With very little direct biological data of HIV-1 from before the 1980s, far-reaching evolutionary and epidemiological inferences regarding the long prediscovery phase of this pandemic are based on ...extrapolations by phylodynamic models of HIV-1 genomic sequences gathered mostly over recent decades. Here, using a very sensitive multiplex RT-PCR assay, we screened 1,645 formalin-fixed paraffin-embedded tissue specimens collected for pathology diagnostics in Central Africa between 1958 and 1966. We report the near-complete viral genome in one HIV-1 positive specimen from Kinshasa, Democratic Republic of Congo (DRC), from1966 (“DRC66”)— a nonrecombinant sister lineage to subtype C that constitutes the oldest HIV-1 near full-length genome recovered to date. Root-to-tip plots showed the DRC66 sequence is not an outlier as would be expected if dating estimates from more recent genomes were systematically biased; and inclusion of the DRC66 sequence in tip-dated BEAST analyses did not significantly alter root and internal node age estimates based on post-1978 HIV-1 sequences. There was larger variation in divergence time estimates among datasets that were subsamples of the available HIV-1 genomes from 1978 to 2014, showing the inherent phylogenetic stochasticity across subsets of the real HIV-1 diversity. Our phylogenetic analyses date the origin of the pandemic lineage of HIV-1 to a time period around the turn of the 20th century (1881 to 1918). In conclusion, this unique archival HIV-1 sequence provides direct genomic insight into HIV-1 in 1960s DRC, and, as an ancient-DNA calibrator, it validates our understanding of HIV-1 evolutionary history.
The emergence of HIV-1 group M subtype B in North American men who have sex with men was a key turning point in the HIV/AIDS pandemic. Phylogenetic studies have suggested cryptic subtype B ...circulation in the United States (US) throughout the 1970s and an even older presence in the Caribbean. However, these temporal and geographical inferences, based upon partial HIV-1 genomes that postdate the recognition of AIDS in 1981, remain contentious and the earliest movements of the virus within the US are unknown. We serologically screened >2,000 1970s serum samples and developed a highly sensitive approach for recovering viral RNA from degraded archival samples. Here, we report eight coding-complete genomes from US serum samples from 1978-1979-eight of the nine oldest HIV-1 group M genomes to date. This early, full-genome 'snapshot' reveals that the US HIV-1 epidemic exhibited extensive genetic diversity in the 1970s but also provides strong evidence for its emergence from a pre-existing Caribbean epidemic. Bayesian phylogenetic analyses estimate the jump to the US at around 1970 and place the ancestral US virus in New York City with 0.99 posterior probability support, strongly suggesting this was the crucial hub of early US HIV/AIDS diversification. Logistic growth coalescent models reveal epidemic doubling times of 0.86 and 1.12 years for the US and Caribbean, respectively, suggesting rapid early expansion in each location. Comparisons with more recent data reveal many of these insights to be unattainable without archival, full-genome sequences. We also recovered the HIV-1 genome from the individual known as 'Patient 0' (ref. 5) and found neither biological nor historical evidence that he was the primary case in the US or for subtype B as a whole. We discuss the genesis and persistence of this belief in the light of these evolutionary insights.
