Ultrasonographic diagnosis of femoral fractures in large animals.

OBJECTIVE: Femoral fractures are often catastrophic in large animals. Radiographic diagnosis is limited by patient size and feasibility, especially in ambulatory settings. Ultrasonography is widely available and may provide an alternative to radiography for definitive diagnosis. ANIMALS: 12 large animals (6 horses, 5 cattle, and 1 elephant). PROCEDURES: Retrospective analysis of large animal patients diagnosed with femoral fracture by use of femoropelvic ultrasonography (2000 to 2019). RESULTS: 5 of 12 cases were ≤ 1 year of age. The remaining 7 cases were 2 to 33 years of age (median, 13 years). All patients developed severe acute lameness after falling (n = 4), limb entrapment (2), dystocia (1), vehicular collision (1), ipsilateral full limb casting (1), or unknown events (3). All were non-weight-bearing or lame at the walk, including 2 recumbent cattle. Ten cases showed upper limb swelling that was variable in location, and 3 had nonspecific upper limb crepitus. Ultrasonography revealed evidence of diaphyseal (n = 6), greater trochanteric (2), capital physeal (2), and distal femoral (2) fractures. Fracture movement during limb manipulation or weight shifting was sonographically visualized in 5 animals. Radiography confirmed fractures in 3 of 8 animals: 2 bovines with distal femoral fractures detected on standing projections and 1 capital physeal fracture that required ventrodorsal projections under general anesthesia. All animals were euthanized (11) or slaughtered (1 bovine). Postmortem examination confirmed ultrasonographic findings in 10 of 10 necropsied animals. CLINICAL RELEVANCE: Femoral fractures were not localized nor confirmed in any case prior to ultrasonography. Study findings supported the use of ultrasonography for rapid patient-side diagnosis, prognostication, and decision-making in suspect cases.

An Investigation of Early Syphilis Among Men Who have Sex with Men: Alaska, 2018: Findings from a 2018 Rapid Ethnographic Assessment.

The state of Alaska had a sharp increase in cases of primary and secondary syphilis among gay, bisexual, and other men who have sex with men (GBMSM) in 2018, centered in Anchorage. A rapid ethnographic assessment was conducted in October 2018 to examine contextual factors contributing to local increases in syphilis. The assessment team conducted qualitative interviews with 64 (N=49 interviews) key informants in Anchorage and Matanuska-Susitna Valley identified through the STD/HIV program at the Alaska Department of Health and Social Services, Division of Public Health (ADPH):  ADPH staff (n = 11; 22%) Medical Providers (n = 18; 37%), Community-Based Organizations/Partners (n = 9; 18%), and GBMSM Community Members (n = 11; 22%). This project was deemed exempt from IRB review. Primary factors affecting syphilis transmission, care, and treatment among GBMSM were: (1) Low awareness about the current syphilis outbreak and ambivalence about syphilis and other STIs; (2) Aspects of sexual partnering such as travel, tourism, and the use of online sites and apps to facilitate anonymous sex and multiple (both sequential/concurrent) partnering; (3) The synergistic effects of substance use, homelessness, and transactional sex; (4) Choosing condomless sex; and (5) Challenges accessing healthcare, including the ability to find appropriate and culturally competent care. Syphilis increases may have been influenced by factors which spanned multiple sectors of the Anchorage community, including individual behavior, community-level risk and protective factors, and use of and interactions with resources offered by ADPH, community-based organizations, and medical providers.

Existing drugs as broad-spectrum and potent inhibitors for Zika virus by targeting NS2B-NS3 interaction.

Recent outbreaks of Zika virus (ZIKV) highlight an urgent need for therapeutics. The protease complex NS2B-NS3 plays essential roles during flaviviral polyprotein processing, and thus represents an attractive drug target. Here, we developed a split luciferase complementation-based high-throughput screening assay to identify orthosteric inhibitors that directly target flavivirus NS2B-NS3 interactions. By screening a total of 2 816 approved and investigational drugs, we identified three potent candidates, temoporfin, niclosamide, and nitazoxanide, as flavivirus NS2B-NS3 interaction inhibitors with nanomolar potencies. Significantly, the most potent compound, temoporfin, not only inhibited ZIKV replication in human placental and neural progenitor cells, but also prevented ZIKV-induced viremia and mortality in mouse models. Structural docking suggests that temoporfin potentially binds NS3 pockets that hold critical NS2B residues, thus inhibiting flaviviral polyprotein processing in a non-competitive manner. As these drugs have already been approved for clinical use in other indications either in the USA or other countries, they represent promising and easily developed therapies for the management of infections by ZIKV and other flaviviruses.

