Search

Abstract Background In young people with spastic diplegic cerebral palsy weakness of the hip extensor muscles are associated with limitations in activity. It is important that clinicians can reliably measure hip extensor muscle strength to monitor changes over time and the effects of any interventions. Previous research has demonstrated high reliability for measuring strength of all muscles of the lower limb, with the exception of the hip extensors. Therefore the aim of this study was to examine the retest reliability of measuring hip extensor strength in young people with cerebral palsy. Methods Using a test-retest reliability research design, 19 participants with spastic diplegic cerebral palsy (Gross Motor Function Classification System Levels II and III) (mean 19 y 2 mo [S D 2 y 5 mo]) attended two testing sessions held 12 weeks apart. Three trials with a hand-held dynamometer were taken at each testing session in supine, prone and standing. Retest reliability was calculated with Intraclass Correlation Coefficients (ICC(2,1)) and with units of measurement (kilograms) converted to a percentage strength change. Results ICC values ranged from .74 to .78 in supine, .75 to .80 in prone, and .73 to .75 in standing. To be 95% confident that real change had occurred, an individual's strength would need to increase 55 to 60% in supine, 86 to 102% in prone, and 102 to 105% in standing. To be 95% confident that real change had occurred across groups, strength would need to increase 4 to 8% in supine, 22 to 31% in prone, and 32% to 34% in standing. Higher ICC values were observed when three trials were used for testing. Conclusions The supine testing position was more reliable than the prone or standing testing positions. It is possible to measure hip extensor strength with sufficient reliability to be able monitor change within groups using the supine position provided three trials are used during testing. However, there is insufficient reliability to monitor changes in hip extensor strength in individuals with cerebral palsy unless they exhibit very large strength increases.

Abstract Background Balance dysfunction and falls are common problems in later stages of dementia. Exercise is a well-established intervention to reduce falls in cognitively intact older people, although there is limited randomised trial evidence of outcomes in people with dementia. The primary objective of this study is to evaluate whether a home-based balance exercise programme improves balance performance in people with mild to moderate severity Alzheimer's disease. Methods/design Two hundred and fourteen community dwelling participants with mild to moderate severity Alzheimer's disease will be recruited for the randomised controlled trial. A series of laboratory and clinical measures will be used to evaluate balance and mobility performance at baseline. Participants will then be randomized to receive either a balance training home exercise programme (intervention group) from a physiotherapist, or an education, information and support programme from an occupational therapist (control group). Both groups will have six home visits in the six months following baseline assessment, as well as phone support. All participants will be re-assessed at the completion of the programme (after six months), and again in a further six months to evaluate sustainability of outcomes. The primary outcome measures will be the Limits of Stability (a force platform measure of balance) and the Step Test (a clinical measure of balance). Secondary outcomes include other balance and mobility measures, number of falls and falls risk measures, cognitive and behavioural measures, and carer burden and quality of life measures. Assessors will be blind to group allocation. Longitudinal change in balance performance will be evaluated in a sub-study, in which the first 64 participants of the control group with mild to moderate severity Alzheimer's disease, and 64 age and gender matched healthy participants will be re-assessed on all measures at initial assessment, and then at 6, 12, 18 and 24 months. Discussion By introducing a balance programme at an early stage of the dementia pathway, when participants are more likely capable of safe and active participation in balance training, there is potential that balance performance will be improved as dementia progresses, which may reduce the high falls risk at this later stage. If successful, this approach has the potential for widespread application through community based services for people with mild to moderate severity Alzheimer's disease. Trial registration The protocol for this study is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12608000040369).

Crimean-Congo hemorrhagic fever virus (CCHFV) is a widely distributed tickborne zoonotic agent that infects a variety of host species. There is a lack of information on the true geographic distribution of the prevalence and risk of CCHFV in West Africa. A countrywide cross-sectional study involving 1413 extensively managed indigenous small ruminants and cattle at livestock sales markets and in village herds, respectively, was carried out in The Gambia. In sheep, an overall anti-CCHFV antibody prevalence of 18.9% (95% CI: 15.5-22.8%), goats 9.0% (95% CI: 6.7-11.7%), and cattle 59.9% (95% CI: 54.9-64.7%) was detected. Significant variation ( p < 0.05) in the prevalence of anti-CCHFV antibodies at sites in the five administrative regions (sheep: 4.8-25.9%; goats: 1.8-17.1%) and three agroecological zones (sheep: 8.9-32.9%; goats: 4.1-18.0%) was also observed. Comparatively, higher anti-CCHFV antibody prevalence was detected in cattle (33.3-84.0%) compared to small ruminants (1.8-8.1%). This study represents the first countrywide investigation of the seroprevalence of CCHFV in The Gambia, and the results suggest potential circulation and endemicity of the virus in the country. These data provide critical information vital to the development of informed policies for the surveillance, diagnosis, and control of CCFHV infection in The Gambia and the region.

