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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the infectious disease COVID-19, which has rapidly become an international pandemic with significant impact on healthcare systems and the global economy. To assist antiviral therapy and vaccine development efforts, we performed a natural history/time course study of SARS-CoV-2 infection in ferrets to characterise and assess the suitability of this animal model. Ten ferrets of each sex were challenged intranasally with 4.64 × 104 TCID50 of SARS-CoV-2 isolate Australia/VIC01/2020 and monitored for clinical disease signs, viral shedding, and tissues collected post-mortem for histopathological and virological assessment at set intervals. We found that SARS-CoV-2 replicated in the upper respiratory tract of ferrets with consistent viral shedding in nasal wash samples and oral swab samples up until day 9. Infectious SARS-CoV-2 was recovered from nasal washes, oral swabs, nasal turbinates, pharynx, and olfactory bulb samples within 3–7 days post-challenge; however, only viral RNA was detected by qRT-PCR in samples collected from the trachea, lung, and parts of the gastrointestinal tract. Viral antigen was seen exclusively in nasal epithelium and associated sloughed cells and draining lymph nodes upon immunohistochemical staining. Due to the absence of clinical signs after viral challenge, our ferret model is appropriate for studying asymptomatic SARS-CoV-2 infections and most suitable for use in vaccine efficacy studies. [ABSTRACT FROM AUTHOR]

Many RNA viruses are displaying great promise in the field of oncolytic virotherapy. Previously, we reported that the picornavirus Coxsackievirus A21 (CVA21) possessed potent oncolytic activity against cultured malignant melanoma cells and melanoma xenografts in mice. In the present study, we demonstrate that three additional Group A Coxsackieviruses; Coxsackievirus A13 (CVA13), Coxsackievirus A15 (CVA15) and Coxsackievirus A18 (CVA18), also have similar oncolytic activity against malignant melanoma. Each of the viruses grew quickly to high titers in cancer cells expressing ICAM-1 and intratumoral injection of preformed subcutaneous SK-Mel-28 xenografts in mice with CVA13, CVA15 and CVA18 resulted in significant tumor volume reduction. As preexisting immunity could potentially hinder oncolytic virotherapy, sera from stage IV melanoma patients and normal controls were tested for levels of protective antibody against the panel of oncolytic Coxsackieviruses. Serum neutralization assays revealed that 3 of 21 subjects possessed low levels of anti-CVA21 antibodies, while protective antibodies for CVA13, CVA15 and CVA18 were not detected in any sample. Serum from individuals who were seropositive for CVA21 failed to exhibit cross-neutralization of CVA13, CVA15 and CVA18. From these studies it can be concluded that the administration of CVA13, CVA15 or CVA18 could be employed as a potential multivalent oncolytic therapy against malignant melanoma. [ABSTRACT FROM AUTHOR]

The dissemination of malignant gastric cells to the peritoneum occurs frequently, usually as an early event in disease, and results in poor patient prognosis. Surgery and chemotherapy offer limited therapeutic success. The low-pathogenic human enterovirus, Echovirus 1 (EV1), is an oncolytic virus that selectively targets and destroys malignant prostate and ovarian cancer xenografts in vivo. Lytic EV1 infection requires the cell surface expression of α2β1, an integrin involved in the dissemination of gastric cancer cells to the peritoneum. Herein, we evaluated the capacity of EV1 for anti-neoplastic cell action in gastric peritoneal carcinomatosis. Flow cytometric analysis demonstrated that α2β1 was abundantly surface expressed on a panel of gastric cancer cell lines, rendering the majority of lines highly susceptible to in vitro lytic EV1 infection and supportive of efficient viral progeny production. A bioluminescent MKN-45-Luc SCID mouse model of peritoneal dissemination was developed to allow real-time non-invasive monitoring of peritoneal tumor burden. Employing this mouse model, we demonstrated a therapeutic dose-response for escalating oncolytic EV1 doses. Taken together, these results emphasize the exciting potential for EV1 as a single or adjunct therapy for the control of the peritoneal dissemination of gastric cancer. [ABSTRACT FROM AUTHOR]