Point mutations in the amyloid-β (Aβ) coding region produce a combination of mutant and WT Aβ isoforms that yield unique clinicopathologies in familial Alzheimer’s disease (fAD) and cerebral amyloid ...angiopathy (fCAA) patients. Here, we report a method to investigate the structural variability of amyloid deposits found in fAD, fCAA, and sporadic AD (sAD). Using this approach, we demonstrate that mutant Aβ determines WT Aβ conformation through prion template-directed misfolding. Using principal component analysis of multiple structure-sensitive fluorescent amyloid-binding dyes, we assessed the conformational variability of Aβ deposits in fAD, fCAA, and sAD patients. Comparing many deposits from a given patient with the overall population, we found that intrapatient variability is much lower than interpatient variability for both disease types. In a given brain, we observed one or two structurally distinct forms. When two forms coexist, they segregate between the parenchyma and cerebrovasculature, particularly in fAD patients. Compared with sAD samples, deposits from fAD patients show less intersubject variability, and little overlap exists between fAD and sAD deposits. Finally, we examined whether E22G (Arctic) or E22Q (Dutch) mutants direct the misfolding of WT Aβ, leading to fAD-like plaques in vivo. Intracerebrally injecting mutant Aβ40 fibrils into transgenic mice expressing only WT Aβ induced the deposition of plaques with many biochemical hallmarks of fAD. Thus, mutant Aβ40 prions induce a conformation of WT Aβ similar to that found in fAD deposits. These findings indicate that diverse AD phenotypes likely arise from one or more initial Aβ prion conformations, which kinetically dominate the spread of prions in the brain.
The spore-forming, anaerobic Gram positive pathogen Clostridium perfringens encodes many of its disease-causing toxins on closely related conjugative plasmids. Studies of the tetracycline resistance ...plasmid pCW3 have identified many of the genes involved in conjugative transfer, which are located in the tcp conjugation locus. Upstream of this locus is an uncharacterised region (the cnaC region) that is highly conserved. This study examined the importance in pCW3 conjugation of several highly conserved proteins encoded in the cnaC region. Conjugative mating studies suggested that the SrtD, TcpN and Dam proteins were required for efficient pCW3 transfer between C. perfringens cells from the same strain background. The requirement of these proteins for conjugation was amplified in matings between C. perfringens cells of different strain backgrounds. Additionally, the putative collagen adhesin protein, CnaC, was only required for the optimal transfer of pCW3 between cells of different strain backgrounds. Based on these studies we postulate that CnaC, SrtD, TcpN and Dam are involved in enhancing the transfer frequency of pCW3. These studies have led to a significant expansion of the tcp conjugation locus, which now encompasses a 19 kb region.
•The cnaC region of pCW3 is conserved in Tcp family plasmids.•The CnaC protein was shown to be required for optimal interstrain transfer of pCW3.•Genetic studies provide evidence that the SrtD, TcpN and Dam proteins are required for both intrastrain and interstrain conjugative transfer.
Plasmids that encode the same replication machinery are generally unable to coexist in the same bacterial cell. However, Clostridium perfringens strains often carry multiple conjugative toxin or ...antibiotic resistance plasmids that are closely related and encode similar Rep proteins. In many bacteria, plasmid partitioning upon cell division involves a ParMRC system; in C. perfringens plasmids, there are approximately 10 different ParMRC families, with significant differences in amino acid sequences between each ParM family (15% to 54% identity). Since plasmids carrying genes belonging to the same ParMRC family are not observed in the same strain, these families appear to represent the basis for plasmid compatibility in C. perfringens. To understand this process, we examined the key recognition steps between ParR DNA-binding proteins and their parC binding sites. The ParR proteins bound to sequences within a parC site from the same ParMRC family but could not interact with a parC site from a different ParMRC family. These data provide evidence that compatibility of the conjugative toxin plasmids of C. perfringens is mediated by their parMRC-like partitioning systems. This process provides a selective advantage by enabling the host bacterium to maintain separate plasmids that encode toxins that are specific for different host targets. IMPORTANCE Toxins produced by the Gram-positive pathogen Clostridium perfringens are primarily encoded by genes found on different conjugative plasmids. These plasmids encode highly similar replication proteins and therefore should be incompatible, but they are often found to coexist within the same isolate. In this study, we showed that a series of phylogenetically related ParMRC plasmid partitioning systems, structures that are normally responsible for ensuring that plasmids segregate correctly at cell division, dictate which toxin plasmid combinations can coexist within the same bacterial cell. We dissected the recognition steps between the DNA-binding ParMRC component, ParR, and the plasmid-derived centromere, parC. Our data suggested a mechanism by which plasmids encoding ParMRC systems from the same family are incompatible, whereas plasmids encoding ParMRC systems from distinct families are compatible. This work provides insight into how these cells can maintain multiple highly similar toxin plasmids, which is a critical first step in understanding how to limit the disease-causing potential of C. perfringens.