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 µM, 11.4 µM, and 4.8 µM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 µM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.

Strengthening pre-service training for skilled birth attendance - An evaluation of the maternal and child health aide training programme in Sierra Leone.

BACKGROUND: The high maternal mortality rate in Sierra Leone combined with an ongoing shortage of midwives has led to the introduction of new cadres of healthcare workers. Maternal and Child Health Aides are one such cadre and now provide 56% of patient care. The quality of the education training programme for MCHA is therefore of paramount importance if high quality maternal care is to be provided. OBJECTIVE: To conduct an evaluation of the MCHAide training programme in Sierra Leone. DESIGN: Mapping of programme and focus group discussions (FGDs) with key informants. Analysis of data using a thematic approach and formulation of recommendations for national, district and individual levels. SETTING: All 14 MCHAide schools across Sierra Leone. PARTICIPANTS: The National Coordinator, Coordinators from 14 MCHAide schools and District Health Sisters from District Health Management Teams. METHODS: Focus group discussions were held with tutors facilitated by a group member to encourage a free flowing discussion. Participants were divided into 4 groups, one for each province, with 5-8 participants per group and 50min for the discussion. RESULTS: Strengths, weaknesses and opportunities of the MCHAide training programme were identified. Four major themes were identified; the need for autonomy and support within the programme from stakeholders; the effect of poor infrastructure on teaching and student learning; the need to ensure rigorous academic quality including teaching quality, curricula content and the academic ability of the students; and the benefits of community support. CONCLUSIONS: It is important that the key personnel be involved in the development and introduction of training programmes for new cadres of staff from the earliest stages of development. On-going programme review and development is essential and those implementing the programme are the best placed to lead and contribute to this. Gathering the experiences and perceptions of key informants helps provide an in-depth examination that can inform recommendations.

'Women and babies are dying but not of Ebola': the effect of the Ebola virus epidemic on the availability, uptake and outcomes of maternal and newborn health services in Sierra Leone.

BACKGROUND: We sought to determine the impact of the Ebola virus epidemic on the availability, uptake and outcome of routine maternity services in Sierra Leone. METHODS: The number of antenatal and postnatal visits, institutional births, availability of emergency obstetric care (EmOC), maternal deaths and stillbirths were assessed by month, by districts and by level of healthcare for 10 months during, and 12 months prior to, the Ebola virus disease (EVD) epidemic. All healthcare facilities designated to provide comprehensive (n=13) or basic (n=67) EmOC across the 13 districts of Sierra Leone were included. RESULTS: Preservice students were not deployed during the EVD epidemic. The number of healthcare providers in facilities remained constant (incidence rate ratio (IRR) 1.03, 95% CI 1.00 to 1.07). Availability of antibiotics, oxytocics, anticonvulsants, manual removal of placenta, removal of retained products of conception, blood transfusion and caesarean section were not affected by the EVD epidemic. Across Sierra Leone, following the onset of the EVD epidemic, there was a 18% decrease in the number of women attending for antenatal (IRR 0.82, 95% CI 0.79 to 0.84); 22% decrease in postnatal attendance (IRR 0.78, 95% CI 0.75 to 0.80) visits and 11% decrease in the number of women attending for birth at a healthcare facility (IRR 0.89, 95% CI 0.87 to 0.91). There was a corresponding 34% increase in the facility maternal mortality ratio (IRR 1.34, 95% CI 1.07 to 1.69) and 24% increase in the stillbirth rate (IRR 1.24, 95% CI 1.14 to 1.35). CONCLUSIONS: During the EVD epidemic, fewer pregnant women accessed healthcare. For those who did, an increase in maternal mortality and stillbirth was observed. In the post-Ebola phase, 'readiness' (or not) of the global partners for large-scale epidemics has been the focus of debate. The level of functioning of the health system with regard to ability to continue to provide high-quality effective routine care needs more attention.

