donations at traffic lights'97 at some of the busiest intersections in the city and county'97 is an effective, viable fund-raising technique.
Actually, the practice is annoying and, at times, can get downright dangerous.
Two of the youngsters, a girl about 11 or 12 and a boy about 8 or 9, approached my car for a donation. I explained to them that I carried no cash.
I could have added that even if I had cash, I probably wouldn't have given it to them. I get in my car to drive from point A to point B, not to dole out cash when I'm stopped at traffic lights.
Another boy, about 10 or 11, followed the first boy and the girl. He stepped in front of my car and walked into traffic on Wabash. That's when a car coming in the lane to my immediate right missed him by about six inches.
Had the boy tried to cross Wabash only a split second earlier, you might have been reading or watching news reports of the tragic, possibly fatal accident that occurred in the city.
And it could have been avoided. All Mayor Vroom Vroom has to do is urge the city council to pass a law to make soliciting funds at busy intersections illegal.
Baltimore County Executive Kevin Kamenetz can urge the Baltimore County Council to do the same.
There are better'97 and safer'97 ways to raise funds for whatever the pet cause du jour is for this group or that one. And people need to be urged to use them.
There'92s one guy that regularly camps out in front of the M&T Bank in the 5400 block of Baltimore National Pike. One day he tried to solicit some money from me for a project his son was involved in, but, as usual, I had no cash. But the next day he was in front of the Giant supermarket on Old Court Road. I figured it must have been kismet. I made it a point to get some cash to give to the guy.
Persistence pays off. Soliciting cash from motorists stopped at red lights isn'92t persistence, it's stupidity!
When Mayor Vroom Vroom asks the City Council to pass that law banning soliciting cash from busy intersections, she can revisit that issue of what to do about dirt bike riders. Why?
About two feet. That's how close the idiot riding a dirt bike, the wrong way on a one-way street, came to my car as I was driving home last week.
Current city policy is that Baltimore police will not chase dirt-bike riding morons. It'92s a safety issue, we'92re told.
How safe is it for one of these cretins to come speeding toward my car doing a wheelie? THAT'S safe?
It most certainly is not safe. Dirt bike riders, almost by definition, are a menace to themselves and law-abiding motorists. Something needs to be done to stop them.
These idiots speed around doing their wheelies and almost daring someone to kill them. What will be the reaction of Mayor Vroom Vroom and the police department when some motorist obliges one of them?


text:
function [xx,yy] = chebpts2(nx,ny,varargin)
%CHEBPTS2 Chebyshev tensor points
%
% [XX YY] = CHEBPTS2(N) constructs an N by N grid of Chebyshev
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% [XX YY] = CHEBPTS2(NX,NY) constructs an NX by NY grid of
% Chebyshev tensor points on [-1 1]^2.
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% [XX YY] = CHEBPTS2(NX,NY,D) constructs an NX by NY grid of
% Chebyshev tensor points on the rectangle [a b] x [c d], where
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%
% See also CHEBPTS.

% Copyright 2013 by The University of Oxford and The Chebfun Developers.
% See http://www.maths.ox.ac.uk/chebfun/ for Chebfun information.

if ( nargin > 2 )  % third argument should be a domain.
   D = varargin{1};
   D = D(:).';  % make a row vector.
   if ( ~all( size(D) == [1 4] ) )
        error('CHEBFUN2:CHEBPTS2:DOMAIN','Unrecognised domain');
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else  % default to the domain in preferences.
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    D = [xdom ydom];
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if nargin == 1 % make it a square chebyshev grid.
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end

x = chebpts(nx,D(1:2)); y = chebpts(ny,D(3:4));
[xx yy] = meshgrid(x,y);   % tensor product.

