Generation of Novel Severe Acute Respiratory Syndrome Coronavirus 2 Variants on the B.1.1.7 Lineage in 3 Patients With Advanced Human Immunodeficiency Virus-1 Disease.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is of public health concern in case of vaccine escape. Described are 3 patients with advanced human immunodeficiency virus (HIV)-1 and chronic SARS-CoV-2 infection in whom there is evidence of selection and persistence of novel mutations that are associated with increased transmissibility and immune escape.

Patient characteristics and severity of human rhinovirus infections in children.

BACKGROUND: It is increasingly recognized that human rhinoviruses (HRV) can be associated with severe infections. However, conflicting results have been reported on the relative prevalence and severity of the three HRV species. OBJECTIVES: The relative prevalence and clinical characteristics of HRV-A, B and C, in children attending a South London teaching hospital were investigated retrospectively. STUDY DESIGN: Children aged<16 years with episodes of respiratory tract infections and detectable entero/rhinovirus RNA in respiratory samples between November 2009 and December 2010 were investigated. Retrospective case review was performed and patients' characteristics recorded. RESULTS: Entero/rhinoviruses were the commonest viral pathogens (498/2316; 21.5%). Amongst 204 infection episodes associated with entero/rhinovirus, 167 were typed HRV, HRV-C was the most prevalent (99/167, 59.3%) followed by HRV-A (60/167; 35.9%) and HRV-B (8/167, 4.8%). The severity spectrum of HRV-A and HRV-C infections were similar and affected all parts of the respiratory tract. Co-pathogens were observed in 54 (26.5%) episodes. Severity was increased in patients with non-viral co-pathogens and those with an underlying respiratory condition. Univariate and multiple regression analyses of potential prognostic variables including age, co-pathogens and underlying respiratory illnesses showed that mono-infection with HRV-C, as compared with other HRV species, was associated with more severe disease in young children<3 years. CONCLUSIONS: HRV-C was the most prevalent species and on its own was associated with severe disease in children<3 years. The association between infection with HRV species and clinical presentation is complex and affected by many confounding factors.

Lineages, sub-lineages and variants of enterovirus 68 in recent outbreaks.

Enterovirus 68 (EV68) was first isolated in 1962. Very few cases of EV68 infection were described over the ensuing 40 years. However, in the past few years, an increase in severe respiratory tract infections associated with EV68 has been reported. We identified two clusters of EV68 infection in South London, UK, one each in the autumn/winters of 2009 and 2010. Sequence comparison showed significant homology of the UK strains with those from other countries including the Netherlands, Japan and the Philippines, which reported EV68 outbreaks between 2008 and 2010. Phylogenetic analysis of all available VP1 sequences indicated the presence of two modern EV68 lineages. The 2010 UK strains belonged to lineage 2. Lineage 1 could be further divided into two sub-lineages: some Japanese and Dutch strains collected between 2004 and 2010 form a distinct sub-lineages (sub-lineage 1.1), whereas other strains from the UK, Japan, Netherlands and Philippines collected between 2008 and 2010 represent sub-lineage 1.2. The UK 2009 strains together with several Dutch and Japanese strains from 2009/2010 represents one variant (1.2.1), whereas those from the Philippines a second variant (1.2.2). Based on specific deletions and substitutions, we suggest rules for the assignment of lineages and sub-lineages. Molecular epidemiological analysis indicates rapid recent evolution of EV68 and this may explain the recent findings of a global resurgence of EV68. Continuous global monitoring of the clinical and molecular epidemiology of EV68 is recommended.

Human papillomavirus detection in dysplastic and malignant oral verrucous lesions.

The role of human papillomaviruses (HPV) in dysplastic and malignant oral verrucous lesions is controversial since there is a wide range in the incidence of virus detection. This study used a multi-tiered method of HPV detection using DNA in-situ hybridisation (ISH) for low- and high-risk subtypes, consensus PCR, and HPV genotype analysis in archival tissue from 20 cases of dysplastic and malignant oral verrucous lesions. The biological significance of HPV DNA detection was assessed by p16 immunohistochemistry (IHC). While 1/7 carcinomas and 5/13 dysplasias contained HPV DNA by consensus PCR and genotype analysis, all specimens were negative for low- and high-risk HPV ISH and negative for p16 IHC. Results show that although high-risk HPV DNA is detectable in a subset of these lesions, the lack of p16 overexpression suggests that the oncogenic process is not driven by HPV oncoproteins.