The sustained spread of African swine fever (ASF) virus throughout much of the world has made ASF a global animal health priority, with an increased emphasis on enhancing preparedness to prevent, detect and respond to a potential outbreak of ASF virus (ASFV). In the event of ASFV entry to the North American swine population, enhanced surveillance and diagnostic testing strategies will be critical to facilitate progressive response and eradication of the disease. Compared to individual animal sampling, pen-based oral fluid collection for active surveillance is a non-invasive alternative that is less resource and time-intensive. To evaluate the feasibility of using rope-based oral fluid for early detection of ASFV, four independent animal experiments were conducted in weaned pigs housed in numbers that mimic the industry settings, utilising either highly virulent ASFV Georgia 2007/1 strain or moderately virulent ASFV Malta'78 strain. Pen-based oral fluid and individual oropharyngeal swabs were collected daily and blood samples from each animal were collected every other day. All samples were subsequently tested for ASFV by real-time PCR. ASFV genome was detected in individual blood samples as early as one day post-infection and detected in oral fluids at low-to-moderate levels as early as 3-5 days post-infection in all four independent experiments. These results suggest that pen-based oral fluid samples may be used to supplement the use of traditional samples for rapid detection of ASFV during ASF surveillance., (© 2021 Her Majesty the Queen in Right of Canada. Transboundary and Emerging Diseases © 2021 Wiley-VCH GmbH. Reproduced with the permission of the Minister of Agriculture and Agri food Canada. This article has been contributed to by US Government employees and their work is in the public domain in the USA.)

An outbreak of rabbit hemorrhagic disease virus 2 (RHDV2)-associated disease occurred in the southwestern United States following its first detection in New Mexico in March 2020. The disease spread throughout several states and was diagnosed for the first time in California on May 11, 2020, in a black-tailed jackrabbit ( Lepus californicus ). The following day, the California Department of Food and Agriculture (CDFA) issued an order banning the entrance into California of several lagomorph species and their products from any state in which the disease had been detected in the last 12 mo. RHDV2 is a threat to wild lagomorph species in California, including the endangered riparian brush rabbit ( Sylvilagus bachmani riparius ). Therefore, the California Department of Fish and Wildlife (CDFW) started tracking any mortality event in wild lagomorph populations. As of August 9, 2020, RHDV2 had been detected in wild and domestic lagomorphs of several counties in southern California that were submitted to the California Animal Health and Food Safety laboratory system by the CDFA or the CDFW. These positive cases included 2 additional black-tailed jackrabbits and 3 desert cottontail rabbits ( Sylvilagus audubonii ). In addition, the infection spilled over to domestic populations, whereby it was confirmed on July 10, 2020, in a domestic rabbit ( Oryctolagus cuniculus ).

Five rabbit hemorrhagic disease virus type 2 (RHDV2) coding-complete genome sequences were obtained from the livers of domestic and wild rabbits during the 2020 outbreak in the United States. These represent the first available RHDV2 sequences from the United States.

Binary ethylenimine (BEI) has been widely used as a virucide to inactivate viruses. For regulatory exclusion of a select agent, the United States Federal Select Agent Program (FSAP) requires an inactivation procedure that renders a select agent non-viable but allows the select agent to retain antigenic characteristics for future use must be validated, and the inactivated agent must be confirmed by a viability testing. In this curve-based validation study, we examined impacts of BEI concentration, treatment temperature, and time on our in-house inactivation procedures of Foot-and-Mouth Disease Virus (FMDV), Vesicular Stomatitis Virus (VSV), and Swine Vesicular Disease Virus (SVDV). The inactivation efficacy was confirmed by virus titration and 3 consecutive blind passages on the monolayers of susceptible cells. A linear correlation between the virus titer reduction and BEI concentration, treatment time, and temperature was established. The results confirmed our in-house BEI inactivation procedure of two doses of 1.5 mM BEI treatment at 37 °C, 1st dose for 24 h, then 2nd dose for 6 more hours for a total of 30 h BEI contact time, can ensure complete inactivation of FMDV, VSV, and SVDV., (Published by Elsevier B.V.)

Real-time PCR assays are highly sensitive, specific and rapid techniques for the identification of ASF virus (ASFV) (Section 3.8, OIE Terrestrial Manual, 2019). Although an ASFV p72 gene-based real-time PCR assay (a.k.a. the Zsak assay) (Journal of Clinical Microbiology, 2005, 43, 112) has been widely used for ASFV detection, several more ASFV whole genome sequences have become available in the 15 years since the design of the Zsak assay. In this study, we developed a new ASFV p72 gene-based real-time PCR after analysis of all currently available sequences of the p72 gene and multiplexed the new assay with a modified Zsak assay aiming to have a broader coverage of ASFV strain/isolates. To reduce false-negative detections, porcine house-keeping gene, beta actin (ACTB), was applied as an internal control. Eight ACTB sequences from the GenBank and 61 partial ACTB sequences generated in this study, and 1,012 p72 sequences from the GenBank and 23 p72 sequences generated at FADDL, were used for ACTB and ASFV primer and probe designs, respectively, to ensure broader host and ASFV coverage. Multiplexing ACTB in the reaction did not affect ASFV amplification. The multiplex assay was evaluated for strain/isolate coverage, sensitivity and specificity. The in silico analysis showed high ASFV strain/isolate coverage: 98.4% (978/994) of all p72 sequences currently available. The limit of detection (LOD) was 6 plasmid copies or 0.1-1 TCID 50 /ml of ASFV isolates per reaction. Only targeted ASFV isolates and the viruses in the positive clinical samples were detected, indicating that the assay is highly specific (100% specificity). The test results of 26 ASFV isolates with different country origins showed that this newly developed multiplex assay performed better than the Zsak assay that has been widely accepted and used worldwide, indicating that it may be used as an alternative assay for ASFV detection., (© 2020 Blackwell Verlag GmbH.)

