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.

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.

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.

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.

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.

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.

Antiviral activity of itraconazole against type I feline coronavirus infection.

Feline coronaviruses (FCoVs) are the causative agents of severe systemic disease (feline infectious peritonitis: FIP) in domestic and wild cats. FCoVs have been classified into serotypes I and II. Type I FCoV is the dominant serotype (approximately 70-90%) worldwide. Therefore, it is necessary to provide antiviral agents for type I FCoV infection. In this study, we demonstrated that itraconazole (ICZ), practically used for fungal infections in cats, inhibits the type I FCoV infection. ICZ also exhibited antiviral effect in cells after viral infection, suggesting that ICZ could potentially be used as a therapeutic.

Novel single-stranded, circular DNA virus identified in cats in Japan.

We detected a novel feline stool-associated circular DNA virus (FeSCV) in fecal samples from cats with diarrhea using consensus primers matching those of circovirus and cyclovirus. FeSCV is a circular DNA virus containing a genome with a total length of 2,046 nt encoding 2 open reading frames. Phylogenetic analyses indicated that FeSCV is classified into a clade different from that of circovirus and cyclovirus. Since the FeSCVs detected in several cats in the same household had genetically similar genomes, these viruses are most likely derived from the same origin.

Viral shedding and clinical status of feline-norovirus-infected cats after reinfection with the same strain.

Norovirus (NoV) infection is the most common cause of acute gastroenteritis in humans of all ages worldwide. When cats are experimentally infected with feline norovirus (FNoV), they develop symptoms of acute gastroenteritis. Therefore, FNoV infection may serve as an animal model for the disease caused by human norovirus infection. In this study, we examined whether FNoV of cats infected with genogroup GVI are protected from reinfection with the same strain. The blood anti-FNoV IgG level was inversely correlated with the viral load in stool samples and the clinical score of FNoV-infected cats, but complete prevention of reinfection was not observed. These findings were similar to the results of a reinfection experiment with NoV in human volunteers.

Antibody-dependent enhancement of serotype II feline enteric coronavirus infection in primary feline monocytes.

Feline coronavirus (FCoV) has been classified into two biotypes: avirulent feline coronavirus (feline enteric coronavirus: FECV) and virulent feline coronavirus (feline infectious peritonitis virus: FIPV). In FIPV infection, antibody-dependent enhancement (ADE) has been reported and was shown to be associated with severe clinical disease. On the other hand, the potential role of ADE in FECV infection has not been examined. In this study, using laboratory strains of serotype II FIPV WSU 79-1146 (FIPV 79-1146) and serotype II FECV WSU 79-1683 (FECV 79-1683), we investigated the relationship between ADE and induction of inflammatory cytokines, which are pathogenesis-related factors, for each strain. As with ADE of FIPV 79-1146 infection, a monoclonal antibody against the spike protein of FCoV (mAb 6-4-2) enhanced FECV 79-1683 replication in U937 cells and primary feline monocytes. However, the ADE activity of FECV 79-1683 was lower than that of FIPV 79-1146. Moreover, mRNA levels of inflammatory cytokines (TNF-α, IL-1ß, and IL-6) significantly increased with ADE of FIPV 79-1146 infection in primary feline monocytes, but FECV 79-1683 did not demonstrate an increase in these levels. In conclusion, infection of monocytes by FECV was enhanced by antibodies, but the efficiency of infection was lower than that of FIPV.

Genetic characterization of feline bocavirus detected in cats in Japan.

Feline bocavirus (FBoV) has been classified into three genotypes (FBoV1-FBoV3). FBoVs are mainly detected in feces. In the present study, we collected rectal swabs from cats in Japan and examined the samples for the presence of FBoV. The FBoV infection rate was 9.9 % in 101 cats. No significant association was observed between FBoV infection and clinical symptoms. Based on the full-length NS1 protein, the three strains of FBoVs detected in the present study shared high homologies with the genotype 2 FBoV POR1 strain. This is the first study to report FBoV in Japan.

