Study of P. falciparum-infected erythrocytes and induced anisotropies under optical and fluid forces

Background & objectives: The effect of P. falciparum on erythrocytes has been studied for a long time at the population level but actual studies at the single cell level remain largely unexplored. The aim of this study was to address the host-parasite relationship at the single cell level under two different kinds of forces, an optical force and a fluid force. The questions addressed were about the basic host-parasite interactions, but our findings have larger implications in diverse fields of parasite biology. Methods: Erythrocytes were monitored under optical forces (using optical tweezers) and fluid forces (using microfluidic chambers) and dynamical images were captured in real-time video clips. These videos were then split into their respective frames so as to yield temporal information and various parameters pertaining to membrane structure, ionic imbalance and interaction with different forces were studied. Results: The results of this study mainly bring to fore the inherent differences between infected and normal cell populations at the single cell level under various external forces. We probed three different criteria folding times, rotation speeds and rolling frequency to show inherent difference in various cell populations and also the dependence of the above to the cycle of the parasite. Interpretation & conclusion: This study portrays the importance of single cell observations pertaining to the host-parasite relationship. It shows the effect the malarial parasite has on erythrocytes and how the intrinsic property of the infected and its neighbouring uninfected cells change as compared to normal erythrocytes. There are thus implications in the fields of cytoadherence, parasite invasions and host immune evasion

Cluster of Symptomatic Graft-to-Host Transmission of Herpes Simplex Virus Type 1 in an Endothelial Keratoplasty Setting

PURPOSE: Descemet's membrane endothelial keratoplasty (DMEK) is becoming the gold standard to treat corneal endothelial dysfunctions worldwide. Compared with conventional penetrating keratoplasty, infectious complications after DMEK are ill defined. We describe the clinical picture of 2 DMEK recipients, operated on the same day and in the same clinic, who developed atypical herpes simplex virus type 1 (HSV-1) infection in the transplant recipient eye within days post-DMEK. Because recipients received cornea tissue from 2 different donors prepared by the same eye bank, the likelihood of a common HSV-1 source was determined. DESIGN: Case series. PARTICIPANTS: Two DMEK recipients who developed atypical intraocular HSV-1 disease shortly after surgery and surplus cornea specimens of 6 donors. METHODS: Surplus cornea donor (pre-DMEK cornea remnants and conditioned cornea storage and transport media) and recipient samples (post-DMEK aqueous humor) were assayed for HSV-1 DNA and infectious virus by real-time polymerase chain reaction (RT-PCR) and cell culture, respectively. Target-enriched whole viral genome sequencing was performed on HSV-1 DNA–positive ocular specimens. MAIN OUTCOMES MEASURES: Clinical picture of atypical intraocular HSV-1 infection post-DMEK and presence and homology of HSV-1 genomes between ocular specimens of DMEK donors and recipients. RESULTS: Herpes simplex virus type 1 DNA was detected in aqueous humor and donor cornea specimens of both DMEK cases, but not in the cornea remnants of 6 randomly selected donors processed by the same eye bank. Infectious HSV-1 was isolated from the cornea remnant and corresponding culture medium of 1 cornea donor. Notably, whole-genome sequencing of virus DNA-positive specimens demonstrated exceptionally high genetic similarity between HSV-1 strains in recipient and donor specimens of both DMEK cases. CONCLUSIONS: Data indicate cross-contamination of cornea grafts during DMEK preparation with subsequent graft-to-host HSV-1 transmission that caused atypical sight-threatening herpetic eye disease shortly after DMEK. Ophthalmologists should be aware that HSV-1 transmission by DMEK is possible and can lead to atypical ocular disease, a condition that can easily be prevented by taking appropriate technical and clinical measures at both eye bank and surgical levels

Haplotype assignment of longitudinal viral deep-sequencing data using co-variation of variant frequencies

Longitudinal deep sequencing of viruses can provide detailed information about intra-host evolutionary dynamics including how viruses interact with and transmit between hosts. Many analyses require haplotype reconstruction, identifying which variants are co-located on the same genomic element. Most current methods to perform this reconstruction are based on a high density of variants and cannot perform this reconstruction for slowly evolving viruses. We present a new approach, HaROLD (HAplotype Reconstruction Of Longitudinal Deep sequencing data), which performs this reconstruction based on identifying co-varying variant frequencies using a probabilistic framework. We illustrate HaROLD on both RNA and DNA viruses with synthetic Illumina paired read data created from mixed human cytomegalovirus and norovirus genomes, and clinical datasets of human cytomegalovirus and norovirus samples, demonstrating high accuracy, especially when longitudinal samples are available

On the effective depth of viral sequence data.

