Translational arrest and mRNA decay are independent activities of alphaherpesvirus virion host shutoff proteins.

The herpes simplex virus 1 (HSV1) virion host shutoff (vhs) protein is an endoribonuclease that regulates the translational environment of the infected cell, by inducing the degradation of host mRNA via cellular exonuclease activity. To further understand the relationship between translational shutoff and mRNA decay, we have used ectopic expression to compare HSV1 vhs (vhsH) to its homologues from four other alphaherpesviruses - varicella zoster virus (vhsV), bovine herpesvirus 1 (vhsB), equine herpesvirus 1 (vhsE) and Marek's disease virus (vhsM). Only vhsH, vhsB and vhsE induced degradation of a reporter luciferase mRNA, with poly(A)+ in situ hybridization indicating a global depletion of cytoplasmic poly(A)+ RNA and a concomitant increase in nuclear poly(A)+ RNA and the polyA tail binding protein PABPC1 in cells expressing these variants. By contrast, vhsV and vhsM failed to induce reporter mRNA decay and poly(A)+ depletion, but rather, induced cytoplasmic G3BP1 and poly(A)+ mRNA- containing granules and phosphorylation of the stress response proteins eIF2α and protein kinase R. Intriguingly, regardless of their apparent endoribonuclease activity, all vhs homologues induced an equivalent general blockade to translation as measured by single-cell puromycin incorporation. Taken together, these data suggest that the activities of translational arrest and mRNA decay induced by vhs are separable and we propose that they represent sequential steps of the vhs host interaction pathway.

A systematic search and narrative review of Aboriginal and Torres Strait Islander women and men pelvic health care: demonstrated need for improvement.

Warning:This article contains terms, descriptions and opinions that may be culturally sensitive for Aboriginal and Torres Strait Islander peoples. BACKGROUND: Pelvic health conditions among Aboriginal and Torres Strait Islander women and men are under-recognised and under-reported despite indication of the significant burden of these conditions. Access to effective management provided in a culturally safe manner appears lacking. The study aims were to: (1) summarise the burden of pelvic health conditions among Aboriginal and Torres Strait Islander peoples; (2) explore barriers and enablers to Aboriginal and Torres Strait Islander peoples accessing pelvic healthcare services; and (3) provide considerations on how to implement culturally safe pelvic healthcare services for Aboriginal and Torres Strait Islander peoples. METHODS: This study involved a systematic literature search informing a narrative review. RESULTS: Fourteen specific articles were identified. Burden related to pelvic health conditions was identified for both women and men, noting high likelihood of under-reporting. Barriers to effective culturally safe treatment included racism, shame and stigma associated with women's and men's business, lack of culturally safe services and geographical distance. Enablers included building trust with local communities, using yarning principles and codesign of pelvic health services with Aboriginal health workers and services. Strategies to improve pelvic health care for Aboriginal and Torres Strait Islander peoples were described at the systems, health service and clinician levels. CONCLUSION: Efforts are needed to improve the diagnosis and management of pelvic health conditions for Aboriginal and Torres Strait Islander peoples. Actions are required to engage with local Aboriginal Community Controlled Aboriginal Community Controlled Health Organisations, professionals overseeing service provision and healthcare providers to address the burden of these conditions among Aboriginal and Torres Strait Islander peoples.

Pathogenesis of Rift Valley Fever Virus in a BALB/c Mouse Model Is Affected by Virus Culture Conditions and Sex of the Animals.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen causing disease in livestock and humans. Whilst initially restricted to the African continent, recent spread to the Arabian Peninsula has highlighted the likelihood of entry into new regions. Due to the absence of a regulatory-approved human vaccine, work is ongoing to develop and assess countermeasures. As such, small animal models play a pivotal role in providing information on disease pathogenesis and elucidating which intervention strategies confer protection. To develop and establish the BALB/c mouse model, we challenged mice with RVFV grown from two separate cell lines: one derived from mosquitoes (C6/36) and the other mammalian derived (Vero E6). Following infection, we assessed the clinical course of disease progression at days 1 and 3 post-challenge and evaluated viral tropism and immune analytes. The results demonstrated that RVFV infection was affected by the cell line used to propagate the challenge virus, with those grown in insect cells resulting in a more rapid disease progression. The lowest dose that caused uniform severe disease remained the same across both virus preparations. In addition, to demonstrate reproducibility, the lowest dose was used for a subsequent infection study using male and female animals. The results further demonstrated that male mice succumbed to infection more rapidly than their female counterparts. Our results establish an RVFV mouse model and key parameters that affect the course of disease progression in BALB/c mice.