end

redpajama_set_name: RedPajamaGithub


The Nobel Prize--winning novelist Orhan Pamuk reminisces on starting to be up at the banks of the mysterious Bosphorus in Istanbul.
From the ghostly yalis, excellent waterside mansions outfitted by way of the nice Ottoman households through the eighteenth and 19th centuries, to the crowds of vessels--Russian frigates, rickety fishing boats, and ferries--that plied its waters, Pamuk takes readers on a journey of the good river. a variety from the shimmering and evocative Istanbul: stories and town, "Along the Bosphorus" is the basic consultant to the city's watery approach.
Ahead of his dying in 1993, Vincent expense used to be participating with Mr. Joel Eisner (author of the over 100,000 replica bestselling reliable Batman Batbook in regards to the '60's Adam West Batman tv sequence) to build a definitive, professional biography of his lifestyles and occupation in motion pictures. this can be that booklet.
The 1st quantity of a magisterial biography: the definitive portrait of the existence and paintings of 1 of the main abidingly influential--and controversial--men in sleek heritage.
Here is a revelatory paintings of biography that takes us from Gandhi's start in 1869 via his upbringing in Gujarat, his 2 years as a scholar in London, and his 2 a long time as a attorney and group organizer in South Africa. Ramachandra Guha has exposed a myriad of formerly untapped records, together with: inner most papers of Gandhi's contemporaries and associates; modern newspapers and court docket files; the writings of Gandhi's young ones; mystery records stored by way of British Empire functionaries. utilizing this wealth of fabric in a brilliantly nuanced narrative, Guha describes the social, political and private worlds within which Gandhi started his trip to develop into the fashionable era's most vital and influential political actor. And Guha makes transparent that Gandhi's paintings in South Africa--far from being a trifling prelude to his accomplishments in India--was profoundly influential on his evolution as a political philosopher, social reformer and cherished chief.
A clean examine this astute, likably quirky statesman, through the writer of the Pulitzer Award-winning Founding Brothers and the nationwide publication Award profitable American Sphinx. "The most lovely and such a lot laughable, the warmest and probably the wisest of the founding fathers, John Adams knew himself as few males do and preserved his wisdom in a voluminous correspondence that also resonates.
McGarr is the executive Inspector of Detectives, a place created to entice him clear of Interpol. sooner than that McGarr have been with legal Justice in Paris and is a bit of respected in his domestic of eire. whilst a very younger and gorgeous Irish lass named may well Quirk is located murdered with a pitchfork alongside the roadside McGarr tackles a case that may contain the I. R. A. and lead him the entire strategy to big apple and again to capture a killer.
Gill writes a fancy secret jam-packed with surroundings for a stunning kingdom the place a few tawdry issues occur. He indicates the sympathetic nature of so much in eire for the I. R. A. although their equipment are frowned upon. it sounds as if may possibly Quirk could in simple terms have lower back to her native land to reveal the monetary pipeline of the I. R. A. for her paper in New York.
McGarr is a fleshed out personality with a spouse, Noreen, 21 years more youthful than he, and a love for tremendous nutrients and eire. As he attempts to unravel might Quirk's demise an explosive try and take out McGarr and a excessive rating I. R. A. member will supply him a steely get to the bottom of to determine even if love or politics used to be on the backside of her death.
Gill subtly makes us believe as if we actually are in eire all through this whole secret. no matter if describing the beautiful Irish nation-state or coast, Gill completely captures the attraction of a rustic the place a pleasant Irish pub is a social membership whereas whilst displaying political division.
This is an efficient sequence with finely drawn characters, nice surroundings and barely enough enjoyable jumbled together to make this an excellent learn. Open up this McGarr secret and revel in a few nice nutrients in scenic eire.
On the other hand, I thought war an insanity . D. was in the country in October, living in a cottage lent by Ford (quite possibly the one with the 'leaking thatch' and 'creakin g latch' Egoists, 1914 37 mentioned in Part X of Pound's 'Hugh Selwyn Mauberly'). Aldington joined her there at week-ends. Lewis, busy decorating Ford's study at South Lodge, or painting the drawing-room bright red and converting it into a Vorticist salon,45 invited the Aldingtons to visit him. Using his own work for Ford as excuse Aldington declined, adding: Apropos, if you are publishing a manifesto in the new number of Blast I would be glad if you would exclude my name-that is supposing you thought of including it.
In one such 'bun shop', in 'the Royal Borough of Kensington' around April 19125 occurred the now near-legendary meeting which marks the official founding of the 'Imagist movement'. At Pound's instigation, the three agreed on the principles of good writing recorded in 'A Retrospect': In the spring or early summer of 1912, ' H. ', Richard Aldington and myself decided that we were agreed upon the three principles following: 1. Direct treatment of the ' thing' whether subjective or objective. 2. To use absolutely no word that does not contribute to the presentation.
D. need hardly surprise us. D. remembers, 'we went soon after to Hampstead to a larger flat that a friend had found US,. 29 After that they saw far less of what she calls 'the Kensington group', meaning chiefly the Pounds and Ford. Perhaps as part of the 'dynamism in the air' John Coumos recollected of that 'hectic summer', July 1914 seems to have been propitious for artists' dinner gatherings. Yet another took place on 30 July, in Lowell's suite at the Berkeley. Guest of honour, seated next to the hostess and that evening won over to be at least a fellow-traveller of the Imagists, was D.