Oncolytic viruses are attractive biological agents for the control of human malignancy. This study assessed the capacity of Coxsackievirus A21 (CVA21) to target and destroy multiple myeloma (MM) and precursor aberrant plasma cells in vitro using established MM cell lines and 15 patient bone marrow (BM) biopsies [ n = 10 MM and five monoclonal gammopathy of undetermined significance (MGUS)]. Cell surface analysis revealed that all tumour cells lines expressed high levels of intercellular adhesion molecule-1 (ICAM-1) and decay-accelerating factor (DAF), the receptor molecules to which CVA21 can bind, leading to subsequent cell-entry and infection. MM cell lines were remarkably susceptible to CVA21 lytic infection, producing 100–1000-fold increases in viral progeny within 24 h. In contrast, normal peripheral blood cells were refractile to CVA21 infection. Furthermore, challenge of patient BM biopsies with CVA21 for 48 h resulted in specific purging of up to 98·7% of CD138+ plasma cells, with no significant decrease in progenitor cell function. Data generated in this study suggests that CVA21 virotherapy may have potential applications as a systemic anti-tumour agent for MM, or in the ex vivo purging of malignant plasma cells prior to autologous stem cell transplantation. [ABSTRACT FROM AUTHOR]

The thymidine 5′-triphosphate analogue containing a methylene group in place of the 5′ oxygen atom can be prepared using modifications of published procedures and can substitute for the natural thymidine triphosphate in chain extension reactions catalyzed by Moloney-MLV reverse transcriptase. Using rabbit β-globin mRNA as the template together with an appropriate primer, the enzyme readily makes full-length DNA transcripts in which all thymidine 5′ oxygen atoms have been replaced with methylene groups. In sequence analyses using the partial depurination procedure, the analogue DNA transcript produces electrophoretic gel patterns identical with those of the corresponding natural DNA transcript. Experiments on second strand synthesis using the four regular triphosphates show that the analogue DNA transcript, like the natural transcript, can serve as a template. The two DNA duplexes (natural/natural and analogue/natural) formed by these reactions produce similar electrophoretic cleavage patterns when treated with either of the endonucleases HaeIII and EcoRI. However, further studies on template properties indicate that, while the enzyme makes a full-length product when using the analogue substrate with a natural DNA strand as template, it appears unable to use the analogue transcript as template with the analogue triphosphate as substrate during second strand synthesis. Preliminary experiments have also been carried out with a DNA polymerase. No products are detected in reactions using Taq polymerase with PCR protocols containing the analogue triphosphate as the only source of thymidine. [ABSTRACT FROM AUTHOR]

Examines the tachykinin NK [sub 1] and NK [sub 3] receptors in prefrontal cortex of the human brain. Role of tachykinins on the inflammation and pain mechanisms and some automatic reflexes; Appearance of NK [sub 1] in glia limitans and pial surface; Observation of astrocytes and beaded fibers of NK [sub 3] receptor immunoreactivity in blood vessels.

Sarbecoviruses such as SARS and SARS-CoV-2 have been responsible for two major outbreaks in humans, the latter resulting in a global pandemic. While sarbecoviruses primarily cause an acute respiratory infection, they have been shown to infect the nervous system. However, mechanisms of sarbecovirus neuroinvasion and neuropathogenesis remain unclear. In this study, we examined the infectivity and trans-synaptic transmission potential of the sarbecoviruses SARS and SARS-CoV-2 in human stem cell–derived neural model systems. We demonstrated limited ability of sarbecoviruses to infect and replicate in human stem cell–derived neurons. Furthermore, we demonstrated an inability of sarbecoviruses to transmit between synaptically connected human stem cell–derived neurons. Finally, we determined an absence of SARS-CoV-2 infection in olfactory neurons in experimentally infected ferrets. Collectively, this study indicates that sarbecoviruses exhibit low potential to infect human stem cell–derived neurons, lack an ability to infect ferret olfactory neurons, and lack an inbuilt molecular mechanism to utilise retrograde axonal trafficking and trans-synaptic transmission to spread within the human nervous system. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) is an emerging virus that has caused significant human morbidity and mortality since its detection in late 2019. With the rapid emergence has come an unprecedented programme of vaccine development with at least 300 candidates under development. Ferrets have proven to be an appropriate animal model for testing safety and efficacy of SARS‐CoV‐2 vaccines due to quantifiable virus shedding in nasal washes and oral swabs. Here, we outline our efforts early in the SARS‐CoV‐2 outbreak to propagate and characterize an Australian isolate of the virus in vitro and in an ex vivo model of human airway epithelium, as well as to demonstrate the susceptibility of domestic ferrets (Mustela putorius furo) to SARS‐CoV‐2 infection following intranasal challenge. [ABSTRACT FROM AUTHOR]

