Characterization of an African Swine Fever Virus Field Isolate from Vietnam with Deletions in the Left Variable Multigene Family Region.

In this paper, we report the characterization of a genetically modified live-attenuated African swine fever virus (ASFV) field strain isolated from Vietnam. The isolate, ASFV-GUS-Vietnam, belongs to p72 genotype II, has six multi-gene family (MGF) genes deleted, and an Escherichia coli GusA gene (GUS) inserted. When six 6-8-week-old pigs were inoculated with ASFV-GUS-Vietnam oro-nasally (2 × 105 TCID50/pig), they developed viremia, mild fever, lethargy, and inappetence, and shed the virus in their oral and nasal secretions and feces. One of the pigs developed severe clinical signs and was euthanized 12 days post-infection, while the remaining five pigs recovered. When ASFV-GUS-Vietnam was inoculated intramuscularly (2 × 103 TCID50/pig) into four 6-8 weeks old pigs, they also developed viremia, mild fever, lethargy, inappetence, and shed the virus in their oral and nasal secretions and feces. Two contact pigs housed together with the four intramuscularly inoculated pigs, started to develop fever, viremia, loss of appetite, and lethargy 12 days post-contact, confirming horizontal transmission of ASFV-GUS-Vietnam. One of the contact pigs died of ASF on day 23 post-contact, while the other one recovered. The pigs that survived the exposure to ASFV-GUS-Vietnam via the mucosal or parenteral route were fully protected against the highly virulent ASFV Georgia 2007/1 challenge. This study showed that ASFV-GUS-Vietnam field isolate is able to induce complete protection in the majority of the pigs against highly virulent homologous ASFV challenge, but has the potential for horizontal transmission, and can be fatal in some animals. This study highlights the need for proper monitoring and surveillance when ASFV live-attenuated virus-based vaccines are used in the field for ASF control in endemic countries.

Trophectoderm biopsy of blastocysts following IVF and embryo culture increases epigenetic dysregulation in a mouse model.

STUDY QUESTION: Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? SUMMARY ANSWER: TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. WHAT IS KNOWN ALREADY: Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. STUDY DESIGN, SIZE, DURATION: We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). PARTICIPANTS/MATERIALS, SETTING, METHODS: Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. LARGE SCALE DATA: Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. LIMITATIONS, REASONS FOR CAUTION: This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

Molecular and Pathological Characterization of Classical Swine Fever Virus Genotype 2 Strains Responsible for the 2013-2018 Outbreak in Colombia.

Classical swine fever (CSF) is a highly contagious transboundary viral disease of domestic and wild pigs. Despite mass vaccination and continuous eradication programs, CSF remains endemic in Asia, some countries in Europe, the Caribbean and South America. Since June 2013, Northern Colombia has reported 137 CSF outbreaks, mostly in backyard production systems with low vaccination coverage. The purpose of this study was to characterize the virus responsible for the outbreak. Phylogenetic analysis based on the full-length E2 sequence shows that the virus is closely related to CSF virus (CSFV) genotype 2.6 strains circulating in Southeast Asia. The pathotyping experiment suggests that the virus responsible is a moderately virulent strain. The 190 nucleotide stretch of the E2 hypervariable region of these isolates also shows high similarity to the CSFV isolates from Colombia in 2005 and 2006, suggesting a common origin for the CSF outbreaks caused by genotype 2.6 strains. The emergence of genotype 2.6 in Colombia suggests a potential transboundary spread of CSFV from Asia to the Americas, complicating the ongoing CSF eradication efforts in the Americas, and emphasizes the need for continuous surveillance in the region.

Generation and characterization of a monoclonal antibody against an African swine fever virus protein encoded by the A137R gene.

The ongoing African swine fever (ASF) pandemic continues to have a major impact on global pork production and trade. Since ASF cannot be distinguished from other swine hemorrhagic fevers clinically, ASF-specific laboratory diagnosis is critical. Thus ASF virus (ASFV)-specific monoclonal antibodies (mAbs) are critical for the development of laboratory diagnostics. In this study, we report one ASFV-specific mAb, F88ASF-55, that was generated and characterized. This mAb recognizes the ASFV A137R-encoded protein (pA137R). Epitope mapping results revealed a highly conserved linear epitope recognized by this mAb, corresponding to amino acids 111-125 of pA137R. We explored the potential use of this mAb in diagnostic applications. Using F88ASF-55 as the detection antibody, six ASFV strains were detected in an enzyme-linked immunosorbent assay (ELISA) with low background. In immunohistochemistry (IHC) assays, this mAb specifically recognized ASFV antigens in the submandibular lymph nodes of animals experimentally infected with different ASFV strains. Although not all ASFV genotypes were tested in this study, based on the conserved ASFV epitope targeted by F88ASF-55, it has the potential to detect multiple ASFV genotypes. In conclusion, this newly generated ASFV pA137R-specific mAb has potential value in ASF diagnostic tool development. It can be used in ELISA, IHC, and possibly-immunochromatographic strip assays for ASFV detection. It also suggests that pA137R may be a good target for diagnostic assays to detect ASFV infection.