Genome sequence data are of great value in describing evolutionary processes in viral populations. However, in such studies, the extent to which data accurately describes the viral population is a matter of importance. Multiple factors may influence the accuracy of a dataset, including the quantity and nature of the sample collected, and the subsequent steps in viral processing. To investigate this phenomenon, we sequenced replica datasets spanning a range of viruses, and in which the point at which samples were split was different in each case, from a dataset in which independent samples were collected from a single patient to another in which all processing steps up to sequencing were applied to a single sample before splitting the sample and sequencing each replicate. We conclude that neither a high read depth nor a high template number in a sample guarantee the precision of a dataset. Measures of consistency calculated from within a single biological sample may also be insufficient; distortion of the composition of a population by the experimental procedure or genuine within-host diversity between samples may each affect the results. Where it is possible, data from replicate samples should be collected to validate the consistency of short-read sequence data

A large population sample of African HIV genomes from the 1980s reveals a reduction in subtype D over time associated with propensity for CXCR4 tropism

We present 109 near full-length HIV genomes amplified from blood serum samples obtained during early 1986 from across Uganda, which to our knowledge is the earliest and largest population sample from the initial phase of the HIV epidemic in Africa. Consensus sequences were made from paired-end Illumina reads with a target-capture approach to amplify HIV material following poor success with standard approaches. In comparisons with a smaller 'intermediate' genome dataset from 1998 to 1999 and a 'modern' genome dataset from 2007 to 2016, the proportion of subtype D was significantly higher initially, dropping from 67% (73/109), to 57% (26/46) to 17% (82/465) respectively (p < 0.0001). Subtype D has previously been shown to have a faster rate of disease progression than other subtypes in East African population studies, and to have a higher propensity to use the CXCR4 co-receptor ("X4 tropism"); associated with a decrease in time to AIDS. Here we find significant differences in predicted tropism between A1 and D subtypes in all three sample periods considered, which is particularly striking the 1986 sample: 66% (53/80) of subtype D env sequences were predicted to be X4 tropic compared with none of the 24 subtype A1. We also analysed the frequency of subtype in the envelope region of inter-subtype recombinants, and found that subtype A1 is over-represented in env, suggesting recombination and selection have acted to remove subtype D env from circulation. The reduction of subtype D frequency over three decades therefore appears to be a result of selective pressure against X4 tropism and its higher virulence. Lastly, we find a subtype D specific codon deletion at position 24 of the V3 loop, which may explain the higher propensity for subtype D to utilise X4 tropism

High prevalence of integrase mutation L74I in West African HIV-1 subtypes prior to integrase inhibitor treatment.

OBJECTIVES: HIV-1 integrase inhibitors are recommended as first-line therapy by WHO, though efficacy and resistance data for non-B subtypes are limited. Two recent trials have identified the integrase L74I mutation to be associated with integrase inhibitor treatment failure in HIV-1 non-B subtypes. We sought to define the prevalence of integrase resistance mutations, including L74I, in West Africa. METHODS: We studied a Nigerian cohort of recipients prior to and during receipt of second-line PI-based therapy, who were integrase inhibitor-naive. Illumina next-generation sequencing with target enrichment was used on stored plasma samples. Drug resistance was interpreted using the Stanford Resistance Database and the IAS-USA 2019 mutation lists. RESULTS: Of 115 individuals, 59.1% harboured CRF02_AG HIV-1 and 40.9% harboured subtype G HIV-1. Four participants had major IAS-USA integrase resistance-associated mutations detected at low levels (2%-5% frequency). Two had Q148K minority variants and two had R263K (one of whom also had L74I). L74I was detected in plasma samples at over 2% frequency in 40% (46/115). Twelve (26.1%) had low-level minority variants of between 2% and 20% of the viral population sampled. The remaining 34 (73.9%) had L74I present at >20% frequency. L74I was more common among those with subtype G infection (55.3%, 26/47) than those with CRF02_AG infection (29.4%, 20/68) (P = 0.005). CONCLUSIONS: HIV-1 subtypes circulating in West Africa appear to have very low prevalence of major integrase mutations, but significant prevalence of L74I. A combination of in vitro and clinical studies is warranted to understand the potential implications.K.E.B. is supported by Wellcome Trust award number 170461. N.N. is supported by NIH R01 AI147331-01. R.K.G. is supported by a Wellcome Trust Senior Fellowship in Clinical Science (WT108082AIA). This study was supported by the President’s Emergency Plan for AIDS Relief (PEPFAR) through the Centers for Disease Control and Prevention (CDC) under the terms of U2G GH002099-01 and PA GH17-1753 (ACHIEVE)