Novel Broad Spectrum Inhibitors Targeting the Flavivirus Methyltransferase.

The flavivirus methyltransferase (MTase) is an essential enzyme that sequentially methylates the N7 and 2'-O positions of the viral RNA cap, using S-adenosyl-L-methionine (SAM) as a methyl donor. We report here that small molecule compounds, which putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function, were identified by using virtual screening. In vitro methylation experiments demonstrated significant MTase inhibition by 13 of these compounds, with the most potent compound displaying sub-micromolar inhibitory activity. The most active compounds showed broad spectrum activity against the MTase proteins of multiple flaviviruses. Two of these compounds also exhibited low cytotoxicity and effectively inhibited viral replication in cell-based assays, providing further structural insight into flavivirus MTase inhibition.

Identification and Characterization of Novel Broad-Spectrum Inhibitors of the Flavivirus Methyltransferase.

Flavivirus methyltransferase (MTase) is essential for viral replication. Here we report the identification of small molecules through virtual screening that putatively bind to the SAM-binding site of flavivirus MTase and inhibit its function. Six of these computationally predicted binders were identified to show significant MTase inhibition with low micromolar inhibitory activity. The most active compounds showed broad-spectrum activity against the MTase proteins of other flaviviruses. Two of these compounds also showed low cytotoxicity and high antiviral efficacy in cell-based assays. Competitive binding analyses indicated that the inhibitors performed their inhibitory function through competitive binding to the SAM cofactor binding site of the MTase. The crystal structure of the MTase-inhibitor complex further supports the mode of action and provides routes for their further optimization as flavivirus MTase inhibitors.

S-adenosyl-homocysteine is a weakly bound inhibitor for a flaviviral methyltransferase.

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.

Quadraplex qRT-PCR assay for the simultaneous detection of Eastern equine encephalitis virus and West Nile virus.

In order to increase testing throughput and reduce cost, we developed a multiplex real-time assay that identifies both Eastern equine encephalitis virus and West Nile virus. The assay allows for the screening for the presence of both the nonstructural and envelope genes of both viruses simultaneously allowing for confirmatory testing to be done in a single assay. We utilized newly designed primers and probes, each labeled with a unique fluorescent label allowing for differentiation using an ABI 7500 real-time PCR machine. The use of Quanta Biosciences qScript XLT One-Step RT-qPCR® Toughmix allowed for a quadraplex assay without loss of sensitivity when compared to the previously run singleplex reaction as seen with viral RNA PFU control dilution series. There was no cross reactivity between the viruses within the reaction, and upon utilization of the assay during surveillance, there was no cross reactivity with other historically encountered arthropod-borne viruses. The results from the quantitative Reverse Transcriptase - Polymerase Chain Reaction were comparable to those achieved by cell culture which was performed on a subset of the field mosquito pools screened during the 2012 surveillance season. The multiplex assay resulted in savings in both time and resources for the lab and faster turn-around of results.

Prevalence and mutation analysis of short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD) detected on newborn screening in Wisconsin.