Dysregulated metabolism of the late herpes simplex virus 1 transcriptome through the vhs-VP22 axis uncouples virus cytopathic effect and virus production.

Herpes simplex virus 1 (HSV1) expresses its genes in a classical cascade culminating in the production of large amounts of structural proteins to facilitate virus assembly. HSV1 lacking the virus protein VP22 (Δ22) exhibits late translational shutoff, a phenotype that has been attributed to the unrestrained activity of the virion host shutoff (vhs) protein, a virus-encoded endoribonuclease which induces mRNA degradation during infection. We have previously shown that vhs is also involved in regulating the nuclear-cytoplasmic compartmentalisation of the virus transcriptome, and in the absence of VP22 a number of virus transcripts are sequestered in the nucleus late in infection. Here we show that despite expressing minimal amounts of structural proteins and failing to plaque on human fibroblasts, the strain 17 Δ22 virus replicates and spreads as efficiently as Wt virus, but without causing cytopathic effect (CPE). Nonetheless, CPE-causing virus spontaneously appeared on Δ22-infected human fibroblasts, and four viruses isolated in this way had all acquired point mutations in vhs which rescued late protein translation. However, unlike a virus deleted for vhs, these viruses still induced the degradation of both cellular and viral mRNA suggesting that vhs mutation in the absence of VP22 is necessary to overcome a more complex disturbance in mRNA metabolism than mRNA degradation alone. The ultimate outcome of secondary mutations in vhs is therefore the rescue of virus-induced CPE caused by late protein synthesis, and while there is a clear selective pressure on HSV1 to mutate vhs for optimal production of late structural proteins, the purpose of this is over and above that of virus production.

Physiotherapists could detect changes of 12 degrees or more in single-plane movement when observing forward bending, squat or hand-over-head: A cross-sectional experiment.

BACKGROUND: The visual accuracy of physiotherapists to detect changes in dynamic joint angles is currently unknown. OBJECTIVE: To investigate (i) the smallest detectable change in movement that physiotherapists could visually observe, and (ii) whether visual accuracy was associated with the functional activity observed or characteristics of the physiotherapist. METHODS: Thirty-four physiotherapists viewed and rated videos of squat, hand-over-head, forward bend functional activities and an artificial test condition (a reference movement followed by subsequent movements showing random differences in peak angle from 0° to 15°, so 18 sets of paired videos per functional activity). They rated each range of movement (same/more/less) relative to the reference movement, while their visual tracking was continuously monitored. Accuracy was calculated (multilevel regression) using two thresholds - two correct out of three viewings (2/3) and three correct out of three viewings (3/3). RESULTS: More than 80% of physiotherapists were able to detect 9° difference using the 2/3 threshold and 12° using the 3/3 threshold. There was no association (p > 0.05) between visual accuracy and experience, sex, or movement type, except when viewing shoulder abduction compared with knee flexion using the 3/3 threshold. The only association between accuracy and visual tracking characteristics was for assessing lumbar flexion, where use of more visual fixation areas and a shorter fixation time per area were more accurate. CONCLUSION: Physiotherapists were consistently accurate at detecting changes of ≥12° in single-plane, low-speed functional activities. Visual accuracy was not explained by experience or sex, and rarely associated with functional activity type or visual fixation.

Herpes Simplex Virus 1 Expressing GFP-Tagged Virion Host Shutoff (vhs) Protein Uncouples the Activities of RNA Degradation and Differential Nuclear Retention of the Virus Transcriptome.