Evaluation of human papillomavirus testing for squamous cell carcinoma of the tonsil in clinical practice.

BACKGROUND: Oncogenic human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (SCC) is a subtype of head-and-neck cancer with a distinct clinical and prognostic profile. While there are calls to undertake HPV testing for oropharyngeal SCCs within the diagnostic setting and for clinical trials, there are currently no internationally accepted standards. METHODS: 142 tonsil SCCs were tested using p16 immunohistochemistry (IHC), high-risk HPV DNA in situ hybridisation (ISH) and HPV DNA polymerase chain reaction (PCR; GP5+/6+ primers). RESULTS: There were high levels of agreement between pathologists for p16 IHC and HPV ISH scoring; however, around 10% of HPV ISH cases showed some interobserver discrepancy that was resolved by slide review. The combination of p16 IHC and HPV ISH classified 53% of the samples as HPV-positive, whereas the combination of p16 IHC and HPV PCR classified 61% of the samples as HPV-positive. By employing a three-tiered, staged algorithm (p16 IHC/HPV ISH/HPV PCR), the authors were able to classify 98% of the cases as either HPV-positive (p16 IHC+/HPV DNA+; 62%) or HPV-negative (p16 IHC-/HPV DNA-; 35%). CONCLUSIONS: The current study suggests that using a combination of p16 IHC/HPV ISH/HPV PCR, in a three-tiered, staged algorithm, in conjunction with consensus reporting of HPV ISH, leads to less equivocal molecular classification. In order to ensure consistent reporting of this emerging disease, it is increasingly important for the head-and-neck oncology community to define the minimum requirements for assigning a diagnosis of 'HPV-related' oropharyngeal SCC in order to inform prognosis and for stratification in clinical trials.

Evolutionary genetics of human enterovirus 71: origin, population dynamics, natural selection, and seasonal periodicity of the VP1 gene.

Human enterovirus 71 (EV-71) is one of the major etiologic causes of hand, foot, and mouth disease (HFMD) among young children worldwide, with fatal instances of neurological complications becoming increasingly common. Global VP1 capsid sequences (n = 628) sampled over 4 decades were collected and subjected to comprehensive evolutionary analysis using a suite of phylogenetic and population genetic methods. We estimated that the common ancestor of human EV-71 likely emerged around 1941 (95% confidence interval [CI], 1929 to 1952), subsequently diverging into three genogroups: B, C, and the now extinct genogroup A. Genealogical analysis revealed that diverse lineages of genogroup B and C (subgenogroups B1 to B5 and C1 to C5) have each circulated cryptically in the human population for up to 5 years before causing large HFMD outbreaks, indicating the quiescent persistence of EV-71 in human populations. Estimated phylogenies showed a complex pattern of spatial structure within well-sampled subgenogroups, suggesting endemicity with occasional lineage migration among locations, such that past HFMD epidemics are unlikely to be linked to continuous transmission of a single strain of virus. In addition, rises in genetic diversity are correlated with the onset of epidemics, driven in part by the emergence of novel EV-71 subgenogroups. Using subgenogroup C1 as a model, we observe temporal strain replacement through time, and we investigate the evidence for positive selection at VP1 immunogenic sites. We discuss the consequences of the evolutionary dynamics of EV-71 for vaccine design and compare its phylodynamic behavior with that of influenza virus.

Molecular epidemiology of human enterovirus 71 in the United Kingdom from 1998 to 2006.