Short/branched chain acyl-CoA dehydrogenase deficiency (SBCADD), also called 2-methylbutyryl CoA dehydrogenase deficiency (2-MBCDD), is a disorder of l-isoleucine metabolism of uncertain clinical significance. SBCADD is inadvertently detected on expanded newborn screening by elevated 2-methylbutyrylcarnitine (C5), which has the same mass to charge (m/s) on tandem mass spectrometry (MS/MS) as isovalerylcarnitine (C5), an analyte that is elevated in isovaleric acidemia (IVA), a disorder in leucine metabolism. SBCADD cases identified in the Hmong-American population have been found in association with the c.1165 A>G mutation in the ACADSB gene. The purposes of this study were to: (a) estimate the prevalence of SBCADD and carrier frequency of the c.1165 A>G mutation in the Hmong ethnic group; (b) determine whether the c.1165 A>G mutation is common to all Hmong newborns screening positive for SBCADD; and (c) evaluate C5 acylcarnitine cut-off values to detect and distinguish between SBCADD and IVA diagnoses. During the first 10years of expanded newborn screening using MS/MS in Wisconsin (2001-2011), 97 infants had elevated C5 values (≥0.44µmol/L), of whom five were Caucasian infants confirmed to have IVA. Of the remaining 92 confirmed SBCADD cases, 90 were of Hmong descent. Mutation analysis was completed on an anonymous, random sample of newborn screening cards (n=1139) from Hmong infants. Fifteen infants, including nine who had screened positive for SBCADD based on a C5 acylcarnitine concentration ≥0.44µmol/L, were homozygous for the c.1165 A>G mutation. This corresponds to a prevalence in this ethnic group of being homozygous for the mutation of 1.3% (95% confidence interval 0.8-2.2%) and of being heterozygous for the mutation of 21.8% (95% confidence interval 19.4-24.3%), which is consistent with the Hardy-Weinberg equilibrium. Detection of homozygous individuals who were not identified on newborn screening suggests that the C5 screening cut-off would need to be as low as 0.20µmol/L to detect all infants homozygous for the ACADSB c.1165 A>G mutation. However, lowering the screening cut-off to 0.20 would also result in five "false positive" (non-homozygous) screening results in the Hmong population for every c.1165 A>G homozygote detected. Increasing the cut-off to 0.60µmol/L and requiring elevated C5/C2 (acetylcarnitine) and C5/C3 (propionylcarnitine) ratios to flag a screen as abnormal would reduce the number of infants screening positive, but would still result in an estimated 5 infants with SBCADD per year who would require follow-up and additional biochemical testing to distinguish between SBCADD and IVA diagnoses. Further research is needed to determine the clinical outcomes of SBCADD detected on newborn screening and the c.1165 A>G mutation before knowing whether the optimal screening cut-off would minimize true positives or false negatives for SBCADD associated with this mutation.

Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs.

The flavivirus methyltransferase (MTase) sequentially methylates the N-7 and 2'-O positions of the viral RNA cap (GpppA-RNA→m(7)GpppA-RNA→m(7)GpppAm-RNA), using S-adenosyl-l-methionine (SAM) as a methyl donor. We report here the synthesis and biological evaluation of a series of novel nucleoside analogs. Two of these compounds can effectively and competitively inhibit the WNV MTase with IC50 values in micromolar range and, more importantly, do not inhibit human MTase. The compounds can also suppress the WNV replication in cell culture.

Characterization of Rabensburg virus, a flavivirus closely related to West Nile virus of the Japanese encephalitis antigenic group.

Rabensburg virus (RABV), a Flavivirus with ∼76% nucleotide and 90% amino acid identity with representative members of lineage one and two West Nile virus (WNV), previously was isolated from Culex pipiens and Aedes rossicus mosquitoes in the Czech Republic, and phylogenetic and serologic analyses demonstrated that it was likely a new lineage of WNV. However, no direct link between RABV and human disease has been definitively established and the extent to which RABV utilizes the typical WNV transmission cycle is unknown. Herein, we evaluated vector competence and capacity for vertical transmission (VT) in Cx. pipiens; in vitro growth on avian, mammalian, and mosquito cells; and infectivity and viremia production in birds. RABV infection and replication only were detected on mosquito cells. Experimentally inoculated birds did not become infected. Cx. pipiens had poor peroral vector competence and a higher VT rate as compared to US-WNV in Cx. pipiens. As a result, we postulate that RABV is an intermediate between the mosquito-specific and horizontally transmitted flaviviruses.

Adaptation of two flaviviruses results in differences in genetic heterogeneity and virus adaptability.

West Nile virus (WNV) is a mosquito-borne flavivirus that was first introduced into the USA in the New York City area in 1999. Since its introduction, WNV has steadily increased both its host and geographical ranges. Outbreaks of the closely related flavivirus, St. Louis encephalitis virus (SLEV), occur in the USA periodically, but levels of activity and host range are more restricted than those of WNV. Understanding the selective pressures that drive arbovirus adaptation and evolution in their disparate mosquito and avian hosts is crucial to predicting their ability to persist and re-emerge. Here, we evaluated the in vivo phenotypes of mosquito cell-adapted WNV and SLEV. Results indicated that in vitro adaptations did not translate to in vivo adaptations for either virus, yet SLEV displayed attenuated growth in both mosquitoes and chickens, while WNV generally did not. In vitro growth analyses also indicated that WNV adaptations could be generalized to cell cultures derived from other mosquito species, while SLEV could not. Analysis of genetic diversity for passaged SLEV revealed a highly homogeneous population that differed significantly from previous results of high levels of diversity in WNV. We hypothesize that this difference in genetic diversity is directly related to the viruses' success in new and changing environments in the laboratory and that differences in a viruses' ability to produce and maintain heterogeneous populations in nature may in some instances explain the variable levels of success seen among arboviruses.

