GALDAR: A genetically encoded galactose sensor for visualizing sugar metabolism in vivo.

Sugar metabolism plays a pivotal role in sustaining life. Its dynamics within organisms is less understood compared to its intracellular metabolism. Galactose, a hexose stereoisomer of glucose, is a monosaccharide transported via the same transporters with glucose. Galactose feeds into glycolysis and regulates protein glycosylation. Defects in galactose metabolism are lethal for animals. Here, by transgenically implementing the yeast galactose sensing system into Drosophila, we developed a genetically encoded sensor, GALDAR, which detects galactose in vivo. Using this heterologous system, we revealed dynamics of galactose metabolism in various tissues. Notably, we discovered that intestinal stem cells do not uptake detectable levels of galactose or glucose. GALDAR elucidates the role for galactokinase in metabolism of galactose and a transition of galactose metabolism during the larval period. This work provides a new system that enables analyses of in vivo sugar metabolism.

Clinical characteristics of patients with COVID-19 harboring detectable intracellular SARS-CoV-2 RNA in peripheral blood cells.

OBJECTIVES: Although SARS-CoV-2 RNAemia has been reported to strongly impact patients with severe COVID-19, the clinical characteristics of patients with COVID-19 harboring detectable intracellular SARS-CoV-2 RNA remain unknown. METHODS: We included adult patients who had developed COVID-19 between February and September 2020. Total white blood cells derived from the buffy coat of peripheral whole blood were used to detect SARS-CoV-2 RNA using the Illumina COVIDSeq test. We compared the clinical characteristics between patients with and without detected viral RNA (detected and undetected groups). RESULTS: Among the 390 patients included, 17 harbored SARS-CoV-2 RNA in peripheral white blood cells. All 17 patients required oxygen support during the disease course and had higher intensive care unit admission (52.9% vs 28.9%, P = 0.035), mortality (17.7% vs 3.5%, P = 0.004), kidney dysfunction (severe, 23.5% vs 6.4%, P = 0.029), and corticosteroid treatment rates (76.5% vs 46.5%, P = 0.016) than those of patients in the undetected group. CONCLUSION: We propose that patients with circulating intracellular SARS-CoV-2 RNA in the peripheral blood exhibited the most severe disease course.

Oncogenic stress-induced Netrin is a humoral signaling molecule that reprograms systemic metabolism in Drosophila.

Cancer exerts pleiotropic, systemic effects on organisms, leading to health deterioration and eventually to organismal death. How cancer induces systemic effects on remote organs and the organism itself still remains elusive. Here we describe a role for NetrinB (NetB), a protein with a particularly well-characterized role as a tissue-level axon guidance cue, in mediating oncogenic stress-induced organismal, metabolic reprogramming as a systemic humoral factor. In Drosophila, Ras-induced dysplastic cells upregulate and secrete NetB. Inhibition of either NetB from the transformed tissue or its receptor in the fat body suppresses oncogenic stress-induced organismal death. NetB from the dysplastic tissue remotely suppresses carnitine biosynthesis in the fat body, which is critical for acetyl-CoA generation and systemic metabolism. Supplementation of carnitine or acetyl-CoA ameliorates organismal health under oncogenic stress. This is the first identification, to our knowledge, of a role for the Netrin molecule, which has been studied extensively for its role within tissues, in humorally mediating systemic effects of local oncogenic stress on remote organs and organismal metabolism.

Prophylactic administration of ivermectin attenuates SARS-CoV-2 induced disease in a Syrian Hamster Model.

COVID-19, caused by SARS-CoV-2 infection, is currently among the most important public health concerns worldwide. Although several effective vaccines have been developed, there is an urgent clinical need for effective pharmaceutical treatments for treatment of COVID-19. Ivermectin, a chemical derivative of avermectin produced by Streptomyces avermitilis, is a macrocyclic lactone with antiparasitic activity. Recent studies have shown that ivermectin inhibits SARS-CoV-2 replication in vitro. In the present study, we investigated the in vivo effects of ivermectin in a hamster model of SARS-CoV-2 infection. The results of the present study demonstrate oral administration of ivermectin prior to SARS-CoV-2 infection in hamsters was associated with decreased weight loss and pulmonary inflammation. In addition, the administration of ivermectin reduced pulmonary viral titers and mRNA expression level of pro-inflammatory cytokines associated with severe COVID-19 disease. The administration of ivermectin rapidly induced the production of virus-specific neutralizing antibodies in the late stage of viral infection. Zinc concentrations leading to immune quiescence were also significantly higher in the lungs of ivermectin-treated hamsters compared to controls. These results indicate that ivermectin may have efficacy in reducing the development and severity of COVID-19 by affecting host immunity in a hamster model of SARS-CoV-2 infection.