We report the genomes of three vesicular stomatitis Indiana virus (VSIV) isolates collected from naturally infected bovines in Wyoming and Colorado during the 2019 outbreak in the United States. These genomes support molecular diagnostic efforts and provide data on the spread and ecology of VSIV in the United States.

African swine fever (ASF) is one of the most complex and lethally haemorrhagic viral diseases of swine, affecting all breeds and ages of pigs. In the absence of ASF vaccines, reliable laboratory diagnosis and restricted biosecurity are critical for disease prevention and control. A detection of ASF-specific antibodies in an unvaccinated pig is a good marker for the diagnosis of ASF. The immunoperoxidase test (IPT) is a sensitive test for detecting ASF virus (ASFV) antibodies. However, due to the complexity of the procedure, the IPT is only suitable to be used as a confirmatory test. The ASFV p30 protein-based enzyme-linked immunosorbent assay (ELISA) is widely used for ASFV antibody screening, but the sensitivity is not comparable to the IPT. It is essential to have a better understanding of the antigenic properties of ASFV p30 to improve p30-based serologic tests. In this study, we developed a panel of 21 monoclonal antibodies (mAbs) against ASFV p30. With 14 out of the 21 mAbs, we defined 4 antigenic regions that contain at least 4 linear epitopes. Nine of the 14 mAbs mapped to antigenic regions 3 and 4 reacted with p30 in all serologic methods tested in this study, such as indirect immunofluorescence assay (IFA), ELISA and Western blot. The antigenic regions 3 and 4 are highly conserved and immunodominant in host antibody response. These mAbs and the defined p30 antigenic regions 3 and 4 provide valuable tools for the development and improvement of ASF serologic assays., (© 2020 Blackwell Verlag GmbH.)

African swine fever virus (ASFV) is the causative agent of a severe and highly contagious viral disease of pigs that poses serious economic consequences to the swine industry due to the high mortality rate and impact on international trade. There is no effective vaccine to control African swine fever (ASF), and therefore, efficient disease control is dependent on early detection and diagnosis of ASFV. The large size of the ASFV genome (∼180 kb) has historically hindered efforts to rapidly obtain a full-genome sequence. Rapid acquisition of data is critical for characterization of the isolate and to support epidemiological efforts. Here, we investigated the capacity of the Oxford Nanopore MinION sequence sensing device to act as a rapid sequencing tool. When coupled with our novel companion software script, the African swine fever fast analysis sequencing tool (ASF-FAST), the analysis of output data was performed in real time. Complete ASFV genome sequences were generated from cell culture isolates and blood samples obtained from experimentally infected pigs. Removal of the host-methylated DNA from the extracted nucleic acid facilitated rapid ASFV sequence identification, with reads specific to ASFV detected within 6 min after the initiation of sequencing. Regardless of the starting material, sufficient sequence was available for complete genome resolution (up to 100%) within 10 min. Overall, this paper highlights the use of Nanopore sequencing technology in combination with the ASF-FAST software for the purpose of rapid and real-time resolution of the full ASFV genome from a diagnostic sample.