In December of 2019, a novel coronavirus, SARS-CoV-2, emerged in the city of Wuhan, China, causing severe morbidity and mortality. Since then, the virus has swept across the globe, causing millions ...of confirmed infections and hundreds of thousands of deaths. To better understand the nature of the pandemic and the introduction and spread of the virus in Arizona, we sequenced viral genomes from clinical samples tested at the TGen North Clinical Laboratory, the Arizona Department of Health Services, and those collected as part of community surveillance projects at Arizona State University and the University of Arizona. Phylogenetic analysis of 84 genomes from across Arizona revealed a minimum of 11 distinct introductions inferred to have occurred during February and March. We show that >80% of our sequences descend from strains that were initially circulating widely in Europe but have since dominated the outbreak in the United States. In addition, we show that the first reported case of community transmission in Arizona descended from the Washington state outbreak that was discovered in late February. Notably, none of the observed transmission clusters are epidemiologically linked to the original travel-related case in the state, suggesting successful early isolation and quarantine. Finally, we use molecular clock analyses to demonstrate a lack of identifiable, widespread cryptic transmission in Arizona prior to the middle of February 2020.
As the COVID-19 pandemic swept across the United States, there was great differential impact on local and regional communities. One of the earliest and hardest hit regions was in New York, while at the same time Arizona (for example) had low incidence. That situation has changed dramatically, with Arizona now having the highest rate of disease increase in the country. Understanding the roots of the pandemic during the initial months is essential as the pandemic continues and reaches new heights. Genomic analysis and phylogenetic modeling of SARS-COV-2 in Arizona can help to reconstruct population composition and predict the earliest undetected introductions. This foundational work represents the basis for future analysis and understanding as the pandemic continues.
Abstract
Background
Plasmodium malariae is considered a minor malaria parasite, although its global disease burden is underappreciated. The aim of this study was to develop an induced blood-stage ...malaria (IBSM) model of P. malariae to study parasite biology, diagnostic assays, and treatment.
Methods
This clinical trial involved 2 healthy subjects who were intravenously inoculated with cryopreserved P. malariae–infected erythrocytes. Subjects were treated with artemether-lumefantrine after development of clinical symptoms. Prior to antimalarial therapy, mosquito-feeding assays were performed to investigate transmission, and blood samples were collected for rapid diagnostic testing and parasite transcription profiling. Serial blood samples were collected for biomarker analysis.
Results
Both subjects experienced symptoms and signs typical of early malaria. Parasitemia was detected 7 days after inoculation, and parasite concentrations increased until antimalarial treatment was initiated 25 and 21 days after inoculation for subjects 1 and 2 respectively (peak parasitemia levels, 174 182 and 50 291 parasites/mL, respectively). The parasite clearance half-life following artemether-lumefantrine treatment was 6.7 hours. Mosquito transmission was observed for 1 subject, while in vivo parasite transcription and biomarkers were successfully profiled.
Conclusions
An IBSM model of P. malariae has been successfully developed and may be used to study the biology of, diagnostic testing for, and treatment of this neglected malaria species.
Clinical Trials Registration
ACTRN12617000048381.
A human model of Plasmodium malariae infection has been successfully developed and may be used to study the biology of, diagnostic testing for, and treatment of this neglected malaria species.