Both Feline Coronavirus Serotypes 1 and 2 Infected Domestic Cats Develop Cross-Reactive Antibodies to SARS-CoV-2 Receptor Binding Domain: Its Implication to Pan-CoV Vaccine Development.

The current study was initiated when our specific-pathogen-free laboratory toms developed unexpectedly high levels of cross-reactive antibodies to human SARS-CoV-2 (SCoV2) receptor binding domain (RBD) upon mating with feline coronavirus (FCoV)-positive queens. Multi-sequence alignment analyses of SCoV2 Wuhan RBD and four strains each from FCoV serotypes 1 and 2 (FCoV1 and FCoV2) demonstrated an amino acid sequence identity of 11.5% and a similarity of 31.8% with FCoV1 RBD (12.2% identity and 36.5% similarity for FCoV2 RBD). The sera from toms and queens cross-reacted with SCoV2 RBD and reacted with FCoV1 RBD and FCoV2 spike-2, nucleocapsid, and membrane proteins, but not with FCoV2 RBD. Thus, the queens and toms were infected with FCoV1. Additionally, the plasma from six FCoV2-inoculated cats reacted with FCoV2 and SCoV2 RBDs, but not with FCoV1 RBD. Hence, the sera from both FCoV1-infected cats and FCoV2-infected cats developed cross-reactive antibodies to SCoV2 RBD. Furthermore, eight group-housed laboratory cats had a range of serum cross-reactivity to SCoV2 RBD even 15 months later. Such cross-reactivity was also observed in FCoV1-positive group-housed pet cats. The SCoV2 RBD at a high non-toxic dose and FCoV2 RBD at a 60-400-fold lower dose blocked the in vitro FCoV2 infection, demonstrating their close structural conformations essential as vaccine immunogens. Remarkably, such cross-reactivity was also detected by the peripheral blood mononuclear cells of FCoV1-infected cats. The broad cross-reactivity between human and feline RBDs provides essential insights into developing a pan-CoV vaccine.

DOCK2 is involved in the host genetics and biology of severe COVID-19.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge1-5. Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target.

Immunodominant B-Cell Linear Epitope on the VP1 P Domain of a Feline Norovirus Cat Model.

Norovirus (NoV) infection remains a major public health concern worldwide. Appropriate animal models are essential for the development of effective NoV vaccines. We previously established the feline NoV (FNoV)-cat model as a surrogate animal model for human NoV infection. In the present study, we analyzed the B-cell linear epitope in the P domain of FNoV to confirm the basic immunological features of the FNoV-cat model. B-cell linear epitopes were present in the P2 subdomain. We compared antibody levels to peptides containing the B-cell linear epitope (P-10) in three FNoV-infected cats with time-course changes in viral load and symptom scoring. After FNoV infection, viral shedding and clinical symptoms were shown to improve by elevated levels of antibodies against P-10 in the plasma. This report provides important information for understanding NoV infections in humans and cats.

Erebosis, a new cell death mechanism during homeostatic turnover of gut enterocytes.

Many adult tissues are composed of differentiated cells and stem cells, each working in a coordinated manner to maintain tissue homeostasis during physiological cell turnover. Old differentiated cells are believed to typically die by apoptosis. Here, we discovered a previously uncharacterized, new phenomenon, which we name erebosis based on the ancient Greek word erebos ("complete darkness"), in the gut enterocytes of adult Drosophila. Cells that undergo erebosis lose cytoskeleton, cell adhesion, organelles and fluorescent proteins, but accumulate Angiotensin-converting enzyme (Ance). Their nuclei become flat and occasionally difficult to detect. Erebotic cells do not have characteristic features of apoptosis, necrosis, or autophagic cell death. Inhibition of apoptosis prevents neither the gut cell turnover nor erebosis. We hypothesize that erebosis is a cell death mechanism for the enterocyte flux to mediate tissue homeostasis in the gut.

In vitro antiviral effects of GS-441524 and itraconazole combination against feline infectious peritonitis virus.

Feline infectious peritonitis virus (FIPV: virulent feline coronavirus) causes a fatal disease called feline infectious peritonitis (FIP) in wild and domestic cat species. Recent studies identified several antiviral drugs that are effective against FIPV. Drug combination is one of the important strategies in the development of novel treatments for viral infections. GS-441524, a nucleoside analog, and itraconazole, a triazole antifungal drug, have been reported that have antiviral effect against FIPV. This study aims to investigate whether the combination of GS-441524 and itraconazole has synergic antiviral effect against FIPV. The antiviral effect was measured by plaque reduction assay using felis catus whole fatus-4 cell. The plaque reduction of GS-441524 against type I FIPVs increased as the concentration of itraconazole increased. The similar result was obtained for type II FIPV. In addition, the calculated combination index (CI) demonstrated that there was a strong synergy between GS-441524 and itraconazole. It is concluded that the combination of GS-441524 and itraconazole may enhance the individual effect of each drug against replication of type I FIPVs and may contribute to development more effective treatment strategy for FIP.