The skin/implant interface of osseointegrated (OI) implants is susceptible to infection, causing excess pain, increased morbidity, and possibly implant removal. Novel distal femoral OI implants with binary nitride coatings have been developed with little physiological modeling to collect microbiological evidence of resistance to bacterial attachment. This in vitro study evaluated a Ti-6Al-4V alloy coated with TiNbN and treated with low plasticity burnishing (LPB) to assess attachment and biofilm formation of methicillin-resistant Staphylococcus aureus (MRSA) under physiologically modeling conditions compared to standard Ti-6Al-4V alloy materials with a polished ("Color Buff") or non-polished finish ("Satin Finish"). Washability of the materials were also assessed and compared. It was hypothesized that the TiNbN/LPB treatments would resist bacterial adhesion and biofilm formation to a greater degree than the other two materials, and have a higher degree of bacterial removal following a clinically relevant wash regimen. Material types were exposed to a constant flow of broth containing MRSA and were analyzed using bacterial quantification, surface coverage analysis, and SEM imaging. Quantification data showed no difference in bacterial attachment among the varying material types both with and without the wash regimen. Surface coverage and SEM analysis confirmed results. The wash regimen led to an approximately 3 log 10 reduction in bacteria for all material types. Though the results did not support the hypothesis that a TiNbN coating/LPB treatment might resist bacterial attachment/biofilm formation more than other alloys, or have less bacteria after cleaning, results did support the potential importance of a daily wound-hygiene regimen at the skin/implant interface of OI materials. Published 2018. This article is a U.S. Government work and is in the public domain in the USA., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Background: Changes in the mechanical behaviour of the Achilles and patellar tendons in tendinopathy could affect muscle performance, and have implications for injury prevention and rehabilitation strategies., Objectives: To determine the effect of clinically diagnosed tendinopathy on the mechanical and material properties of the Achilles tendon (AT) and patellar tendon (PT)., Design: Systematic review with meta-analysis., Methods: A search of electronic databases (SPORTDiscus, CINAHL, PubMed, ScienceDirect and Google Scholar) was conducted to identify research articles that reported local and global in vivo mechanical (e.g. strain, stiffness) and/or material properties (e.g. modulus) of the AT and/or PT in people with and without tendinopathy. Effect sizes and corresponding 95% confidence intervals for individual studies were calculated for tendon strain, stiffness, modulus and cross-sectional area., Results: Eighteen articles met the inclusion criteria (AT only = 11, PT only = 5, AT and PT = 2). There was consistent evidence that the reported AT strain was higher in people with tendinopathy, compared to asymptomatic controls. People with Achilles tendinopathy had a lower AT global stiffness, lower global modulus and lower local modulus, compared to asymptomatic controls. In contrast, there was no clear and consistent evidence that the global or local mechanical or material properties of the PT are altered in tendinopathy., Conclusions: The in vivo mechanical and material properties of the Achilles tendon-aponeurosis are altered in tendinopathy, compared to asymptomatic tendons. Despite a similar clinical presentation to Achilles tendinopathy, patellar tendinopathy does not appear to alter the tensile behaviour of the PT in vivo.

During the Ebola virus outbreak of 2013-2016, the Viral Special Pathogens Branch field laboratory in Sierra Leone tested approximately 26 000 specimens between August 2014 and October 2015. Analysis of the B2M endogenous control Ct values showed its utility in monitoring specimen quality, comparing results with different specimen types, and interpretation of results. For live patients, blood is the most sensitive specimen type and oral swabs have little diagnostic utility. However, swabs are highly sensitive for diagnostic testing of corpses., (Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Hantavirus pulmonary syndrome (HPS) is a severe disease caused by hantavirus infection of pulmonary microvascular endothelial cells leading to microvascular leakage, pulmonary edema, pleural effusion and high case fatality. Previously, we demonstrated that Andes virus (ANDV) infection caused up-regulation of vascular endothelial growth factor (VEGF) and concomitant downregulation of the cellular adhesion molecule VE-cadherin leading to increased permeability. Analyses of human HPS-patient sera have further demonstrated increased circulating levels of VEGF. Here we investigate the impact of a small molecule antagonist of the VEGF receptor 2 (VEGFR-2) activation in vitro, and overall impact on survival in the Syrian hamster model of HPS., (Copyright © 2016. Published by Elsevier B.V.)

Animal models recapitulating human Ebola virus disease (EVD) are critical for insights into virus pathogenesis. Ebola virus (EBOV) isolates derived directly from human specimens do not, without adaptation, cause disease in immunocompetent adult rodents. Here, we describe EVD in mice engrafted with human immune cells (hu-BLT). hu-BLT mice developed EVD following wild-type EBOV infection. Infection with high-dose EBOV resulted in rapid, lethal EVD with high viral loads, alterations in key human antiviral immune cytokines and chemokines, and severe histopathologic findings similar to those shown in the limited human postmortem data available. A dose- and donor-dependent clinical course was observed in hu-BLT mice infected with lower doses of either Mayinga (1976) or Makona (2014) isolates derived from human EBOV cases. Engraftment of the human cellular immune system appeared to be essential for the observed virulence, as nonengrafted mice did not support productive EBOV replication or develop lethal disease. hu-BLT mice offer a unique model for investigating the human immune response in EVD and an alternative animal model for EVD pathogenesis studies and therapeutic screening., (Published by Oxford University Press for the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

In August 2014, the Viral Special Pathogens Branch of the US Centers for Disease Control and Prevention established a field laboratory in Sierra Leone in response to the ongoing Ebola virus outbreak. Through March 2015, this laboratory tested >12 000 specimens from throughout Sierra Leone. We describe the organization and procedures of the laboratory located in Bo, Sierra Leone., (Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Many U.S. medical schools have developed curricula in geriatric medicine to address the growing older adult population. At our university, the authors have integrated an assisted living facility (ALF) program into a required first-year clinical skills course. During the 2011 to 2012 academic year, an electronic survey was distributed to 109 first-year medical students prior to and after the program. Eighty-eight percent and 85% of students completed the pre- and postintervention survey, respectively. Students reported a positive attitude toward caring for older adults (92.5% post- vs. 80.2% preintervention), an understanding of the medical and social needs of older adults (89.2% post- vs. 38.5% preintervention), an acquisition of the skills to assess the health of older adults (71% post- vs. 14.5% preintervention), and an understanding of ALFs as nonmedical supportive housing (92.5% post- vs. 70.8% preintervention). The authors' curriculum offers an innovative method to integrate geriatrics education early in medical education and to involve medical students in their community.