Hendra virus (HeV) is lethal to horses and a zoonotic threat to humans in Australia, causing severe neurological and/or respiratory disease with high mortality. An equine vaccine has been available since 2012. Foals acquire antibodies from their dams by ingesting colostrum after parturition, therefore it is assumed that foals of mares vaccinated against HeV will have passive HeV antibodies circulating during the first several months of life until they are actively vaccinated. However, no studies have yet examined passive or active immunity against HeV in foals. Here, we investigated anti-HeV antibody levels in vaccinated mares and their foals. Testing for HeV neutralising antibodies is cumbersome due to the requirement for Biosafety level 4 (BSL-4) containment to conduct virus neutralisation tests (VNT). For this study, a subset of samples was tested for HeV G-specific antibodies by both an authentic VNT with infectious HeV and a microsphere-based immunoassay (MIA), revealing a strong correlation. An indicative neutralising level was then applied to the results of a larger sample set tested using the MIA. Mares had high levels of HeV-specific neutralising antibodies at the time of parturition. Foals acquired high levels of maternal antibodies which then waned to below predictive protective levels in most foals by 6 months old when vaccination commenced. Foals showed a suboptimal response to vaccination, suggesting maternal antibodies may interfere with active vaccination. The correlation analysis between the authentic HeV VNT and HeV MIA will enable further high throughput serological studies to inform optimal vaccination protocols for both broodmares and foals. • Luminex® microsphere immunoassay can indicate neutralising Hendra Virus antibodies. • Foals of mares vaccinated for Hendra Virus acquire protective passive antibodies. • Passive antibodies in foals waned to below protective levels by 3–6 months of age. • Foals from vaccinated mares had a suboptimal response to Hendra Virus vaccination. [ABSTRACT FROM AUTHOR]

The host response to SARS-CoV-2 infection provide insights into both viral pathogenesis and patient management. The host-encoded microRNA (miRNA) response to SARS-CoV-2 infection, however, remains poorly defined. Here we profiled circulating miRNAs from ten COVID-19 patients sampled longitudinally and ten age and gender matched healthy donors. We observed 55 miRNAs that were altered in COVID-19 patients during early-stage disease, with the inflammatory miR-31-5p the most strongly upregulated. Supervised machine learning analysis revealed that a three-miRNA signature (miR-423-5p, miR-23a-3p and miR-195-5p) independently classified COVID-19 cases with an accuracy of 99.9%. In a ferret COVID-19 model, the three-miRNA signature again detected SARS-CoV-2 infection with 99.7% accuracy, and distinguished SARS-CoV-2 infection from influenza A (H1N1) infection and healthy controls with 95% accuracy. Distinct miRNA profiles were also observed in COVID-19 patients requiring oxygenation. This study demonstrates that SARS-CoV-2 infection induces a robust host miRNA response that could improve COVID-19 detection and patient management. Author summary: While it is recognized that the host response to infection plays a critical role in determining the severity and outcome of COVID-19, the host microRNA (miRNA) response to SARS-CoV-2 infection is poorly defined. Here we have used next-generation sequencing and bioinformatics to profile circulating miRNAs in 10 COVID-19 patients that were sampled longitudinally over time. COVID-19 was associated with altered expression of 55 plasma miRNAs, with miR-776-3p and miR-1275 among the most strongly down-regulated, and miR-4742-3p, miR-31-5p and miR-3215-3p the most up-regulated. An artificial intelligence methodology was used to identify a miRNA signature, consisting of miR423-5p, miR-23a-3p, miR-195-5p, which could independently classify COVID-19 patients from healthy controls with 99.9% accuracy. When applied to the ferret model of COVID-19, the same signature classified COVID-19 cases with 99.8% accuracy and could distinguish between COVID-19 and influenza A(H1N1) infection with >95% accuracy. In summary this study profiles the host miRNA response to COVID-19 and suggests that the measurement of select host molecules may have potential to independently detect disease cases. [ABSTRACT FROM AUTHOR]