Virion host shutoff (vhs) protein is an endoribonuclease encoded by herpes simplex virus 1 (HSV1). vhs causes several changes to the infected cell environment that favor the translation of late (L) virus proteins: cellular mRNAs are degraded, immediate early (IE) and early (E) viral transcripts are sequestered in the nucleus with polyA binding protein (PABPC1), and dsRNA is degraded to help dampen the PKR-dependent stress response. To further our understanding of the cell biology of vhs, we constructed a virus expressing vhs tagged at its C terminus with GFP. When first expressed, vhs-GFP localized to juxtanuclear clusters, and later it colocalized and interacted with its binding partner VP16, and was packaged into virions. Despite vhs-GFP maintaining activity when expressed in isolation, it failed to degrade mRNA or relocalise PABPC1 during infection, while viral transcript levels were similar to those seen for a vhs knockout virus. PKR phosphorylation was also enhanced in vhs-GFP infected cells, which is in line with a failure to degrade dsRNA. Nonetheless, mRNA FISH revealed that as in Wt but not Dvhs infection, IE and E, but not L transcripts were retained in the nucleus of vhs-GFP infected cells at late times. These results revealed that the vhs-induced nuclear retention of IE and E transcripts was dependent on vhs expression but not on its endoribonuclease activity, uncoupling these two functions of vhs. IMPORTANCE Like many viruses, herpes simplex virus 1 (HSV1) expresses an endoribonuclease, the virion host shutoff (vhs) protein, which regulates the RNA environment of the infected cell and facilitates the classical cascade of virus protein translation. It does this by causing the degradation of some mRNA molecules and the nuclear retention of others. Here, we describe a virus expressing vhs tagged at its C terminus with a green fluorescent protein (GFP) and show that the vhs-GFP fusion protein retains the physical properties of native vhs but does not induce the degradation of mRNA. Nonetheless, vhs-GFP maintains the ability to trap the early virus transcriptome in the nucleus to favor late protein translation, proving for the first time that mRNA degradation is not a prerequisite for vhs effects on the nuclear transcriptome. This virus, therefore, has uncoupled the nuclear retention and degradation activities of vhs, providing a new understanding of vhs during infection.

Association between pelvic pain bothersomeness and pain sensitivity: A community-based cross-sectional study of young adult females in the Raine Study.

OBJECTIVE: Pelvic pain has been associated with augmented nociceptive processing, but large studies controlling for multiple potential confounding factors are lacking. This study investigated the association between pelvic pain bothersomeness and pain sensitivity in young adult women, accounting for potential confounding factors. DESIGN: Cross-sectional study. SETTING: Community-dwelling sample. POPULATION: The Raine Study Gen2-22 year follow-up (n = 475). MAIN OUTCOME MEASURES: The experience of bothersomeness related to pelvic pain was determined from a question in the Urogenital Distress Inventory short form. Pain sensitivity was measured using pressure pain and cold pain thresholds. Potential confounding factors included ethnicity, marital status, highest level of education, income, waist-hip ratio, level of activity, sleep quality, smoking, comorbidity history, C-reactive protein level, musculoskeletal pain experience and psychological distress. RESULTS: Three hundred and sixty-two women (76.2%) reported no pelvic pain bothersomeness, 74 (15.6%) reported mild pelvic pain bothersomeness and 39 (8.2%) reported moderate-severe pelvic pain bothersomeness. After adjusting for marital status (and test site), moderate-severe pelvic pain bothersomeness was associated with a lower pressure pain threshold (i.e. greater pressure pain sensitivity) (coefficient -51.46, 95% CI -98.06 to -4.86, p = 0.030). After adjusting for smoking, moderate-severe pelvic pain bothersomeness was also associated with a higher cold pain threshold (i.e. greater cold pain sensitivity) (coefficient 4.35, 95% CI 0.90-7.79, p = 0.014). CONCLUSIONS: This study suggests augmented nociceptive processing as a contributing factor in pelvic pain bothersomeness for some women. Thorough assessment of women who present clinically with pelvic pain should consider pain sensitivity as a potential contributing factor to their presentation.

Reverse-Transcription Loop-Mediated Isothermal Amplification Has High Accuracy for Detecting Severe Acute Respiratory Syndrome Coronavirus 2 in Saliva and Nasopharyngeal/Oropharyngeal Swabs from Asymptomatic and Symptomatic Individuals.