Correction: Genetic interactions among ADAMTS metalloproteases and basement membrane molecules in cell migration in Caenorhabditis elegans.

[This corrects the article DOI: 10.1371/journal.pone.0240571.].

Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.

Predominance of canine parvovirus 2b in Japan: an epidemiological study during 2014-2019.

Canine parvovirus 2 (CPV-2) is an important pathogen of domestic dogs and wild canids. In Japan, CPV-2 infection is one of the most common infectious diseases of dogs. We analyzed samples collected between 2014 and 2019 to identify antigenic variants of CPV-2 in dogs in Japan. Our results demonstrated that the CPV-2b variant was predominant. The CPV-2c variant was not found among our samples. Our findings demonstrate that the distribution of CPV-2 antigenic variants in Japan was more similar to that in Australia than to that in neighboring countries in Asia.

white regulates proliferative homeostasis of intestinal stem cells during ageing in Drosophila.

Tissue integrity is contingent on maintaining stem cells. Intestinal stem cells (ISCs) over-proliferate during ageing, leading to tissue dysplasia in Drosophila melanogaster. Here we describe a role for white, encoding the evolutionarily conserved ATP-binding cassette transporter subfamily G, with a particularly well-characterized role in eye colour pigmentation, in ageing-induced ISC proliferation in the midgut. ISCs increase expression of white during ageing. ISC-specific inhibition of white suppresses ageing-induced ISC dysregulation and prolongs lifespan. Of the proteins that form heterodimers with White, Brown mediates ISC dysregulation during ageing. Metabolomics analyses reveal previously unappreciated, profound metabolic impacts of white inhibition on organismal metabolism. Among the metabolites affected by White, tetrahydrofolate is transported by White, is accumulated in ISCs during ageing and is indispensable for ageing-induced ISC over-proliferation. Since Thomas Morgan's isolation of a white mutant as the first Drosophila mutant, white mutants have been used extensively as genetic systems and often as controls. Our findings provide insights into metabolic regulation of stem cells mediated by the classic gene white.

Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in Drosophila.

Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. Here, we reveal that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. Our findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. We also exemplify a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism.

Detection of feline norovirus using commercial real-time RT-PCR kit for the diagnosis of human norovirus infection.

Feline noroviruses (FNoVs) are potential clinical pathogens in cats. To perform an epidemiological study of FNoV infection, it is necessary to develop a simple and effective method for virus detection. We investigated whether a commercial human NoV quantitative RT-PCR kit for the detection of human NoVs used in medical practice can be applied for FNoV detection. This kit was capable of detecting the FNoV gene regardless of the genogroup (GIV and GVI) in experimental and field samples. Based on the above findings, it is possible to detect FNoVs using human NoV tests. The relationship between FNoV infection and gastroenteritis in cats may be clarified by applying these methods to an epidemiological survey of FNoVs.

Possible Antiviral Activity of 5-Aminolevulinic Acid in Feline Infectious Peritonitis Virus (Feline Coronavirus) Infection.

Feline infectious peritonitis (FIP) is a life-threatening infectious disease of cats caused by virulent feline coronavirus (FIP virus: FIPV). For the treatment of FIP, several effective antivirals were recently reported, but many of these are not available for practical use. 5-amino levulinic acid (5-ALA) is a low-molecular-weight amino acid synthesized in plant and animal cells. 5-ALA can be synthesized in a large amount, and it is widely applied in the medical and agricultural fields. We hypothesized that 5-ALA inhibits FIPV infection. Therefore, we evaluated its antiviral activity against FIPV in felis catus whole fetus-4 cells and feline primary macrophages. FIPV infection was significantly inhibited by 250 µM 5-ALA. Our study suggested that 5-ALA is applicable for the treatment and prevention of FIPV infection.

Genetic interactions among ADAMTS metalloproteases and basement membrane molecules in cell migration in Caenorhabditis elegans.