Background: Kyasanur Forest disease virus (KFDV) and Alkhurma hemorrhagic fever virus (AHFV) are closely related members of the Flavivirus genus and are important causes of human disease in India and the Arabian Peninsula, respectively. Despite high genetic similarity, the viruses have distinctly different host ranges and ecologies. Human cases of KFDV or AHFV develop a spectrum of disease syndromes ranging from liver pathology to neurologic disease. Case reports suggest KFDV is more commonly associated with hepatic and gastrointestinal manifestations whereas AHFV is more commonly associated with neurologic disease., Methodology/principal Findings: Inoculation of three immunocompetent laboratory mouse strains revealed that KFDV was consistently more lethal than AHFV. In subsequent studies utilizing C57BL/6J mice, we demonstrated that KFDV infection was associated with higher viral loads and significantly higher mortality. KFDV-infected mice rapidly developed more severe disease than AHFV-infected mice, as evidenced by significant abnormalities on clinical chemistry panels and more severe pathology in the brain and gastrointestinal tract., Conclusions/significance: Infections of C57BL/6J mice with KFDV or AHFV resulted in clinical disease syndromes that closely approximate the diseases seen in human cases. Despite high genetic similarity, there were clear differences in survival, viral kinetics, clinical chemistry data and histology. These results suggest that distinct mouse models for AHFV and KFDV are necessary in order to gain a better understanding of the unique pathogenesis of each virus, as well as to provide platforms for testing promising vaccines and therapeutics.

Background: Rift Valley fever virus (RVFV) causes outbreaks of severe disease in livestock and humans throughout Africa and the Arabian Peninsula. In people, RVFV generally causes a self-limiting febrile illness but in a subset of individuals, it progresses to more serious disease. One manifestation is a delayed-onset encephalitis that can be fatal or leave the afflicted with long-term neurologic sequelae. In order to design targeted interventions, the basic pathogenesis of RVFV encephalitis must be better understood., Methodology/principal Findings: To characterize the host immune responses and viral kinetics associated with fatal and nonfatal infections, mice were infected with an attenuated RVFV lacking NSs (ΔNSs) that causes lethal disease only when administered intranasally (IN). Following IN infection, C57BL/6 mice developed severe neurologic disease and succumbed 7-9 days post-infection. In contrast, inoculation of ΔNSs virus subcutaneously in the footpad (FP) resulted in a subclinical infection characterized by a robust immune response with rapid antibody production and strong T cell responses. IN-inoculated mice had delayed antibody responses and failed to clear virus from the periphery. Severe neurological signs and obtundation characterized end stage-disease in IN-inoculated mice, and within the CNS, the development of peak virus RNA loads coincided with strong proinflammatory responses and infiltration of activated T cells. Interestingly, depletion of T cells did not significantly alter survival, suggesting that neurologic disease is not a by-product of an aberrant immune response., Conclusions/significance: Comparison of fatal (IN-inoculated) and nonfatal (FP-inoculated) ΔNSs RVFV infections in the mouse model highlighted the role of the host immune response in controlling viral replication and therefore determining clinical outcome. There was no evidence to suggest that neurologic disease is immune-mediated in RVFV infection. These results provide important insights for the future design of vaccines and therapeutic options.

No antiviral therapies are available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). We tested compounds reported to have antiviral activity against members of the Flaviviridae family for their ability to inhibit AHFV replication. 6-Azauridine (6-azaU), 2'-C-methylcytidine (2'-CMC), and interferon alpha 2a (IFN-α2a) inhibited the replication of AHFV and also KFDV, OHFV, and Powassan virus. The combination of IFN-α2a and 2'-CMC exerted an additive antiviral effect on AHFV, and the combination of IFN-α2a and 6-azaU was moderately synergistic. The combination of 2'-CMC and 6-azaU was complex, being strongly synergistic but with a moderate level of antagonism. The antiviral activity of 6-azaU was reduced by the addition of cytidine but not guanosine, suggesting that it acted by inhibiting pyrimidine biosynthesis. To investigate the mechanism of action of 2'-CMC, AHFV variants with reduced susceptibility to 2'-CMC were selected. We used a replicon system to assess the substitutions present in the selected AHFV population. A double NS5 mutant, S603T/C666S, and a triple mutant, S603T/C666S/M644V, were more resistant to 2'-CMC than the wild-type replicon. The S603T/C666S mutant had a reduced level of replication which was increased when M644V was also present, although the replication of this triple mutant was still below that of the wild type. The S603 and C666 residues were predicted to lie in the active site of the AHFV NS5 polymerase, implicating the catalytic center of the enzyme as the binding site for 2'-CMC., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. Human RVFV infections generally manifest as a self-limiting febrile illness, but in some individuals, the disease can progress to a fatal encephalitis or hemorrhagic syndrome. Little is known about the host characteristics that predispose development of more severe disease. Early in infection, interferon-mediated antiviral responses are critical for controlling RVFV replication, but the roles of downstream adaptive immune responses in determining clinical outcome have not been examined. Here, using a C57BL/6 mouse disease model, we evaluated the roles of B cells and T cells in RVFV pathogenesis. Given the profound inhibition of the innate response by the viral NSs protein and rapid course of wild-type infection, we utilized an attenuated RVFV lacking NSs to examine host responses following primary infection. Experiments utilizing B-cell-deficient mice or targeted T cell depletions of wild-type mice demonstrated that B cells and CD4(+) T cells, but not CD8(+) T cells, were critical for mediating viral clearance, even in the presence of a functional innate response. One-third of CD4-depleted mice developed severe neurologic disease following infection, in contrast to virus-infected mock-depleted mice that showed no clinical signs. CD4(+) T cells were required for robust IgG and neutralizing antibody responses that correlated with RVFV clearance from peripheral tissues. Furthermore, CD4-depleted mice demonstrated significantly stronger proinflammatory responses relative to controls, suggesting CD4(+) T cells regulate immune responses to RVFV infection. Together, these results indicate CD4(+) T cells are critical determinants of RVFV pathogenesis and play an important role in preventing onset of neurologic disease.