The last decade witnessed a significant increase in epidemic activity of human enterovirus 71 (EV71) in the Western Pacific Region (WPR). In most European countries, this risk is unrecognized despite occasional cases of severe disease and two severe outbreaks in Eastern Europe 30 years ago. In this study we report the first examination of the molecular epidemiology of EV71 in the United Kingdom from 1998 to 2006. Genomic regions encoding the 1D coat protein (VP1) and 3D polymerase (Pol) from 32 EV71 isolates associated with neurological or cutaneous manifestations were sequenced. Phylogenetic analyses of VP1 and 3D Pol sequences identified genotype C as the dominant strain. Several United Kingdom isolates had genetic linkages with predated C1 or C2 strains from Europe and the WPR. Recombination events were not detected between United Kingdom strains. However, a previously published Taiwanese strain was identified as an intergenotypic recombinant. EV71 genotype C appears to have continuous circulation in the United Kingdom from 1998 to 2006 with repeated introductions of new strains replacing previous strains. It is necessary to continuously monitor the molecular evolution and recombination events of EV71.

Genetic evolution of enterovirus 71: epidemiological and pathological implications.

Since its discovery in the 1970s, enterovirus 71 (EV71) has become one of the most pathogenic enterovirus serotypes causing recurrent outbreaks in different parts of the world. Three waves of outbreaks globally have been recorded over the last three decades and more recently active circulation of EV71 is evident amongst countries in South East Asia and beyond. There is evidence of a continuous evolution in its genetic make up which is likely to impact on its epidemiology and pathological potential. This review examines the molecular genetics and evolution of EV71 in relation to its epidemiological and pathological properties. A thorough understanding of the relationship between the genetic changes and the resulting host-virus interaction is essential for successful control.

Analyses of variant human papillomavirus type-16 E5 proteins for their ability to induce mitogenesis of murine fibroblasts.

BACKGROUND: Human papillomavirus type 16 (HPV-16) E5 protein co-operates with epidermal growth factor to stimulate mitogenesis of murine fibroblasts. Currently, little is known about which viral amino acids are involved in this process. Using sequence variants of HPV-16 E5 we have investigated their effects upon E5 transcription, cell-cycling and cell-growth of murine fibroblasts. RESULTS: We demonstrate that: (i) introduction of Thr64 into the reference E5 sequence of HPV-16 abrogates mitogenic activity: both were poorly transcribed in NIH-3T3 cells; (ii) substitution of Leu44Val65 or, Thr37Leu44Val65 into the HPV-16 E5 reference backbone resulted in high transcription in NIH-3T3 cells, enhanced cell-cycle progression and high cell-growth; and, (iii) inclusion of Tyr8 into the Leu44Val65 backbone inhibited E5 induced cell-growth and repression of p21 expression, despite high transcription levels. CONCLUSION: The effects of HPV-16 E5 variants upon mitosis help to explain why Leu44Val65 HPV-16 E5 variants are most prevalent in 'wild' pathogenic viral populations in the UK.

Identification of HPV variants.

The vast majority of anogenital carcinomas are caused by high-risk human papillomaviruses (HPVs), and among Western nations HPV-16 is usually the most predominant cancer-associated type. As a DNA virus, HPV type 16 has a relatively stable genome that is believed to have co-evolved with its host over the millennia. Nevertheless, among the "wild" populations of HPV-16 that are circulating, a large number of variants have been identified, and these may have considerably different pathogenic potentials. In this chapter, methods for screening and characterizing HPV-16 sequence variants are described. In particular, we describe methods for the identification of variation within the HPV-16 E5 open reading frame and for the detection of the nt 131 A-->G mutation of the E6 ORF, using restriction fragment length polymorphism assays. In addition, we describe approaches for DNA sequencing and analysis. Such methods are likely to be of particular interest to those involved in epidemiological investigations of virus transmission and pathogenicity studies.

Immunoglobulin light-chain genes in the rhesus macaque II: lambda light-chain germline sequences for subgroups IGLV1, IGLV2, IGLV3, IGLV4 and IGLV5.

The combined processes of immunoglobulin (IG) gene rearrangement and somatic hypermutation allow for the creation of an extremely diverse antibody repertoire. Knowledge of the germline sequence of the IG genes is required so that hypermutation and the affinity matured humoral response can be properly studied. Variable region genes can be arranged into subgroups; in humans, there are 11 IGLV subgroups and 6 IGKV subgroups. The rhesus macaque (Macaca mulatta) is a relevant non-human primate model for human immunological systems. A number of macaque IGHV, IGHD and IGHJ genes have already been reported. We have also previously reported a number of macaque IGKV genes. Here we report the isolation of new macaque IGLV genes by polymerase chain reaction amplification from macaque genomic DNA using primers based on the human sequences. Nine IGLV1, 10 IGLV2, 21 IGLV3, 5 IGLV4 and 7 IGLV5 germline genes for the macaque were found, the open-reading frames of which exhibit high homology to their human counterparts (>89.3, >88.6, >89.0, >94.7 and >87.1%, respectively).