Previous studies have described reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal/oropharyngeal swab and saliva samples. This multisite clinical evaluation describes the validation of an improved sample preparation method for extraction-free RT-LAMP and reports clinical performance of four RT-LAMP assay formats for SARS-CoV-2 detection. Direct RT-LAMP was performed on 559 swabs and 86,760 saliva samples and RNA RT-LAMP on extracted RNA from 12,619 swabs and 12,521 saliva samples from asymptomatic and symptomatic individuals across health care and community settings. For direct RT-LAMP, overall diagnostic sensitivity (DSe) was 70.35% (95% CI, 63.48%-76.60%) on swabs and 84.62% (95% CI, 79.50%-88.88%) on saliva, with diagnostic specificity of 100% (95% CI, 98.98%-100.00%) on swabs and 100% (95% CI, 99.72%-100.00%) on saliva, compared with quantitative RT-PCR (RT-qPCR); analyzing samples with RT-qPCR ORF1ab CT values of ≤25 and ≤33, DSe values were 100% (95% CI, 96.34%-100%) and 77.78% (95% CI, 70.99%-83.62%) for swabs, and 99.01% (95% CI, 94.61%-99.97%) and 87.61% (95% CI, 82.69%-91.54%) for saliva, respectively. For RNA RT-LAMP, overall DSe and diagnostic specificity were 96.06% (95% CI, 92.88%-98.12%) and 99.99% (95% CI, 99.95%-100%) for swabs, and 80.65% (95% CI, 73.54%-86.54%) and 99.99% (95% CI, 99.95%-100%) for saliva, respectively. These findings demonstrate that RT-LAMP is applicable to a variety of use cases, including frequent, interval-based direct RT-LAMP of saliva from asymptomatic individuals who may otherwise be missed using symptomatic testing alone.

Overlap Intensity: An ImageJ Macro for Analyzing the HIV-1 In Situ Uncoating Assay.

Capsid uncoating is at the crossroads of early steps in HIV-1 replication. In recent years, the development of novel assays has expanded how HIV-1 uncoating can be studied. In the in situ uncoating assay, dual fluorescently labelled virus allows for the identification of fused viral cores. Antibody staining then detects the amount of capsid associated with each viral core at different times post-infection. Following fixed cell imaging, manual counting can be used to assess the fusion state and capsid signal for each viral core, but this method can introduce bias with increased time of analysis. To address these limitations, we developed the Overlap Intensity macro in ImageJ. This macro automates the detection of viral cores and quantification of overlapping fusion and capsid signals. We demonstrated the high accuracy of the macro by comparing core detection to manual methods. Analysis of an in situ uncoating assay further verified the macro by detecting progressive uncoating as expected. Therefore, this macro improves the accessibility of the in situ uncoating assay by replacing time-consuming manual methods or the need for expensive data analysis software. Beyond the described assay, the Overlap Intensity macro includes adjustable settings for use in other methods requiring quantification of overlapping fluorescent signals.

Diagnostic accuracy of loop-mediated isothermal amplification coupled to nanopore sequencing (LamPORE) for the detection of SARS-CoV-2 infection at scale in symptomatic and asymptomatic populations.

OBJECTIVES: Rapid, high throughput diagnostics are a valuable tool, allowing the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in populations so as to identify and isolate people with asymptomatic and symptomatic infections. Reagent shortages and restricted access to high throughput testing solutions have limited the effectiveness of conventional assays such as quantitative RT-PCR (RT-qPCR), particularly throughout the first months of the coronavirus disease 2019 pandemic. We investigated the use of LamPORE, where loop-mediated isothermal amplification (LAMP) is coupled to nanopore sequencing technology, for the detection of SARS-CoV-2 in symptomatic and asymptomatic populations. METHODS: In an asymptomatic prospective cohort, for 3 weeks in September 2020, health-care workers across four sites (Birmingham, Southampton, Basingstoke and Manchester) self-swabbed with nasopharyngeal swabs weekly and supplied a saliva specimen daily. These samples were tested for SARS-CoV-2 RNA using the Oxford Nanopore LamPORE system and a reference RT-qPCR assay on extracted sample RNA. A second retrospective cohort of 848 patients with influenza-like illness from March 2020 to June 2020 were similarly tested from nasopharyngeal swabs. RESULTS: In the asymptomatic cohort a total of 1200 participants supplied 23 427 samples (3966 swab, 19 461 saliva) over a 3-week period. The incidence of SARS-CoV-2 detection using LamPORE was 0.95%. Diagnostic sensitivity and specificity of LamPORE was >99.5% (decreasing to approximately 98% when clustered estimation was used) in both swab and saliva asymptomatic samples when compared with the reference RT-qPCR test. In the retrospective symptomatic cohort, the incidence was 13.4% and the sensitivity and specificity were 100%. CONCLUSIONS: LamPORE is a highly accurate methodology for the detection of SARS-CoV-2 in both symptomatic and asymptomatic population settings and can be used as an alternative to RT-qPCR.