Many viruses target signal transducers and activators of transcription (STAT) 1 and 2 to antagonise antiviral interferon signalling, but targeting of signalling by other STATs/cytokines, including STAT3/interleukin 6 that regulate processes important to Ebola virus (EBOV) haemorrhagic fever, is poorly defined. We report that EBOV potently inhibits STAT3 responses to interleukin-6 family cytokines, and that this is mediated by the interferon-antagonist VP24. Mechanistic analysis indicates that VP24 effects a unique strategy combining distinct karyopherin-dependent and karyopherin-independent mechanisms to antagonise STAT3-STAT1 heterodimers and STAT3 homodimers, respectively. This appears to reflect distinct mechanisms of nuclear trafficking of the STAT3 complexes, revealed for the first time by our analysis of VP24 function. These findings are consistent with major roles for global inhibition of STAT3 signalling in EBOV infection, and provide new insights into the molecular mechanisms of STAT3 nuclear trafficking, significant to pathogen-host interactions, cell physiology and pathologies such as cancer. Author summary: Ebola virus (EBOV) continues to pose a significant risk to human health globally, causing ongoing disease outbreaks with case-fatality rates between 40 and 65%. Suppression of immune responses is a critical component of EBOV haemorrhagic fever, but understanding of EBOV impact on signalling by cytokines other than interferon is limited. We find that infectious EBOV inhibits interleukin-6 cytokine signalling via antagonism of STAT3. The antagonistic strategy uniquely combines two distinct mechanisms, which appear to reflect differing nuclear trafficking mechanisms of critical STAT3 complexes. This provides fundamental insights into the mechanisms of pathogenesis of a lethal virus, and biology of STAT3, a critical player in immunity, development, growth and cancer. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome. [ABSTRACT FROM AUTHOR]

Background: Early events leading to intrauterine infection and fetal lung injury remain poorly defined, but may hold the key to preventing neonatal and adult chronic lung disease. Our objective was to establish a nonhuman primate model of an early stage of chorioamnionitis in order to determine the time course and mechanisms of fetal lung injury in utero. Methodology/Principal Findings: Ten chronically catheterized pregnant monkeys (Macaca nemestrina) at 118-125 days gestation (term = 172 days) received one of two treatments: 1) choriodecidual and intra-amniotic saline (n = 5), or 2) choriodecidual inoculation of Group B Streptococcus (GBS) 1x106 colony forming units (n = 5). Cesarean section was performed regardless of labor 4 days after GBS or 7 days after saline infusion to collect fetal and placental tissues. Only two GBS animals developed early labor with no cervical change in the remaining animals. Despite uterine quiescence in most cases, blinded review found histopathological evidence of fetal lung injury in four GBS animals characterized by intraalveolar neutrophils and interstitial thickening, which was absent in controls. Significant elevations of cytokines in amniotic fluid (TNF-α, IL-8, IL-1β, IL-6) and fetal plasma (IL-8) were detected in GBS animals and correlated with lung injury (p<0.05). Lung injury was not directly caused by GBS, because GBS was undetectable in amniotic fluid (∼10 samples tested/animal), maternal and fetal blood by culture and polymerase chain reaction. In only two cases was GBS cultured from the inoculation site in low numbers. Chorioamnionitis occurred in two GBS animals with lung injury, but two others with lung injury had normal placental histology. Conclusions/Significance: A transient choriodecidual infection can induce cytokine production, which is associated with fetal lung injury without overt infection of amniotic fluid, chorioamnionitis or preterm labor. Fetal lung injury may, thus, occur silently without symptoms and before the onset of the fetal systemic inflammatory response syndrome. [ABSTRACT FROM AUTHOR]