Reply to Jensen and Kowalik: Consideration of mixed infections is central to understanding HCMV intrahost diversity.

Kowalik and Jensen (1) have reported that intra-host variation in HCMV approaches levels similar to those of HCV, with fast mutation rates mooted as one explanation (2).While we discussed that HCMV mutation rates were postulated as an explanation for high diversity, the focus of our work is on observed inconsistencies in nucleotide diversity between and within patients (3). Kowalik and Jensen did calculate HCMV mutation rates to be similar to MCMV but maintained that this could underestimate the true levels (2). In contrast, our study showed that in the absence of mixed infections, HCMV is no more diverse than other DNA viruses, and considerably less so than chronic RNA viruses. This simple conclusion is different to that of Kowalik and Jensen and had not been stated prior to our publication.N

Mortuary Truck

Final report and team photo for Project 20 of ME450, Winter 2009 semester.At the Komfo Anokye Teaching Hospital (KATH) in Kumasi, Ghana, locally made mortuary trucks are used to transport deceased bodies from the crowded hospital rooms to morgues that are in separate buildings. Multiple bodies are usually transported during a single trip to the morgue. Currently the trucks in use are difficult to maneuver, difficult to operate, and create sanitation issues. We have designed and fabricated a new truck that is inexpensive, easy to maneuver, controls bodily fluids, conceals bodies from public view, and performs well in an indoor and outdoor environment with minimal maintenance.Kathleen Sienko (Mechanical Engineering, U of M), Multidisciplinary Design Minor Specialization in Global Health Design (U of M); Mentor: George Fusanihttp://deepblue.lib.umich.edu/bitstream/2027.42/62474/2/ME450 Winter2009 Team Photo - Project 20 - Mortuary Truck.JPGhttp://deepblue.lib.umich.edu/bitstream/2027.42/62474/1/ME450 Winter2009 Final Report - Project 20 - Mortuary Truck.pd

Mixed cytomegalovirus genotypes in HIV-positive mothers show compartmentalization and distinct patterns of transmission to infants.

Cytomegalovirus (CMV) is the commonest cause of congenital infection and particularly so among infants born to HIV-infected women. Studies of congenital CMV infection (cCMVi) pathogenesis are complicated by the presence of multiple infecting maternal CMV strains, especially in HIV-positive women, and the large, recombinant CMV genome. Using newly developed tools to reconstruct CMV haplotypes, we demonstrate anatomic CMV compartmentalization in five HIV-infected mothers and identify the possibility of congenitally transmitted genotypes in three of their infants. A single CMV strain was transmitted in each congenitally infected case, and all were closely related to those that predominate in the cognate maternal cervix. Compared to non-transmitted strains, these congenitally transmitted CMV strains showed statistically significant similarities in 19 genes associated with tissue tropism and immunomodulation. In all infants, incident superinfections with distinct strains from breast milk were captured during follow-up. The results represent potentially important new insights into the virologic determinants of early CMV infection

A comprehensive characterization of chronic norovirus infection in immunodeficient hosts.

Letter to the edito

High Viral Diversity and Mixed Infections in Cerebral Spinal Fluid From Cases of Varicella Zoster Virus Encephalitis.

Background Varicella zoster virus (VZV) may cause encephalitis, both with and without rash. Here we investigate whether viruses recovered from the central nervous system (CNS; encephalitis or meningitis) differ genetically from those recovered from non-CNS samples. Methods Enrichment-based deep sequencing of 45 VZV genomes from cerebral spinal fluid (CSF), plasma, bronchoalveolar lavage (BAL), and vesicles was carried out with samples collected from 34 patients with and without VZV infection of the CNS. Results Viral sequences from multiple sites in the same patient were identical at the consensus level. Virus from vesicle fluid and CSF in cases of meningitis showed low-level diversity. By contrast, plasma, BAL, and encephalitis had higher numbers of variant alleles. Two CSF-encephalitis samples had high genetic diversity, with variant frequency patterns typical of mixed infections with different clades. Conclusions Low viral genetic diversity in vesicle fluid is compatible with previous observations that VZV skin lesions arise from single or low numbers of virions. A similar result was observed in VZV from cases of VZV meningitis, a generally self-limiting infection. CSF from cases of encephalitis had higher diversity with evidence for mixed clade infections in 2 cases. We hypothesize that reactivation from multiple neurons may contribute to the pathogenesis of VZV encephalitis

Use of Whole-genome Sequencing of Adenovirus in Immunocompromised Paediatric Patients to Identify Nosocomial Transmission and Mixed-genotype Infection