In vitro resistance selection and in vivo efficacy of morpholino oligomers against West Nile virus.

We characterize in vitro resistance to and demonstrate the in vivo efficacy of two antisense phosphorodiamidate morpholino oligomers (PMOs) against West Nile virus (WNV). Both PMOs were conjugated with an Arg-rich peptide. One peptide-conjugated PMO (PPMO) binds to the 5' terminus of the viral genome (5'-end PPMO); the other targets an essential 3' RNA element required for genome cyclization (3' conserved sequence I [3' CSI] PPMO). The 3' CSI PPMO displayed a broad spectrum of antiflavivirus activity, suppressing WNV, Japanese encephalitis virus, and St. Louis encephalitis virus, as demonstrated by reductions in viral titers of 3 to 5 logs in cell cultures, likely due to the absolute conservation of the 3' CSI PPMO-targeted sequences among these viruses. The selection and sequencing of PPMO-resistant WNV showed that the 5'-end-PPMO-resistant viruses contained two to three mismatches within the PPMO-binding site whereas the 3' CSI PPMO-resistant viruses accumulated mutations outside the PPMO-targeted region. The mutagenesis of a WNV infectious clone demonstrated that the mismatches within the PPMO-binding site were responsible for the 5'-end PPMO resistance. In contrast, a U insertion or a G deletion located within the 3'-terminal stem-loop of the viral genome was the determinant of the 3' CSI PPMO resistance. In a mouse model, both the 5'-end and 3' CSI PPMOs (administered at 100 or 200 microg/day) partially protected mice from WNV disease, with minimal to no PPMO-mediated toxicity. A higher treatment dose (300 microg/day) caused toxicity. Unconjugated PMOs (3 mg/day) showed neither efficacy nor toxicity, suggesting the importance of the peptide conjugate for efficacy. The results suggest that a modification of the peptide conjugate composition to reduce its toxicity yet maintain its ability to effectively deliver PMO into cells may improve PMO-mediated therapy.

Cell-specific adaptation of two flaviviruses following serial passage in mosquito cell culture.

West Nile Virus (WNV) is a mosquito-borne flavivirus that was introduced into the U.S. in the New York City area in 1999. Despite its successful establishment and rapid spread in a naive environment, WNV has undergone limited evolution since its introduction. This evolutionary stability has been attributed to compromises made to permit alternating cycles of viral replication in vertebrate hosts and arthropod vectors. Outbreaks of a close relative of WNV, St. Louis encephalitis virus (SLEV), occur in the U.S. periodically and are also characterized by limited genetic change overtime. We measured both phenotypic and genotypic changes in WNV and SLEV serially passaged in mosquito cell culture in order to clarify the role of an individual host cell type in flavivirus adaptation and evolution. Genetic changes in passaged WNV and SLEV were minimal but led to increased relative fitness and replicative ability of the virus in the homologous cell line C6/36 mosquito cells. Similar increases were not measured in the heterologous cell line DF-1 avian cells. These phenotypic changes are consistent with the concept of cell-specific adaptation in flaviviruses.

Virus detection protocols for west nile virus in vertebrate and mosquito specimens.

The recent outbreaks of West Nile virus (WNV) infection in the northeastern United States and other regions of the world have made it essential to develop efficient, sensitive, and rapid protocols for virus surveillance. Laboratory testing is the backbone of any surveillance program. Protocols to detect the presence of WNV have been refined since 1999 for sensitivity, speed, efficiency, and specificity. This paper presents the protocols currently used by the New York State Department of Health to handle vertebrate and mosquito specimens that have been submitted for WNV testing to the Arbovirus Laboratories of the Wadsworth Center.