Characterization of neurotropic HPAI H5N1 viruses with novel genome constellations and mammalian adaptive mutations in free-living mesocarnivores in Canada.

The GsGd lineage (A/goose/Guangdong/1/1996) H5N1 virus was introduced to Canada in 2021/2022 through the Atlantic and East Asia-Australasia/Pacific flyways by migratory birds. This was followed by unprecedented outbreaks affecting domestic and wild birds, with spillover into other animals. Here, we report sporadic cases of H5N1 in 40 free-living mesocarnivore species such as red foxes, striped skunks, and mink in Canada. The clinical presentations of the disease in mesocarnivores were consistent with central nervous system infection. This was supported by the presence of microscopic lesions and the presence of abundant IAV antigen by immunohistochemistry. Some red foxes that survived clinical infection developed anti-H5N1 antibodies. Phylogenetically, the H5N1 viruses from the mesocarnivore species belonged to clade 2.3.4.4b and had four different genome constellation patterns. The first group of viruses had wholly Eurasian (EA) genome segments. The other three groups were reassortant viruses containing genome segments derived from both North American (NAm) and EA influenza A viruses. Almost 17 percent of the H5N1 viruses had mammalian adaptive mutations (E627 K, E627V and D701N) in the polymerase basic protein 2 (PB2) subunit of the RNA polymerase complex. Other mutations that may favour adaptation to mammalian hosts were also present in other internal gene segments. The detection of these critical mutations in a large number of mammals within short duration after virus introduction inevitably highlights the need for continually monitoring and assessing mammalian-origin H5N1 clade 2.3.4.4b viruses for adaptive mutations, which potentially can facilitate virus replication, horizontal transmission and posing pandemic risks for humans.

Quantification of Excited-State Brønsted-Lowry Acidity of Weak Photoacids Using Steady-State Photoluminescence Spectroscopy and a Driving-Force-Dependent Kinetic Theory.

Photoacids and photobases constitute a class of molecules that upon absorption of light undergoes a reversible change in acidity, i.e. pKa. Knowledge of the excited-state pKa value, pKa*, is critical for predicting excited-state proton-transfer behavior. A reasonable approximation of pKa* is possible using the Förster cycle analysis, but only when the ground-state pKa is known. This poses a challenge for the study of weak photoacids (photobases) with less acidic (basic) excited states (pKa* (pKb*) > 7), because ground-state pKa (pKb) values are >14, making it difficult to quantify them accurately in water. Another method to determine pKa* relies on acid-base titrations with photoluminescence detection and Henderson-Hasselbalch analysis. This method requires that the acid dissociation reaction involving the thermally equilibrated electronic excited state reaches chemical quasi-equilibrium, which does not occur for weak photoacids (photobases) due to slow rates of excited-state proton transfer. Herein, we report a method to overcome these limitations. We demonstrate that liquid water and aqueous hydroxide are unique proton-accepting quenchers of excited-state photoacids. We determine that Stern-Volmer quenching analysis is appropriate to extract rate constants for excited-state proton transfer in aqueous solutions from a weak photoacid, 5-aminonaphthalene-1-sulfonate, to a series of proton-accepting quenchers. Analysis of these data by Marcus-Cohen bond-energy-bond-order theory yields an accurate value for pKa* of 5-aminonaphthalene-1-sulfonate. Our method is broadly accessible because it only requires readily available steady-state photoluminescence spectroscopy. Moreover, our results for weak photoacids are consistent with those from previous studies of strong photoacids, each showing the applicability of kinetic theories to interpret driving-force-dependent rate constants for proton-transfer reactions.

Light-driven dissipative self-assembly of a peptide hydrogel.

Light energy provides an attractive fuel source for energy dissipating systems because of the lack of waste production, wavelength tunability and the potential for spatial and temporal resolution. In this work, we describe a peptide-spiropyran conjugate that assembled into a transient nanofiber hydrogel in the presence of visible light, and dissociated when the light source was removed.

Perspectives of Interstitial Lung Disease Patients and Carers During COVID-19.