This adorable monogrammed seersucker dress will claim your sweet baby a spot in the next preppy fashion show! The attention to detail extends down the entire back side with a complete row of buttons. The dainty ruffle trim at the bottom completes the look.


text:
Instant grades and reactions: Portland Trail Blazers confuse everybody by trading for Hassan Whiteside
By Zach Harper Jul 1, 2019 47
The Deal: Portland sends Mo Harkless to the LA Clippers and Meyers Leonard to the Miami Heat in exchange for Hassan Whiteside, according to Adrian Wojnarowski of ESPN.
The Portland Trail Blazers are certainly scratching some heads with their offseason, not even 24 hours for free agency. After letting Al-Farouq Aminu go to Orlando on a very manageable three-year, $29 million deal (assuming Aminu wanted to stay in Portland), the Blazers kept Rodney Hood on a two-year, $16 million contract. That part isn't so confusing. However, it's a bit of a head-scratcher to believe in Hood for two years rather than the ultra-important Aminu for three years at virtually the same money. But maybe Aminu wasn't going to stay for the same contract number in Portland. Maybe he felt the Blazers should value him more than that.
These things happen when it comes to negotiating and players bouncing to new teams for what seems like justifiable money for the previous...


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Vivaldi is now the default browser on Manjaro Linux Cinnamon
The Arch-based Linux distribution Manjaro adds Vivaldi as the default browser in its most popular Community Edition, Cinnamon.
Published on September 9, 2021 – By Team Vivaldi
OSLO, Norway – 09th Sept 2021: The Vivaldi browser is now the default browser on the Arch-based Linux distribution Manjaro on its Cinnamon edition, offering users a fast, reliable, and feature-rich browsing experience.
Cinnamon, one of the most popular Manjaro editions, is an established desktop environment for Linux that works very well with Vivaldi's functionality and design philosophy.
Vivaldi – available on Windows, Mac, and Linux computers, as well as on Android devices – gives users the freedom and flexibility to browse exactly the way they want, keeping their privacy first.
Vivaldi is readily available in most Linux distributions including Linux-based ARM devices, including the Raspberry Pi, and is simple and quick to install.
In our repos, Manjaro always provides the very latest version of Vivaldi, and thanks to direct developer contact we are now also able to include matching default themes for our editions," Co-CEO of Manjaro GmbH & Co. KG, Bernhard Landauer, says.
"To give Vivaldi more of the attention it deserves, I decided to include it as the default browser in our popular Cinnamon Community Edition. With its remarkable browsing speed, exceptional customizability, and especially the way it values user privacy, Vivaldi for me is a perfect match for Manjaro Linux."
Customization and out-of-the-box features for Manjaro users
Theme: Manjaro-Cinnamon
Linux users love to tweak and tune the software to create their own unique experience – and search for just the right software. While most browsers let users tweak only the most basic functionality, that's certainly not the case with Vivaldi. Exploring the breadth and depth of its settings is just what Vivaldi encourages its users to do.
The Vivaldi settings give users a fine-grained control not found anywhere else. With its one-of-a-kind, comprehensive Tab Management, Vivaldi frees users from their tab woes.
Unique workflows can be created with customizable shortcuts, gestures, customizable menus, and Command Chains – especially suited to keyboard-based browsing. Browsing is fast with fully configurable search options and a powerful Bookmark Manager. The UI elements in the browser such as Toolbar, Status bar, Bookmark bar, and Address bar are easily configurable.
With endless design options and predefined and custom Themes, now also including a 'Manjaro-Cinnamon' theme, users can even set the UI to adapt to the colors of the websites they visit.