Conjugative transfer is a major contributor to the dissemination of antibiotic resistance and virulence genes in the human and animal pathogen, Clostridium perfringens. The C. perfringens plasmid ...pCW3 is the archetype of an extensive family of highly related conjugative toxin and antibiotic resistance plasmids found in this bacterium. These plasmids were thought to constitute the only conjugative plasmid family in C. perfringens. Recently, another series of C. perfringens plasmids, the pCP13-like family, have been shown to harbour important toxin genes, including genes that encode the novel binary clostridial enterotoxin, BEC. Based on early bioinformatics analysis this plasmid family was thought to be non-conjugative. Here we demonstrate that pCP13 is in fact conjugative, transfers at high frequency and that the newly defined Pcp conjugation locus encodes putative homologues of a type 4 secretion system (T4SS), one of which, PcpB4, was shown to be essential for transfer. The T4SS of pCP13 also appears to be evolutionarily related to conjugative toxin plasmids from other clostridia-like species, including Paeniclostridium (formerly Clostridium) sordellii, Clostridioides (formerly Clostridium) difficile and Clostridium botulinum. Therefore, it is clear that there are two distinct families of conjugative plasmids in C. perfringens: the pCW3 family and the pCP13 family. This study has significant implications for our understanding of the movement of toxin genes both within C. perfringens, but also potentially to other pathogenic clostridia.
•Members of the pCW3 family were thought to be the only conjugative plasmids in C. perfringens.•We demonstrate that pCP13 represents a new family of conjugative plasmids in this important human and animal pathogen.•The pCP13 conjugation locus represents an evolutionarily conserved transfer region within the clostridial genus.•This study has implications for the dissemination of toxins including the clinically relevant BEC enterotoxin.
Peripheral nerve blockade is associated with excellent patient outcomes after surgery; however, neurologic and other complications can be devastating for the patient. This article reports the ...development and preliminary results of a multicenter audit describing the quality and safety of peripheral nerve blockade.
From January 2006 to May 2008, patients who received peripheral nerve blockade had data relating to efficacy and complications entered into databases. All patients who received nerve blocks performed by all anesthetists during each hospital's contributing period were included. Patients were followed up by phone to detect potential neurologic complications. The timing of follow-up was either at 7 to 10 days or 6 weeks postoperatively, depending on practice location and time period. Late neurologic deficits were defined as a new onset of sensory and/or motor deficit consistent with a nerve/plexus distribution without other identifiable cause, and one of the following: electrophysiologic evidence of nerve damage, new neurologic signs, new onset of neuropathic pain in a nerve distribution area, paresthesia in relevant nerve/plexus distribution area.
A total of 6950 patients received 8189 peripheral nerve or plexus blocks. Of the 6950 patients, 6069 patients were successfully followed up. In these 6069 patients, there were a total of 7156 blocks forming the denominator for late neurologic complications. Thirty patients (0.5%) had clinical features requiring referral for neurologic assessment. Three of the 30 patients had a block-related nerve injury, giving an incidence of 0.4 per 1000 blocks (95% confidence interval, 0.08-1.1:1000). The incidence of systemic local anesthetic toxicity was 0.98 per 1000 blocks (95% confidence interval, 0.42-1.9:1000).
These results indicate that the incidence of serious complications after peripheral nerve blockade is uncommon and that the origin of neurologic symptoms/signs in the postoperative period is most likely to be unrelated to nerve blockade.
1. The extent to which variability in desiccation resistance among ecologically diverse Drosophila species is related to their ability to resist starvation is unknown. Resistance to desiccation and ...starvation was measured in females and males of ecologically and phylogenetically diverse Drosophila species. 2. We measured resistance to both stressors in ecologically and phylogenetically diverse species. In general females exhibited greater resistance to both stressors than males. Correcting for body size produces a highly significant correlation between resistances to both stressors in both sexes. 3. Phylogenetic relatedness, however, appears to have a large influence not only on resistance to both stressors, but also on the observed correlations between stressors. 4. Species of the Drosophila subgenus Sophophora examined in this study tend to be fruit breeders inhabiting more temperate and mesic habitats, whereas many of the species in the other major subgenus, Drosophila, tend to be cactophilic flies living in more xeric environments. 5. The difference between these two major subgenera, the Sophophora and the Drosophila, in the nature of the association we observed between desiccation and starvation resistance suggests that selection may have led to different mechanisms underlying resistance to these stressors in the two groups.
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