SARS-CoV-2 within-host diversity and transmission.

Extensive global sampling and sequencing of the pandemic virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have enabled researchers to monitor its spread and to identify concerning new variants. Two important determinants of variant spread are how frequently they arise within individuals and how likely they are to be transmitted. To characterize within-host diversity and transmission, we deep-sequenced 1313 clinical samples from the United Kingdom. SARS-CoV-2 infections are characterized by low levels of within-host diversity when viral loads are high and by a narrow bottleneck at transmission. Most variants are either lost or occasionally fixed at the point of transmission, with minimal persistence of shared diversity, patterns that are readily observable on the phylogenetic tree. Our results suggest that transmission-enhancing and/or immune-escape SARS-CoV-2 variants are likely to arise infrequently but could spread rapidly if successfully transmitted.

A highly effective reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 infection.

The COVID-19 pandemic has illustrated the importance of simple, rapid and accurate diagnostic testing. This study describes the validation of a new rapid SARS-CoV-2 RT-LAMP assay for use on extracted RNA or directly from swab offering an alternative diagnostic pathway that does not rely on traditional reagents that are often in short supply during a pandemic. Analytical specificity (ASp) of this new RT-LAMP assay was 100% and analytical sensitivity (ASe) was between 1 × 101 and 1 × 102 copies per reaction when using a synthetic DNA target. The overall diagnostic sensitivity (DSe) and specificity (DSp) of RNA RT-LAMP was 97% and 99% respectively, relative to the standard of care rRT-PCR. When a CT cut-off of 33 was employed, above which increasingly evidence suggests there is a low risk of patients shedding infectious virus, the diagnostic sensitivity was 100%. The DSe and DSp of Direct RT-LAMP (that does not require RNA extraction) was 67% and 97%, respectively. When setting CT cut-offs of ≤33 and ≤25, the DSe increased to 75% and 100%, respectively, time from swab-to-result, CT < 25, was < 15 min. We propose that RNA RT-LAMP could replace rRT-PCR where there is a need for increased sample throughput and Direct RT-LAMP as a near-patient screening tool to rapidly identify highly contagious individuals within emergency departments and care homes during times of increased disease prevalence ensuring negative results still get laboratory confirmation.

Experimental Lagos bat virus infection in straw-colored fruit bats: A suitable model for bat rabies in a natural reservoir species.

Rabies is a fatal neurologic disease caused by lyssavirus infection. Bats are important natural reservoir hosts of various lyssaviruses that can be transmitted to people. The epidemiology and pathogenesis of rabies in bats are poorly understood, making it difficult to prevent zoonotic transmission. To further our understanding of lyssavirus pathogenesis in a natural bat host, an experimental model using straw-colored fruit bats (Eidolon helvum) and Lagos bat virus, an endemic lyssavirus in this species, was developed. To determine the lowest viral dose resulting in 100% productive infection, bats in five groups (four bats per group) were inoculated intramuscularly with one of five doses, ranging from 100.1 to 104.1 median tissue culture infectious dose (TCID50). More bats died due to the development of rabies after the middle dose (102.1 TCID50, 4/4 bats) than after lower (101.1, 2/4; 101.1, 2/4) or higher (103.1, 2/4; 104.1, 2/4) doses of virus. In the two highest dose groups, 4/8 bats developed rabies. Of those bats that remained healthy 3/4 bats seroconverted, suggesting that high antigen loads can trigger a strong immune response that abrogates a productive infection. In contrast, in the two lowest dose groups, 3/8 bats developed rabies, 1/8 remained healthy and seroconverted and 4/8 bats remained healthy and did not seroconvert, suggesting these doses are too low to reliably induce infection. The main lesion in all clinically affected bats was meningoencephalitis associated with lyssavirus-positive neurons. Lyssavirus antigen was detected in tongue epithelium (5/11 infected bats) rather than in salivary gland epithelium (0/11), suggesting viral excretion via the tongue. Thus, intramuscular inoculation of 102.1 TCID50 of Lagos bat virus into straw-colored fruit bats is a suitable model for lyssavirus associated bat rabies in a natural reservoir host, and can help with the investigation of lyssavirus infection dynamics in bats.