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is partially maintained among repeated serial samples from the same host, it can transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control

Using Whole Genome Sequences to Investigate Adenovirus Outbreaks in a Hematopoietic Stem Cell Transplant Unit

A recent surge in human mastadenovirus (HAdV) cases, including five deaths, amongst a haematopoietic stem cell transplant population led us to use whole genome sequencing (WGS) to investigate. We compared sequences from 37 patients collected over a 20-month period with sequences from GenBank and our own database of HAdVs. Maximum likelihood trees and pairwise differences were used to evaluate genotypic relationships, paired with the epidemiological data from routine infection prevention and control (IPC) records and hospital activity data. During this time period, two formal outbreaks had been declared by IPC, while WGS detected nine monophyletic clusters, seven were corroborated by epidemiological evidence and by comparison of single-nucleotide polymorphisms. One of the formal outbreaks was confirmed, and the other was not. Of the five HAdV-associated deaths, three were unlinked and the remaining two considered the source of transmission. Mixed infection was frequent (10%), providing a sentinel source of recombination and superinfection. Immunosuppressed patients harboring a high rate of HAdV positivity require comprehensive surveillance. As a consequence of these findings, HAdV WGS is being incorporated routinely into clinical practice to influence IPC policy contemporaneously

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1000 cases of unexplained pediatric hepatitis in children have been reported worldwide, including 278 cases in the UK. Here we report investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator subjects, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in liver, blood, plasma or stool from 27/28 cases. We found low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), in 23/31 and 16/23 respectively of the cases tested. In contrast, AAV2 was infrequently detected at low titre in blood or liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T-cells and B-lineage cells. Proteomic comparison of liver tissue from cases and healthy controls, identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and in severe cases HHV-6B, may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Multivariate Statistical Analyses Demonstrate Unique Host Immune Responses to Single and Dual Lentiviral Infection

Feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) are recently identified lentiviruses that cause progressive immune decline and ultimately death in infected cats and humans. It is of great interest to understand how to prevent immune system collapse caused by these lentiviruses. We recently described that disease caused by a virulent FIV strain in cats can be attenuated if animals are first infected with a feline immunodeficiency virus derived from a wild cougar. The detailed temporal tracking of cat immunological parameters in response to two viral infections resulted in high-dimensional datasets containing variables that exhibit strong co-variation. Initial analyses of these complex data using univariate statistical techniques did not account for interactions among immunological response variables and therefore potentially obscured significant effects between infection state and immunological parameters.Here, we apply a suite of multivariate statistical tools, including Principal Component Analysis, MANOVA and Linear Discriminant Analysis, to temporal immunological data resulting from FIV superinfection in domestic cats. We investigated the co-variation among immunological responses, the differences in immune parameters among four groups of five cats each (uninfected, single and dual infected animals), and the "immune profiles" that discriminate among them over the first four weeks following superinfection. Dual infected cats mount an immune response by 24 days post superinfection that is characterized by elevated levels of CD8 and CD25 cells and increased expression of IL4 and IFNgamma, and FAS. This profile discriminates dual infected cats from cats infected with FIV alone, which show high IL-10 and lower numbers of CD8 and CD25 cells.Multivariate statistical analyses demonstrate both the dynamic nature of the immune response to FIV single and dual infection and the development of a unique immunological profile in dual infected cats, which are protected from immune decline