Arenaviruses are rodent-borne viruses with a bisegmented RNA genome. A genetically unique arenavirus, Lujo virus, was recently discovered as the causal agent of a nosocomial outbreak of acute febrile illness with hemorrhagic manifestations in Zambia and South Africa. The outbreak had a case fatality rate of 80%. A reverse genetics system to rescue infectious Lujo virus from cDNA was established to investigate the biological properties of this virus. Sequencing the genomic termini showed unique nucleotides at the 3' terminus of the S segment promoter element. While developing this system, we discovered that reconstructing infectious Lujo virus using the previously reported L segment intergenic region (IGR), comprising the arenaviral transcription termination signal, yielded an attenuated Lujo virus. Resequencing revealed that the correct L segment IGR was 36 nucleotides longer, and incorporating it into the reconstructed Lujo virus restored the growth rate to that of the authentic clinical virus isolate. These additional nucleotides were predicted to more than double the free energy of the IGR main stem-loop structure. In addition, incorporating the newly determined L-IGR into a replicon reporter system enhanced the expression of a luciferase reporter L segment. Overall, these results imply that an extremely stable secondary structure within the L-IGR is critical for Lujo virus propagation and viral protein production. The technology for producing recombinant Lujo virus now provides a method to precisely investigate the molecular determinants of virulence of this newly identified pathogen.

Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The potential for RVFV introduction outside the area of endemicity highlights the need for fast-acting, safe, and efficacious vaccines. Here, we demonstrate a robust system for the reverse genetics generation of a RVF virus replicon particle (VRP(RVF)) vaccine candidate. Using a mouse model, we show that VRP(RVF) immunization provides the optimal balance of safety and single-dose robust efficacy. VRP(RVF) can actively synthesize viral RNA and proteins but lacks structural glycoprotein genes, preventing spread within immunized individuals and reducing the risk of vaccine-induced pathogenicity. VRP(RVF) proved to be completely safe following intracranial inoculation of suckling mice, a stringent test of vaccine safety. Single-dose subcutaneous immunization with VRP(RVF), although it is highly attenuated, completely protected mice against a virulent RVFV challenge dose which was 100,000-fold greater than the 50% lethal dose (LD(50)). Robust protection from lethal challenge was observed by 24 h postvaccination, with 100% protection induced in as little as 96 h. We show that a single subcutaneous VRP(RVF) immunization initiated a systemic antiviral state followed by an enhanced adaptive response. These data contrast sharply with the much-reduced survivability and immune responses observed among animals immunized with nonreplicating viral particles, indicating that replication, even if confined to the initially infected cells, contributes substantially to protective efficacy at early and late time points postimmunization. These data demonstrate that replicon vaccines successfully bridge the gap between safety and efficacy and provide insights into the kinetics of antiviral protection from RVFV infection.

Lujo virus (LUJV) is a novel member of the Arenaviridae family that was first identified in 2008 after an outbreak of severe hemorrhagic fever (HF). In what was a small but rapidly progressing outbreak, this previously unknown virus was transmitted from the critically ill index patient to 4 attending healthcare workers. Four persons died during this outbreak, for a total case fatality of 80% (4/5). The suspected rodent source of the initial exposure to LUJV remains a mystery. Because of the ease of transmission, high case fatality, and novel nature of LUJV, we sought to establish an animal model of LUJV HF. Initial attempts in mice failed, but infection of inbred strain 13/N guinea pigs resulted in lethal disease. A total of 41 adult strain 13/N guinea pigs were infected with either wild-type LUJV or a full-length recombinant LUJV. Results demonstrated that strain 13/N guinea pigs provide an excellent model of severe and lethal LUJV HF that closely resembles what is known of the human disease. All infected animals experienced consistent weight loss (3-5% per day) and clinical illness characterized by ocular discharge, ruffled fur, hunched posture, and lethargy. Uniform lethality occurred by 11-16 days post-infection. All animals developed disseminated LUJV infection in various organs (liver, spleen, lung, and kidney), and leukopenia, lymphopenia, thrombocytopenia, coagulopathy, and elevated transaminase levels. Serial euthanasia studies revealed a temporal pattern of virus dissemination and increasing severity of disease, primarily targeting the liver, spleen, lungs, and lower gastrointestinal tract. Establishing an animal LUJV model is an important first step towards understanding the high pathogenicity of LUJV and developing vaccines and antiviral therapeutic drugs for this highly transmissible and lethal emerging pathogen.