Oropouche virus cases identified in Ecuador using an optimised qRT-PCR informed by metagenomic sequencing.

Oropouche virus (OROV) is responsible for outbreaks of Oropouche fever in parts of South America. We recently identified and isolated OROV from a febrile Ecuadorian patient, however, a previously published qRT-PCR assay did not detect OROV in the patient sample. A primer mismatch to the Ecuadorian OROV lineage was identified from metagenomic sequencing data. We report the optimisation of an qRT-PCR assay for the Ecuadorian OROV lineage, which subsequently identified a further five cases in a cohort of 196 febrile patients. We isolated OROV via cell culture and developed an algorithmically-designed primer set for whole-genome amplification of the virus. Metagenomic sequencing of the patient samples provided OROV genome coverage ranging from 68-99%. The additional cases formed a single phylogenetic cluster together with the initial case. OROV should be considered as a differential diagnosis for Ecuadorian patients with febrile illness to avoid mis-diagnosis with other circulating pathogens.

Evolutionary Dynamics of Oropouche Virus in South America.

The Amazon basin is home to numerous arthropod-borne viral pathogens that cause febrile disease in humans. Among these, Oropouche orthobunyavirus (OROV) is a relatively understudied member of the genus Orthobunyavirus, family Peribunyaviridae, that causes periodic outbreaks in human populations in Brazil and other South American countries. Although several studies have described the genetic diversity of the virus, the evolutionary processes that shape the OROV genome remain poorly understood. Here, we present a comprehensive study of the genomic dynamics of OROV that encompasses phylogenetic analysis, evolutionary rate estimates, inference of natural selective pressures, recombination and reassortment, and structural analysis of OROV variants. Our study includes all available published sequences, as well as a set of new OROV genome sequences obtained from patients in Ecuador, representing the first set of genomes from this country. Our results show differing evolutionary processes on the three segments that comprise the viral genome. We infer differing times of the most recent common ancestors of the genome segments and propose that this can be explained by cryptic reassortment. We also present the discovery of previously unobserved putative N-linked glycosylation sites, as well as codons that evolve under positive selection on the viral surface proteins, and discuss the potential role of these features in the evolution of OROV through a combined phylogenetic and structural approach.IMPORTANCE The emergence and reemergence of pathogens such as Zika virus, chikungunya virus, and yellow fever virus have drawn attention toward other cocirculating arboviruses in South America. Oropouche virus (OROV) is a poorly studied pathogen responsible for over a dozen outbreaks since the early 1960s and represents a public health burden to countries such as Brazil, Panama, and Peru. OROV is likely underreported since its symptomatology can be easily confounded with other febrile illnesses (e.g., dengue fever and leptospirosis) and point-of-care testing for the virus is still uncommon. With limited data, there is a need to optimize the information currently available. Analysis of OROV genomes can help us understand how the virus circulates in nature and can reveal the evolutionary forces that shape the genetic diversity of the virus, which has implications for molecular diagnostics and the design of potential vaccines.

Isolation of Oropouche Virus from Febrile Patient, Ecuador.

We report identification of an Oropouche virus strain in a febrile patient from Ecuador by using metagenomic sequencing and real-time reverse transcription PCR. Virus was isolated from patient serum by using Vero cells. Phylogenetic analysis of the whole-genome sequence showed the virus to be similar to a strain from Peru.

