Potent protease inhibitors of deadly lagoviruses: rabbit hemorrhagic disease virus and European brown hare syndrome virus

Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family and mainly affect rabbits and hares, respectively. These infectious diseases are associated with high mortality and a serious threat to domesticated (farmed and pet) and wild rabbits and hares, including endangered species such as Riparian brush rabbits. In the US, only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by RHD type 2 virus (RHDV2) was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several US states infecting wild rabbits and hares. Since it is almost impossible to control and eradicate the virus from wild animals, it is highly likely RHD will become endemic in the US. Vaccines are available for RHD, however, there is no specific treatment for these deadly diseases. RHDV and EBHSV encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses that encode 3CLpro including caliciviruses and coronaviruses. Here we established the enzyme assay and cell-based assays for these uncultivable viruses to determine the in vitro activity of 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis, and identified potent inhibitors of RHDV1 and 2 and EBHSV. In addition, structure-activity relationship study and homology modelling of the 3CLpros and inhibitors revealed that lagoviruses share similar structural requirements for 3CLpro inhibition with other caliciviruses.

Effect of Respiratory Training Combined with Core Muscle Training on the Overall Motor Function and Activities of Daily Living of Patients with Early and Midterm Stroke

Stroke is a cerebral ischemic or hemorrhagic disease with sudden onset and rapid progress. To analyze the effect of respiratory training combined with core muscle training on the overall motor function and activities of daily living of patients with early and midterm stroke, 90 cases with early and midterm stroke admitted to the neurological department of our hospital from April 2018 to April 2019 were chosen as the research objects. According to the odd or even hospitalization numbers, they were equally divided into the study group and the reference group. Both groups received basic drug treatment. On this basis, the reference group was given routine rehabilitation training, while the study group was given respiratory training combined with core muscle training. The clinical indexes of both groups before and after intervention were evaluated to analyze the effect of different training methods on the rehabilitation of patients with early and midterm stroke. There was no significant difference in gender ratio, average age, average BMI, average course of disease, stroke types, MAS grading, location of limb dysfunction, and combined disease between the two groups ( P < 0.05 ). The total clinical effective rate of the study group after intervention was obviously higher than that of the reference group ( P < 0.05 ). The MoCA scores of both groups after intervention were obviously higher than those before intervention, and the score of the study group after intervention was obviously higher than that of the reference group. The scores of limb motor function, activities of daily living, and balance function at T2, T3, and T4 in the study group were obviously higher than those in the reference group ( P < 0.001 ). At 4 and 8 weeks after intervention, the 10 m MWS of the study group was obviously higher than that of the reference group ( P < 0.001 ), while the TUGT was obviously lower ( P < 0.001 ). Respiratory training combined with core muscle training can obviously improve the activities of daily living, cognitive function, and limb motor function of patients with early and midterm stroke, which is worth popularizing and using.

Production and application of mouse monoclonal antibodies targeting linear epitopes in pB602L of African swine fever virus

African swine fever (ASF) is an acute hemorrhagic disease of domestic pigs. The causative agent of ASF, ASF virus (ASFV), is a double-stranded DNA virus, the sole member in the family Asfarviridae. The non-structural protein pB602L of ASFV is a molecular chaperone of the major capsid protein p72 and plays a key role in icosahedral capsid assembly. This protein is antigenic and is a target for developing diagnostic tools for ASF. To generate monoclonal antibodies (mAbs) against pB602L, a prokaryotically expressed recombinant pB602L protein was produced, purified, and used as an antigen to immunize mice. A total of eight mouse mAbs were obtained, and their binding epitopes were screened by Western blot using an overlapping set of polypeptides from pB602L. Three linear epitopes were identified and designated epitope 1 (366ANRERYNY373), epitope 2 (415GPDAPGLSI423), and epitope 3 (498EMLNVPDD505). Based on the epitope recognized, the eight mAbs were placed into three groups: group 1 (B2A1, B2F1, and B2D10), group 2 (B2H10, B2B2, B2D8, and B2A3), and group 3 (B2E12). The mAbs B2A1, B2H10, and B2E12, each representing one of the groups, were used to detect pB602L in ASFV-infected porcine alveolar macrophages (PAMs) and pig tissues, using an indirect fluorescence assay (IFA) and immunohistochemical staining, respectively. The results showed that pB602L was detectable with all three mAbs in immunohistochemical staining, but only B2H10 was suitable for detecting the proteins in ASFV-infected PAMs by IFA. In summary, we developed eight anti-pB602L mouse mAbs recognizing three linear epitopes in the protein, which can be used as reagents for basic and applied research on ASFV.