Background: Adenoviruses are significant pathogens for the immunocompromised, arising from primary infection or reinfection. Serotyping is insufficient to support nosocomial transmission investigations. We investigate whether whole-genome sequencing (WGS) provides clinically relevant information on transmission among patients in a paediatric tertiary hospital. Methods: We developed a target-enriched adenovirus WGS technique for clinical samples and retrospectively sequenced 107 adenovirus-positive residual diagnostic samples, including viraemias (>5x104 copies/ml), from 37 patients collected January 2011 - March 2016. WGS was used to determine genotype and for phylogenetic analysis. Results: Adenovirus sequences were recovered from 105/107 samples. Full genome sequences were recovered from all 20 non-species C samples and from 36/85 species C viruses, with partial genome sequences recovered from the rest. Whole genome phylogenetic analysis suggested linkage of three genotype A31 cases and uncovered an unsuspected epidemiological link to an A31 infection first detected on the same ward four years earlier. In nine samples from one patient who died we identified a mixed genotype adenovirus infection. Conclusions: Adenovirus WGS from clinical samples is possible and useful for genotyping and molecular epidemiology. WGS identified likely nosocomial transmission with greater resolution than conventional genotyping, and distinguished between adenovirus disease due to single or multiple genotypes

Mixed cytomegalovirus genotypes in HIV positive mothers show compartmentalization and distinct patterns of transmission to infants

Cytomegalovirus (CMV) is the commonest cause of congenital infection (cCMVi) and particularly so among infants born to HIV-infected women. Studies of cCMVi pathogenesis are complicated by the presence of multiple infecting maternal CMV strains, especially in HIV-positive women, and the large, recombinant CMV genome. Using newly developed tools to reconstruct CMV haplotypes, we demonstrate anatomic CMV compartmentalization in five HIV-infected mothers and identify the possibility of congenitally transmitted genotypes in three of their infants. A single CMV strain was transmitted in each congenitally infected case, and all were closely related to those that predominate in the cognate maternal cervix. Compared to non-transmitted strains, these congenitally transmitted CMV strains showed statistically significant similarities in 19 genes associated with tissue-tropism and immunomodulation. In all infants, incident superinfections with distinct strains from breast milk were captured during follow-up. The results represent potentially important new insights into the virologic determinants of early CMV infection

High Viral Diversity and Mixed Infections in Cerebral Spinal Fluid From Cases of Varicella Zoster Virus Encephalitis.

BACKGROUND: Varicella zoster virus (VZV) may cause encephalitis, both with and without rash. Here we investigate whether viruses recovered from the central nervous system (CNS; encephalitis or meningitis) differ genetically from those recovered from non-CNS samples. METHODS: Enrichment-based deep sequencing of 45 VZV genomes from cerebral spinal fluid (CSF), plasma, bronchoalveolar lavage (BAL), and vesicles was carried out with samples collected from 34 patients with and without VZV infection of the CNS. RESULTS: Viral sequences from multiple sites in the same patient were identical at the consensus level. Virus from vesicle fluid and CSF in cases of meningitis showed low-level diversity. By contrast, plasma, BAL, and encephalitis had higher numbers of variant alleles. Two CSF-encephalitis samples had high genetic diversity, with variant frequency patterns typical of mixed infections with different clades. CONCLUSIONS: Low viral genetic diversity in vesicle fluid is compatible with previous observations that VZV skin lesions arise from single or low numbers of virions. A similar result was observed in VZV from cases of VZV meningitis, a generally self-limiting infection. CSF from cases of encephalitis had higher diversity with evidence for mixed clade infections in 2 cases. We hypothesize that reactivation from multiple neurons may contribute to the pathogenesis of VZV encephalitis.Action Medical research GN2424 This work was supported by a UK MRC New Investigator Award to D. P. D; UCL/UCLH BRC (J. B.); Action Medical Research (grant number GN2424 to C. J. H); Swedish Research Council (P. N. and T. B.). The work was also support by an NIHR Fellowship (grant number DRF-2013-06-168 to F. M.), the Meningitis Research Foundation (grant number 0904.0), an NIHR Programme Grant in Applied Research (grant number RP-PG-0108-10048 to T. S.), and the NIHR Health Protection Research Unit in Emerging and Zoonotic Infections, University of Liverpool

Human cytomegalovirus haplotype reconstruction reveals high diversity due to superinfection and evidence of within-host recombination

Recent sequencing efforts have led to estimates of human cytomegalovirus (HCMV) genome-wide intrahost diversity that rival those of persistent RNA viruses [Renzette N, Bhattacharjee B, Jensen JD, Gibson L, Kowalik TF (2011) PLoS Pathog 7:e1001344]. Here, we deep sequence HCMV genomes recovered from single and longitudinally collected blood samples from immunocompromised children to show that the observations of high within-host HCMV nucleotide diversity are explained by the frequent occurrence of mixed infections caused by genetically distant strains. To confirm this finding, we reconstructed within-host viral haplotypes from short-read sequence data. We verify that within-host HCMV nucleotide diversity in unmixed infections is no greater than that of other DNA viruses analyzed by the same sequencing and bioinformatic methods and considerably less than that of human immunodeficiency and hepatitis C viruses. By resolving individual viral haplotypes within patients, we reconstruct the timing, likely origins, and natural history of superinfecting strains. We uncover evidence for within-host recombination between genetically distinct HCMV strains, observing the loss of the parental virus containing the nonrecombinant fragment. The data suggest selection for strains containing the recombinant fragment, generating testable hypotheses about HCMV evolution and pathogenesis. These results highlight that high HCMV diversity present in some samples is caused by coinfection with multiple distinct strains and provide reassurance that within the host diversity for single-strain HCMV infections is no greater than for other herpesviruses