Aim To gain an understanding of the impact of COVID-19 on the daily life, healthcare needs, mental wellbeing and outlook of patients with Interstitial Lung Disease (ILD) and their caregivers. Methods ILD patients and caregivers were invited to participate in a quantitative survey. Respondents could self-select to then participate in in-depth structured telephone interviews. Survey data was compared to Department of Health COVID-19 public opinion tracker findings for the comparable time period. Results There were 170 survey respones (111 patients and 59 caregivers) and 14 in-depth interview participants. 32% (n=36) of patients and 42% (n=25) of caregivers expressed extreme worry regarding COVID-19 on a 1-10 scale. 83% (n=92) of patients expressed concern about safe hospital access, 33% (n=37) had received a telephone consultation with their clinician, 43% (n=48) reported test delays, 47% (n=52) were exercising less, 23% (n=26) reported worse sleep and 15% (n=17) reported being financially worse off. Carers reported that sleep was worse for 58% (n=34), 42% (n=25) reported being worse off financially, and 40% (n=24) reported a worse diet. Worry (66%, n=39), stress (51%, n=30), anxiety (49%, n=29) were commonly reported by carers. Discussion ILD patients and caregivers reported higher levels of worry regarding COVID-19 compared to the general public. Alternative pathways for quality ILD patient care and interventions to reduce the burden of care on ILD caregivers are required.

Identification of Phenazine-Based MEMO1 Small-Molecule Inhibitors: Virtual Screening, Fluorescence Polarization Validation, and Inhibition of Breast Cancer Migration.

Phosphorylation-dependent protein-protein interactions play a significant role in biological signaling pathways; therefore, small molecules that are capable of influencing these interactions can be valuable research tools and have potential as pharmaceutical agents. MEMO1 (mediator of ErbB2-cell driven motility) is a phosphotyrosine-binding protein that interacts with a variety of protein partners and has been found to be upregulated in breast cancer patients. Herein, we report the first small-molecule inhibitors of MEMO1 interactions identified through a virtual screening platform and validated in a competitive fluorescence polarization assay. Initial structure-activity relationships have been investigated for these phenazine-core inhibitors and the binding sites have been postulated using molecular dynamics simulations. The most potent biochemical inhibitor is capable of disrupting the large protein interface with a KI of 2.7 µm. In addition, the most promising phenazine core compounds slow the migration of breast cancer cell lines in a scratch assay.

Operative Management of Perinatal Lumbar Disc Herniation and Cauda Equina Syndrome: A Case Series

Introduction Perinatal lumbar discectomy for lumbar disc herniation or cauda equina syndrome is a rare clinical scenario. This case series outlines the surgical management of this clinical scenario at a national tertiary referral centre over a 10-year period Methods A retrospective review of all females who underwent discectomy / decompression for lumbar disc herniation or cauda equina syndrome in the perinatal period at a national tertiary referral centre for spine surgery over a 10-year period between January 2008 to December 2017. Results 6 cases required surgical intervention. All patients were successfully managed with surgical decompressive procedures and recovered well in the postoperative period without complication. Conclusions The principles of management remain the same in the pregnant and non-pregnant populations, although treatment options are complicated by the desire to avoid risk to the developing foetus. Surgical intervention is safe to both mother and baby and if performed promptly is associated with an excellent functional outcome.

Cervical spine fracture through a cervical disc replacement.

INTRODUCTION: We report a rare case of cervical spine trauma through a cervical disc replacement and adjacent multilevel disc fusions. Cervical disc replacement (CDR) is a viable option for the surgical treatment of degenerative disc disease however long term follow up data regarding this operative technique is poor specifically relating to traumatic complications. We know of no previous reports of bilateral cervical pedicle fractures occurring adjacent to CDR and anterior cervical spine instrumentation. PRESENTATION OF CASE: A 46 year-old with a history of C6C7 CDR and C4-6 anterior cervical decompression and fusion was an unrestrained driver involved in a road traffic accident and suffered bilateral C7 pedicle fractures and a right C6C7 facet joint fracture-subluxation without neurological deficit. Reduction and fixation via a posterior approach achieved a satisfactory alignment and the patient made an uneventful recovery. DISCUSSION: A significant force coupled with cervical fixation resulted in a bilateral pedicle fracture of the cervical spine with preserved neurological function. CONCLUSION: The protective role of the CDR has not been previously demonstrated but may have played a role in this case. The authors believe the challenges encountered in the treatment of this patient provide valuable lessons in the management of complex cervical spine trauma in the setting of previous instrumentation.

Silencing the buzz: a new approach to population suppression of mosquitoes by feeding larvae double-stranded RNAs.