Preliminary Outbreak Investigation and Spatial Epidemiology of Rabbit Hemorrhagic Disease Outbreak in Nigeria

The outbreak of highly contagious transboundary rabbit hemorrhagic disease (RHD) in Nigeria has a severe socio-economic impact on the rabbit industry. We present the outbreak investigation and spatial epidemiology of the first confirmed RHD outbreak in Nigeria from a field survey of 28 stochastic outbreaks in Kwara State, north-central Nigeria. A total of 1,639 rabbits died from 2,053 susceptible rabbits. The serotype “RHDV-2” was detected in tissue samples from some of the outbreaks. The case fatality rate of the RHDV-2 outbreak was 79.8%. The source of the outbreak is still unknown. Most (71.4%) of the farmers had introduced new rabbits into their farms 1–2 weeks before the outbreak. Most of the farmers practiced biosecurity measures such as farm fencing (83.1%) and routine disinfection of the farm materials (53.6%). However, only 17.8% of the farmers enforced movement restrictions into their farms. Some of the farmers (42.8%) had restocked their farms after being affected by the RHD outbreak and 75% of all those farmers that have restocked had used the RHD vaccine. There was no statistically significant association between adherence to biosecurity measures and the RHD outbreak in affected farms (p = 0.408). However, the introduction of new rabbits into rabbit farms significantly pre-disposed farms to the RHD outbreak (p &lt; 0.001). There is a need for active surveillance of RHD across the country to ensure efficient and effective tracking, monitoring, and control of the disease. Equally, understanding the genetic diversity of the Lagoviruses in Nigeria that cause RHD to aid vaccine development is of utmost importance to prevent future RHD outbreaks.

Rapid Differentiation of Vector Species: Development of Microsatellite Markers for Population Genetics of Biting Midges and a Potential Tool for Species Identification of Culicoides Sonorensis

Background: Proper vector surveillance relies on the ability to identify species of interest accurately and efficiently, though this can be difficult in groups containing cryptic species. Culicoides is a genus of small biting flies responsible for the transmission of numerous pathogens to a multitude of vertebrates. Regarding pathogen transmission, the C. variipennis species complex is of particular interest in North America. Of the six species within this group, only C. sonorensis is a proven vector of bluetongue virus and epizootic hemorrhagic disease virus. Unfortunately, subtle morphological differences, cryptic species, and mitonuclear discordance make species identification in the C. variipennis complex challenging. Recently, a SNP analysis enabled discrimination between the species of this group; however, this demanding approach is not practical for vector surveillance. Methods: The aim of the current study was to develop a reliable and affordable way of differentiating the species within the C. variipennis complex, especially C. sonorensis. Twenty-five putative microsatellite markers were identified using the C. sonorensis genome and tested for amplification within five species of the C. variipennis complex. Machine learning was then used to determine which markers best explain the genetic differentiation between species. This led to the development of a subset of four and seven markers which were also tested for species differentiation.Results: A total of 21 microsatellite markers were successfully amplified in the species tested. Clustering analyses of all of these markers recover the same species-level identification as the previous SNP data. Additionally, the subset of seven markers was equally capable of accurately differentiating the members of the C. variipennis complex as the 21 microsatellite markers. Finally, one microsatellite marker (C508) was found to be species-specific, only amplifying in the vector species C. sonorensis among the samples tested. Conclusions: These microsatellites provide an affordable way in which to differentiate the species of the C. variipennis complex and could lead to a better understanding of the species dynamics within this group. Additionally, after further testing, marker C508 may allow for the identification of C. sonorensis with a single-tube assay, potentially providing a powerful new tool for vector surveillance in North America.