Immunoglobulin light-chain genes in the rhesus macaque I: kappa light-chain germline sequences for subgroups IGKV1, IGKV and IGKV3.

The combined processes of immunoglobulin (IG) gene rearrangement and somatic hypermutation allow for the creation of an extremely diverse antibody repertoire. Knowledge of the germline sequence of the IG genes is required so that hypermutation and the affinity matured humoral response can be properly studied. Variable region genes can be arranged into subgroups; in humans, there are 11 IGLV subgroups and six IGKV subgroups. The rhesus macaque (Macaca mulatta) is a relevant non-human primate model for human immunological systems. A number of macaque IGHV, IGHD and IGHJ genes have already been reported, but only one light-chain germline gene has been published so far. Here we report the isolation of new macaque IGKV genes by polymerase chain reaction (PCR) amplification from macaque genomic DNA using primers based on the human sequences. Twenty-eight IGKV1, 22 IGKV2 and 12 IGKV3 germline genes for the macaque were found, the open reading frames of which exhibit high homology to their human counterparts (>96, >99 and >96%, respectively).

Buccal exposure to human papillomavirus type 16 is a common yet transitory event of childhood.

High-risk human papillomaviruses, such as type 16 (HPV-16), are established etiological agents for cervical carcinoma. In most cases, this virus is transmitted sexually, though can also be spread from mother to infant at delivery. We have demonstrated previously a high prevalence ( approximately 52%) of HPV-16 DNA in the mouths of prepubertal children, albeit with low levels of transcription [Rice et al., 2000]. We investigated whether childhood buccal infections with HPV-16 are persistent or transient and whether children became infected through contact with their immediate family members. Two groups of children were selected: one group were all initially HPV-16 E5 DNA-positive in sensitive polymerase chain reaction tests of swabs from their buccal mucosa (n = 20), and the other group consisted of children who were all HPV-16 E5-negative (n = 19). Thirty months later, a second oral swab was collected from each child and tested for HPV DNA. At this second visit, 40% of the HPV-16-positive group had no detectable HPV-16 DNA; conversely, 63% of children who were originally HPV-16-negative had now acquired the virus. Three months later, a third sample was collected from eight children and their immediate families (seven were HPV-16 E5 DNA-positive at the second visit). Amongst the family samples tested, in two families a single previously untested child was HPV-16 DNA-positive. It is concluded that HPV-16 DNA in the oral cavities of children is a transient event and is most probably acquired from their peers.

IGHV1, IGHV5 and IGHV7 subgroup genes in the rhesus macaque.

The diversity of the antibody response is achieved, in part, by rearrangement of different immunoglobulin (Ig) genes. The Ig heavy chain is made up of a variable region (IGHV), a diversity region (IGHD) and a joining region (IGHJ). Human germline IGHV genes have been grouped into seven multigene subgroups. Size and usage of these subgroups is not equal, the IGHV3 subgroup is the most commonly used (36%), followed by IGHV1/7 (26%), then IGHV4, IGHV5, IGHV2, IGHV6 (15%, 12%, 4%, 3% respectively). The rhesus macaque (Macaca mulatta) is a useful non-human primate model for studies of infection and the database of germline Ig genes for the macaque is gradually growing to become a useful tool in the study of B-cell responses. The proportions of IGHV subgroup usage in the macaque are similar to those in man. Representatives from IGHV3 and IGHV4 subgroups for the macaque have been published, as have germline sequences of the IGHD and IGHJ genes. However, to date there have been no sequences published from the second largest IGHV subgroup, IGHV1. We report the isolation and sequencing of a genomic fragment containing an IGHV1 gene from the macaque. Polymerase chain reaction (PCR) primers designed from this sequence enabled us to amplify and sequence 25 new IGHV1 germline genes. We also isolated two IGHV7 genes, using the same primers, and two IGHV5 genes, using human IGHV5 primers.