BACKGROUND: Mosquito-borne diseases threaten over half the world's human population, making the need for environmentally-safe mosquito population control tools critical. The sterile insect technique (SIT) is a biological control method that can reduce pest insect populations by releasing a large number of sterile males to compete with wild males for female mates to reduce the number of progeny produced. Typically, males are sterilized using radiation, but such methods can reduce their mating competitiveness. The method is also most effective if only males are produced, but this requires the development of effective sex-sorting methods. Recent efforts to use transgenic methods to produce sterile male mosquitoes have increased interest in using SIT to control some of our most serious disease vectors, but the release of genetically modified mosquitoes will undoubtedly encounter considerable delays as regulatory agencies deal with safety issues and public concerns. METHODS: Testis genes in the dengue vector Aedes aegypti were identified using a suppression subtractive hybridization technique. Mosquito larvae were fed double-stranded RNAs (dsRNAs) that targeted both the testis genes and a female sex determination gene (doublesex) to induce RNA interference (RNAi) -mediated sterility and inhibition of female development. Fertility and mating competiveness of the treated males were assessed in small-scale mating competition experiments. RESULTS: Feeding mosquito larvae dsRNAs targeting testis genes produced adult males with greatly reduced fertility; several dsRNAs produced males that were highly effective in competing for mates. RNAi-mediated knockdown of the female-specific isoform of doublesex was also effective in producing a highly male-biased population of mosquitoes, thereby overcoming the need to sex-sort insects before release. CONCLUSIONS: The sequence-specific gene-silencing mechanism of this RNAi technology renders it adaptable for species-specific application across numerous insect species. We envisage its use for traditional large-scale reared releases of mosquitoes and other pest insects, although the technology might also have potential for field-based control of mosquitoes where eggs deposited into a spiked larval site lead to the release of new sterile males.

Paget's disease of the cervical spine: case report and review.

STUDY DESIGN: This article presents a case of Paget's disease of the cervical spine. OBJECTIVES: To report a very rare case of Paget's disease of the cervical spine and review the literature. METHODS: We report a case of Paget's disease of the cervical spine in a 51-year-old male presenting with neck pain post RTA. Routine radiographs revealed enlarged sclerotic bodies of C2-C5 with normal alignment. CT revealed a fracture of the lateral mass of C2 with extensive sclerotic changes in the bodies of C2-C5 with periosteal apposition, endosteal resorption "pumice stone" type focal periosteal apposition consistent with Paget's disease. Laboratory investigations were consistent with a diagnosis of Paget's disease. Isotope bone scan revealed isolated uptake in the cervical spine. He was commenced on biosphosphonate therapy. Following 12 weeks of immobilisation his orthosis was removed and rehabilitation commenced. RESULTS: At 12-month follow-up he is asymptomatic with no radiological change. CONCLUSION: This case illustrates the investigation and successful management of a very rare case of Paget's disease of the cervical spine.

Post-depositional fracturing and subsidence of pumice flow deposits: Lascar Volcano, Chile.

Unconsolidated pyroclastic flow deposits of the 1993 eruption of Lascar Volcano, Chile, have, with time, become increasingly dissected by a network of deeply penetrating fractures. The fracture network comprises orthogonal sets of decimeter-wide linear voids that form a pseudo-polygonal grid visible on the deposit surface. In this work, we combine shallow surface geophysical imaging tools with remote sensing observations and direct field measurements of the deposit to investigate these fractures and their underlying causal mechanisms. Based on ground penetrating radar images, the fractures are observed to have propagated to depths of up to 10 m. In addition, orbiting radar interferometry shows that deposit subsidence of up to 1 cm/year-1 occurred between 1993 and 1996 with continued subsidence occurring at a slower rate thereafter. In situ measurements show that 1 m below the surface, the 1993 deposits remain 5°C to 15°C hotter, 18 years after emplacement, than adjacent deposits. Based on the observed subsidence as well as estimated cooling rates, the fractures are inferred to be the combined result of deaeration, thermal contraction, and sedimentary compaction in the months to years following deposition. Significant environmental factors, including regional earthquakes in 1995 and 2007, accelerated settling at punctuated moments in time. The spatially variable fracture pattern relates to surface slope and lithofacies variations as well as substrate lithology. Similar fractures have been reported in other ignimbrites but are generally exposed only in cross section and are often attributed to formation by external forces. Here we suggest that such interpretations should be invoked with caution, and deformation including post-emplacement subsidence and fracturing of loosely packed ash-rich deposits in the months to years post-emplacement is a process inherent in the settling of pyroclastic material.