We are extremely delighted that Manjaro has shown belief and trust in Vivaldi. Similar to Manjaro, Vivaldi has an incredible community of dedicated Linux users who have shown immense faith and support in what we do," Vivaldi CEO, Jon von Tetzchner, says.
In our experience, people engage and even volunteer when the values of a company are aligned with their own. We hope Manjaro users will enjoy browsing with Vivaldi."
Privacy and Security are paramount
Vivaldi's Start Page
Since both Vivaldi and Manjaro appeal to users who value privacy, bringing the two together will create an elegant desktop experience with the browser tightly integrated. Vivaldi believes that everything that a user does in the browser is no one else's business — which is why Vivaldi never tracks its users, collects their data, or willingly compromises on privacy.
Vivaldi blocks privacy-invasive technologies, keeps browsing data safe with its end-to-end encrypted Sync, and helps to limit unnecessary tracking and block annoying ads with its built-in Ad and Tracker Blocker, on both Android and desktop versions.
The beta versions of Vivaldi Mail with Vivaldi Feed Reader and Vivaldi Calendar on desktop help break away from the locked ecosystems and creepy, surveillance-driven practices of huge corporations.
Continuously updated, Vivaldi is built using Chromium but this is where the similarities with Chrome end. Vivaldi is completely transparent about where its revenue comes from.
Another nice thing, as well, is that Vivaldi treats us Linux users as first-class citizens, right along with all the other users from other operating systems. There's no, "Well, it will come to Linux sometime in the future…maybe." 😃
— Chad McCullough (@chadmccullough) September 2, 2021
Vivaldi Browser on Manjaro Linux
Tightly integrated into the Manjaro Cinnamon desktop, Vivaldi's look and feel have been adapted and developed by Manjaro, making sure they have a browser that fits the desktop and can be trusted by Manjaro's users and community.
Working closely together, Vivaldi and Manjaro are keeping a watchful eye for any issue or request with regards to the browser and worked on by the Vivaldi development team. Manjaro also has access to Vivaldi's advanced configurations so that users get the full Vivaldi experience and speed, right there on their Linux desktop.
How do you get Vivaldi on Manjaro Linux?
Access the internet with Vivaldi Browser
While Vivaldi will now be found as the default browser on any fresh install of the Cinnamon Edition, it is also available in Manjaro's official repos and installation through the package manager just a click away for all users.
About Manjaro
Manjaro, one of the most popular Linux distributions, is based on Archlinux – a fast and customizable rolling-release system. Manjaro has introduced a layer of stability by handling package updates with a process of evaluation and bug fixing through three branches with dedicated tools that greatly simplify installation, hardware, and package management, updates and backups to the point where everyday life with this Linux distribution is now at a level of ease comparable to Windows and Mac.
Offering a variety of install media with pre-configured desktops, Xfce, Gnome, and KDE-Plasma being the flagship editions, Manjaro prides itself on supporting both older and even the newest hardware with the latest available kernels and drivers.
Most recently full support for the btrfs filesystem, including automatic system snapshots and rollbacks with 'timeshift' have been introduced in the 21.1 'Pahvo' release.
Established in 2019, Manjaro GmbH & Co KG offers professional services for tailor-made and optimized desktop and software solutions for specific hardware and commercial needs as well as consulting in the field. Development and support for ARM-based hardware is an important focus, namely close cooperation with Pine64 for their Pinebooks and the PinePhone, which now ship Manjaro as their stock OS.
More details here.