Isolation and determination of Vibrio spp. pathogen from hemorrhagic disease in Sciaenops ocellatus in cage culture

In this study, 18 strains of Vibrio bacteria were identified from 27 samples of Red drum fish (Sciaenops ocellatus) suffering from the haemorrhagic disease from cage culture in Vietnam. The bacterial strains were identified with the 16S rRNA sequencing method and checked for morphological, physiological, and biochemical characteristics by using the API 20E KIT. Twelve strains of V. alginolyticus, three strains of V. fluvialis, and three strains of V. orientalis were recorded. All Vibrio strains have gene similarities with those on the gene bank ranging from 98 to 100%. The biochemical characteristics of these 18 isolates were similar. These bacteria are susceptible to tetracycline and doxycycline and entirely resistant to ampicillin, amoxicillin, and erythromycin.

Spatiotemporally Orchestrated Interactions between Viral and Cellular Proteins Involved in the Entry of African Swine Fever Virus

African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars with a mortality of up to 100%. The causative agent, African swine fever virus (ASFV), is a member of the Asfarviridae family of the nucleocytoplasmic large DNA viruses. The genome size of ASFV ranges from 170 to 194 kb, encoding more than 50 structural and 100 nonstructural proteins. ASFV virions are 260–300 nm in diameter and composed of complex multilayered structures, leading to an intricate internalization pathway to enter host cells. Currently, no commercial vaccines or antivirals are available, due to the insufficient knowledge of the viral receptor(s), the molecular events of ASFV entry into host cells, and the functions of virulence-associated genes. During the early stage of ASFV infection, the fundamental aspects of virus-host interactions, including virus internalization, intracellular transport through the endolysosomal system, and membrane fusion with endosome, are precisely regulated and orchestrated via a series of molecular events. In this review, we summarize the currently available knowledge on the pathways of ASFV entry into host cells and the functions of viral proteins involved in virus entry. Furthermore, we conclude with future perspectives and highlight areas that require further investigation. This review is expected to provide unique insights for further understanding ASFV entry and facilitate the development of vaccines and antivirals.

A pigtailed macaque model of Kyasanur Forest disease virus and Alkhurma hemorrhagic disease virus pathogenesis

Kyasanur Forest disease virus (KFDV) and the closely related Alkhurma hemorrhagic disease virus (AHFV) are emerging flaviviruses that cause severe viral hemorrhagic fevers in humans. Increasing geographical expansion and case numbers, particularly of KFDV in southwest India, class these viruses as a public health threat. Viral pathogenesis is not well understood and additional vaccines and antivirals are needed to effectively counter the impact of these viruses. However, current animal models of KFDV pathogenesis do not accurately reproduce viral tissue tropism or clinical outcomes observed in humans. Here, we show that pigtailed macaques (Macaca nemestrina) infected with KFDV or AHFV develop viremia that peaks 2 to 4 days following inoculation. Over the course of infection, animals developed lymphocytopenia, thrombocytopenia, and elevated liver enzymes. Infected animals exhibited hallmark signs of human disease characterized by a flushed appearance, piloerection, dehydration, loss of appetite, weakness, and hemorrhagic signs including epistaxis. Virus was commonly present in the gastrointestinal tract, consistent with human disease caused by KFDV and AHFV where gastrointestinal symptoms (hemorrhage, vomiting, diarrhea) are common. Importantly, RNAseq of whole blood revealed that KFDV downregulated gene expression of key clotting factors that was not observed during AHFV infection, consistent with increased severity of KFDV disease observed in this model. This work characterizes a nonhuman primate model for KFDV and AHFV that closely resembles human disease for further utilization in understanding host immunity and development of antiviral countermeasures.