Pathogenesis of bat rabies in a natural reservoir: Comparative susceptibility of the straw-colored fruit bat (Eidolon helvum) to three strains of Lagos bat virus.

Rabies is a fatal neurologic disease caused by lyssavirus infection. People are infected through contact with infected animals. The relative increase of human rabies acquired from bats calls for a better understanding of lyssavirus infections in their natural hosts. So far, there is no experimental model that mimics natural lyssavirus infection in the reservoir bat species. Lagos bat virus is a lyssavirus that is endemic in straw-colored fruit bats (Eidolon helvum) in Africa. Here we compared the susceptibility of these bats to three strains of Lagos bat virus (from Senegal, Nigeria, and Ghana) by intracranial inoculation. To allow comparison between strains, we ensured the same titer of virus was inoculated in the same location of the brain of each bat. All bats (n = 3 per strain) were infected, and developed neurological signs, and fatal meningoencephalitis with lyssavirus antigen expression in neurons. There were three main differences among the groups. First, time to death was substantially shorter in the Senegal and Ghana groups (4 to 6 days) than in the Nigeria group (8 days). Second, each virus strain produced a distinct clinical syndrome. Third, the spread of virus to peripheral tissues, tested by hemi-nested reverse transcriptase PCR, was frequent (3 of 3 bats) and widespread (8 to 10 tissues positive of 11 tissues examined) in the Ghana group, was frequent and less widespread in the Senegal group (3/3 bats, 3 to 6 tissues positive), and was rare and restricted in the Nigeria group (1/3 bats, 2 tissues positive). Centrifugal spread of virus from brain to tissue of excretion in the oral cavity is required to enable lyssavirus transmission. Therefore, the Senegal and Ghana strains seem most suitable for further pathogenesis, and for transmission, studies in the straw-colored fruit bat.

Molecular Epidemiology and Evolution of European Bat Lyssavirus 2.

Bat rabies cases in Europe are mainly attributed to two lyssaviruses, namely European Bat Lyssavirus 1 (EBLV-1) and European Bat Lyssavirus 2 (EBLV-2). Prior to the death of a bat worker in Finland in 1985, very few bat rabies cases were reported. Enhanced surveillance in the two subsequent years (1986-1987) identified 263 cases (more than a fifth of all reported cases to date). Between 1977 and 2016, 1183 cases of bat rabies were reported, with the vast majority (>97%) being attributed to EBLV-1. In contrast, there have been only 39 suspected cases of EBLV-2, of which 34 have been confirmed by virus typing and presently restricted to just two bat species; Myotis daubentonii and Myotis dasycneme. The limited number of EBLV-2 cases in Europe prompted the establishment of a network of European reference laboratories to collate all available viruses and data. Despite the relatively low number of EBLV-2 cases, a large amount of anomalous data has been published in the scientific literature, which we have here reviewed and clarified. In this review, 29 EBLV-2 full genome sequences have been analysed to further our understanding of the diversity and molecular evolution of EBLV-2 in Europe. Analysis of the 29 complete EBLV-2 genome sequences clearly corroborated geographical relationships with all EBLV-2 sequences clustering at the country level irrespective of the gene studied. Further geographical clustering was also observed at a local level. There are high levels of homogeneity within the EBLV-2 species with nucleotide identities ranging from 95.5-100% and amino acid identities between 98.7% and 100%, despite the widespread distribution of the isolates both geographically and chronologically. The mean substitution rate for EBLV-2 across the five concatenated genes was 1.65 × 10-5, and evolutionary clock analysis confirms the slow evolution of EBLV-2 both between and within countries in Europe. This is further supported by the first detailed EBLV-2 intra-roost genomic analysis whereby a relatively high sequence homogeneity was found across the genomes of three EBLV-2 isolates obtained several years apart (2007, 2008, and 2014) from M. daubentonii at the same site (Stokesay Castle, Shropshire, UK).

Complete Genomic Sequence of Canine Distemper Virus from an Ethiopian Wolf.

Canine distemper virus (CDV) has been implicated in population declines of wildlife, including many threatened species. Here we present the full genome of CDV from an Ethiopian wolf, Canis simensis, the world's rarest and most endangered canid.