Rift Valley fever virus (RVFV) is a mosquito-borne human and veterinary pathogen causing large outbreaks of severe disease throughout Africa and the Arabian Peninsula. Safe and effective vaccines are critically needed, especially those that can be used in a targeted one-health approach to prevent both livestock and human disease. We report here on the safety, immunogenicity, and efficacy of the ΔNSs-ΔNSm recombinant RVFV (rRVFV) vaccine (which lacks the NSs and NSm virulence factors) in a total of 41 sheep, including 29 timed-pregnant ewes. This vaccine was proven safe and immunogenic for adult animals at doses ranging from 1.0 × 10(3) to 1.0 × 10(5) PFU administered subcutaneously (s.c.). Pregnant animals were vaccinated with 1.0 × 10(4) PFU s.c. at day 42 of gestation, when fetal sensitivity to RVFV vaccine-induced teratogenesis is highest. No febrile reactions, clinical illness, or pregnancy loss was observed following vaccination. Vaccination resulted in a rapid increase in anti-RVFV IgM (day 4) and IgG (day 7) titers. No seroconversion occurred in cohoused control animals. A subset of 20 ewes progressed to full-term delivery after vaccination. All lambs were born without musculoskeletal, neurological, or histological birth defects. Vaccine efficacy was assessed in 9 pregnant animals challenged at day 122 of gestation with virulent RVFV (1.0 × 10(6) PFU intravenously). Following challenge, 100% (9/9) of the animals were protected, progressed to full term, and delivered healthy lambs. As expected, all 3 sham-vaccinated controls experienced viremia, fetal death, and abortion postchallenge. These results demonstrate that the ΔNSs-ΔNSm rRVFV vaccine is safe and nonteratogenic and confers high-level protection in sheep.

Candid1, a live-attenuated Junin virus vaccine strain, was developed during the early 1980s to control Argentine hemorrhagic fever, a severe and frequently fatal human disease. Six amino acid substitutions were found to be unique to this vaccine strain, and their role in virulence attenuation in mice was analyzed using a series of recombinant viruses. Our results indicate that Candid1 is attenuated in mice through a single amino acid substitution in the transmembrane domain of the G2 glycoprotein. This work provides insight into the molecular mechanisms of attenuation of the only arenavirus vaccine currently available.

Background: Alkhurma hemorrhagic fever virus (AHFV) and Kyasanur forest disease virus (KFDV) cause significant human disease and mortality in Saudi Arabia and India, respectively. Despite their distinct geographic ranges, AHFV and KFDV share a remarkably high sequence identity. Given its emergence decades after KFDV, AHFV has since been considered a variant of KFDV and thought to have arisen from an introduction of KFDV to Saudi Arabia from India. To gain a better understanding of the evolutionary history of AHFV and KFDV, we analyzed the full length genomes of 16 AHFV and 3 KFDV isolates., Methodology/principal Findings: Viral genomes were sequenced and compared to two AHFV sequences available in GenBank. Sequence analyses revealed higher genetic diversity within AHFVs isolated from ticks than human AHFV isolates. A Bayesian coalescent phylogenetic analysis demonstrated an ancient divergence of AHFV and KFDV of approximately 700 years ago., Conclusions/significance: The high sequence diversity within tick populations and the presence of competent tick vectors in the surrounding regions, coupled with the recent identification of AHFV in Egypt, indicate possible viral range expansion or a larger geographic range than previously thought. The divergence of AHFV from KFDV nearly 700 years ago suggests other AHFV/KFDV-like viruses might exist in the regions between Saudi Arabia and India. Given the human morbidity and mortality associated with these viruses, these results emphasize the importance of more focused study of these significant public health threats.

Lassa virus (LASV), is a significant cause of severe, often fatal, hemorrhagic fever in humans throughout western Africa, with an estimated 100,000 infections each year. No vaccines are commercially available. We report the development of an efficient reverse genetics system to rescue recombinant LASV and to investigate the contributions of the long 5' and 3' noncoding regions (NCRs) of the S genomic segment to in vitro growth and in vivo virulence. This work demonstrates that deletions of large portions of these NCRs confer an attenuated phenotype and are a first step toward further insights into the high virulence of LASV.

The Arenaviridae are a diverse and globally distributed collection of viruses that are maintained primarily by rodent reservoirs. Junin virus (JUNV) and Lassa virus (LASV) can both cause significant outbreaks of severe and often fatal human disease throughout their respective areas of endemicity. In an effort to improve upon the existing live attenuated JUNV Candid1 vaccine, we generated a genetically homogenous stock of this virus from cDNA copies of the virus S and L segments by using a reverse genetics system. Further, these cDNAs were used in combination with LASV cDNAs to successfully generate two recombinant Candid1 JUNV/LASV chimeric viruses (via envelope glycoprotein [GPC] exchange). It was found that while the GPC extravirion domains were readily exchangeable, homologous stable signal peptide (SSP) and G2 transmembrane and cytoplasmic tail domains were essential for correct GPC maturation and production of infectious chimeric viruses. The switching of the JUNV and LASV G1/G2 ectodomains within the Candid1 vaccine background did not alter the attenuated phenotype of the vaccine strain in a lethal mouse model. These recombinant chimeric viruses shed light on the fundamental requirements of arenavirus GPC maturation and may serve as a strategy for the development of bivalent JUNV and LASV vaccine candidates.