During development of the Caenorhabditis elegans gonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms. The ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family metalloproteases MIG-17 and GON-1 are required for correct DTC migration. Mutations in mig-17 result in misshapen gonads due to the misdirected DTC migration, and mutations in gon-1 result in shortened and swollen gonads due to the premature termination of DTC migration. Although the phenotypes shown by mig-17 and gon-1 mutants are very different from one another, mutations that result in amino acid substitutions in the same basement membrane protein genes, emb-9/collagen IV a1, let-2/collagen IV a2 and fbl-1/fibulin-1, were identified as genetic suppressors of mig-17 and gon-1 mutants. To understand the roles shared by these two proteases, we examined the effects of the mig-17 suppressors on gon-1 and the effects of the gon-1 suppressors and enhancers on mig-17 gonadal defects. Some of the emb-9, let-2 and fbl-1 mutations suppressed both mig-17 and gon-1, whereas others acted only on mig-17 or gon-1. These results suggest that mig-17 and gon-1 have their specific functions as well as functions commonly shared between them for gonad formation. The levels of collagen IV accumulation in the DTC basement membrane were significantly higher in the gon-1 mutants as compared with wild type and were reduced to the wild-type levels when combined with suppressor mutations, but not with enhancer mutations, suggesting that the ability to reduce collagen IV levels is important for gon-1 suppression.

Clinical efficacy of combination therapy of itraconazole and prednisolone for treating effusive feline infectious peritonitis.

A 3-month-old male Scottish Fold kitten with pleural fluid and low ratio of albumin to globulin (A/G ratio) was brought to our small animal hospital. Since RNA from the type I feline coronavirus (FCoV) were detected in drained pleural fluid, the cat was tentatively diagnosed with effusive feline infectious peritonitis (FIP). Following the administration of itraconazole and prednisolone, the A/G ratio increased, and the pleural fluid mostly disappeared. The fecal FCoV levels temporarily decreased. However, the cat showed neurological manifestations and was eventually euthanized due to status epilepticus after 38 days of treatment. In conclusion, itraconazole partly exerted a beneficial effect in a cat with FIP. However, further investigation of a possible role of itraconazole in FIP treatment is warranted.

Antiviral Effects of Hydroxychloroquine and Type I Interferon on In Vitro Fatal Feline Coronavirus Infection.

Feline infectious peritonitis (FIP) is a viral disease with a high morbidity and mortality by the FIP virus (FIPV, virulent feline coronavirus). Several antiviral drugs for FIP have been identified, but many of these are expensive and not available in veterinary medicine. Hydroxychloroquine (HCQ) is a drug approved by several countries to treat malaria and immune-mediated diseases in humans, and its antiviral effects on other viral infections (e.g., SARS-CoV-2, dengue virus) have been confirmed. We investigated whether HCQ in association with interferon-ω (IFN-ω) is effective for FIPV in vitro. A total of 100 µM of HCQ significantly inhibited the replication of types I and II FIPV. Interestingly, the combination of 100 µM of HCQ and 104 U/mL of recombinant feline IFN-ω (rfIFN-ω, veterinary registered drug) increased its antiviral activity against type I FIPV infection. Our study suggested that HCQ and rfIFN-ω are applicable for treatment of FIP. Further clinical studies are needed to verify the combination of HCQ and rIFN-ω will be effective and safe treatment for cats with FIP.

Therapeutic effect of an anti-human-TNF-alpha antibody and itraconazole on feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a fatal disease in wild and domestic cat species. Although several drugs are expected to be useful as treatments for FIP, no drugs are available in clinical practice. In this study, we evaluated the therapeutic effect of combined use of adalimumab (an anti-human-TNF-alpha monoclonal antibody, ADA) and itraconazole (ICZ), which are presently available to veterinarians. The neutralizing activity of ADA against fTNF-alpha-induced cytotoxicity was measured in WEHI-164 cells. Ten specific pathogen-free (SPF) cats were inoculated intraperitoneally with type I FIPV KU-2. To the cats that developed FIP, ADA (10 mg/animal) was administered twice between day 0 and day 4 after the start of treatment. ICZ (50 mg/head, SID) was orally administered daily from day 0 after the start of treatment. ADA demonstrated dose-dependent neutralizing activity against rfTNF-alpha. In an animal experiment, 2 of 3 cats showed improvements in FIP clinical symptoms and blood chemistry test results, an increase in the peripheral blood lymphocyte count, and a decrease in the plasma alpha 1-AGP level were observed after the beginning of treatment. One of the cats failed to respond to treatment and was euthanized, although the viral gene level in ascites temporarily decreased after the start of treatment. ADA was found to have neutralizing activity against rfTNF-alpha. The combined use of ADA and ICZ showed a therapeutic effect for experimentally induced FIP. We consider these drugs to be a treatment option until effective anti-FIPV drugs become available.