The internal FECV→FIPV mutation theory and three of its correlates were tested in four sibs/half-sib kittens, a healthy contact cat, and in four unrelated cats that died of FIP at geographically disparate regions. Coronavirus from feces and extraintestinal FIP lesions from the same cat were always >99% related in accessory and structural gene sequences. SNPs and deletions causing a truncation of the 3c gene product were found in almost all isolates from the diseased tissues of the eight cats suffering from FIP, whereas most, but not all fecal isolates from these same cats had intact 3c genes. Other accessory and structural genes appeared normal in both fecal and lesional viruses. Deliterious mutations in the 3c gene were unique to each cat, indicating that they did not originate in one cat and were subsequently passed horizontally to the others. Compartmentalization of the parental and mutant forms was not absolute; virus of lesional type was sometimes found in feces of affected cats and virus identical to fecal type was occasionally identified in diseased tissues. Although 3c gene mutants in this study were not horizontally transmitted, the parental fecal virus was readily transmitted by contact from a cat that died of FIP to its housemate. There was a high rate of mutability in all structural and accessory genes both within and between cats, leading to minor genetic variants. More than one variant could be identified in both diseased tissues and feces of the same cat. Laboratory cats inoculated with a mixture of two closely related variants from the same FIP cat developed disease from one or the other variant, but not both. Significant genetic drift existed between isolates from geographically distinct regions of the Western US.

In addition to being the major site of cerebrospinal fluid formation, the choroid plexus epithelium emerges as an important source of polypeptides in the brain. Physiologically regulated release of some polypeptides synthesized by the choroid plexus has been shown. The molecular mechanisms underlying this polypeptide secretion have not been characterized, however. In the present study, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein, two membrane fusion proteins playing a critical role in exocytosis in neurons and endocrine cells, were found to be expressed in the choroid plexus epithelium. It was also shown that in choroidal epithelium, synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein stably interact. Two members of the vesicle-associated membrane protein family, vesicle-associated membrane protein-1 and vesicle-associated membrane protein-2, were expressed in the rat choroid plexus at the messenger RNA and protein level. However, their newly discovered isoforms, vesicle-associated membrane protein-1b and vesicle-associated membrane protein-2b, produced by alternative RNA splicing, were not detected in choroidal tissue. Immunohistochemistry demonstrated that vesicle-associated membrane protein is confined to the cytoplasm of choroidal epithelium, whereas synaptosome-associated protein of 25 kDa is associated with plasma membranes, albeit with a varied cellular distribution among species studied. Specifically, in the rat choroid plexus, synaptosome-associated protein of 25 kDa was localized to the basolateral membrane domain of choroidal epithelium and was expressed in small groups of cells. In comparison, in ovine and human choroidal tissues, apical staining for synaptosome-associated protein of 25 kDa was found in the majority of epithelial cells. These species-related differences in cellular synaptosome-associated protein of 25 kDa distribution suggested that the synaptosome-associated protein of 25 kDa homologue, synaptosome-associated protein of 23 kDa, is also expressed in the rat choroid plexus, which was confirmed by reverse-transcriptase polymerase chain reaction. Our findings suggest that synaptosome-associated protein of 25 kDa and vesicle-associated membrane protein are involved in secretion of polypeptides from the choroid plexus epithelium. The presence of synaptosome-associated protein of 25 kDa and its homologue as well as multiple isoforms of vesicle-associated membrane protein in choroidal epithelium may play a role in the apical versus basolateral targeting of secretory vesicles., (Copyright 2003 IBRO)

Vasopressin (VP) levels in cerebrospinal fluid (CSF) change in response to physiological stimuli and under various pathological conditions. The sources of CSF VP have yet to be clarified, however. In the present study, we provide evidence indicating that VP is synthesized in the choroid plexus, the primary site of CSF formation. All experiments were performed on adult male Sprague-Dawley rats. The presence of VP mRNA in choroid plexus epithelium was demonstrated by in situ hybridization histochemistry using the 35S-labeled riboprobe that was complementary to cDNA fragment of rat VP encoding the C-terminus part of proVP. In situ hybridization findings were confirmed by reverse transcriptase-polymerase chain reaction analysis. Immunohistochemistry for VP-associated neurophysin (VP-NP), a polypeptide component of proVP, revealed subapical accumulation of VP-NP-immunopositive product in choroidal epithelial cells. Immunoprecipitation and immunoblotting of choroidal protein extracts with anti-VP-NP antibody demonstrated the presence of a approximately 10-kD polypeptide that was also detected in hypothalamus. We hypothesize that the choroid plexus-derived VP exerts autocrine and/or paracrine effects on tissues near the CSF system.