Differential effect of cholesterol on type I and II feline coronavirus infection.

Feline infectious peritonitis (FIP) is a fatal disease of domestic and wild felidae that is caused by feline coronavirus (FCoV). FCoV has been classified into types I and II. Since type I FCoV infection is dominant in the field, it is necessary to develop antiviral agents and vaccines against type I FCoV infection. However, few studies have been conducted on type I FCoV. Here, we compare the effects of cholesterol on types I and II FCoV infections. When cells were treated methyl-ß-cyclodextrin (MßCD) and inoculated with type I FCoV, the infection rate decreased significantly, and the addition of exogenous cholesterol to MßCD-treated cells resulted in the recovery of the infectivity of type I FCoV. Furthermore, exogenous cholesterol increased the infectivity of type I FCoV. In contrast, the addition of MßCD and exogenous cholesterol had little effect on the efficiency of type II FCoV infection. These results strongly suggest that the dependence of infection by types I and II FCoV on cholesterol differs.

Detection of canine astrovirus in dogs with diarrhea in Japan.

Canine astrovirus (CAstV) is the causative agent of gastroenteritis in dogs. We collected rectal swabs from dogs with or without diarrhea symptoms in Japan and examined the feces for the presence of CAstV by RT-PCR with primers based on a conserved region of the ORF1b gene. The ORF1b gene of CAstV was not detected in the 42 dogs without clinical illness but was present in three pups out of the 31 dogs with diarrhea symptoms. Based on the full-length capsid protein, the CAstV KU-D4-12 strain that we detected in this study shared high homology with the novel virulent CAstV VM-2011 strain.

Differential effects of viroporin inhibitors against feline infectious peritonitis virus serotypes I and II.

Feline infectious peritonitis virus (FIP virus: FIPV), a feline coronavirus of the family Coronaviridae, causes a fatal disease called FIP in wild and domestic cat species. The genome of coronaviruses encodes a hydrophobic transmembrane protein, the envelope (E) protein. The E protein possesses ion channel activity. Viral proteins with ion channel activity are collectively termed "viroporins". Hexamethylene amiloride (HMA), a viroporin inhibitor, can inhibit the ion channel activity of the E protein and replication of several coronaviruses. However, it is not clear whether HMA and other viroporin inhibitors affect replication of FIPV. We examined the effect of HMA and other viroporin inhibitors (DIDS [4,4'-disothiocyano-2,2'-stilbenedisulphonic acid] and amantadine) on infection by FIPV serotypes I and II. HMA treatment drastically decreased the titers of FIPV serotype I strains Black and KU-2 in a dose-dependent manner, but it only slightly decreased the titer of FIPV serotype II strain 79-1146. In contrast, DIDS treatment decreased the titer of FIPV serotype II strain 79-1146 in dose-dependent manner, but it only slightly decreased the titers of FIPV serotype I strains Black and KU-2. We investigated whether there is a difference in ion channel activity of the E protein between viral serotypes using E. coli cells expressing the E protein of FIPV serotypes I and II. No difference was observed, suggesting that a viroporin other than the E protein influences the differences in the actions of HMA and DIDS on FIPV serotypes I and II.

The emerging ST8 methicillin-resistant Staphylococcus aureus clone in the community in Japan: associated infections, genetic diversity, and comparative genomics.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a major concern worldwide. In the United States, ST8 CA-MRSA with SCCmecIVa (USA300) has been predominant, affecting the entire United States. In this study, we investigated Japanese ST8 CA-MRSA with new SCCmecIV1 (designated ST8 CA-MRSA/J), which has emerged in Japan since 2003. Regarding community spread and infections, ST8 CA-MRSA/J spread in 16.2-34.4% as a major genotype in the community in Japan, and was associated with skin and soft tissue infections (SSTIs), colitis, and invasive infections (sepsis, epidural abscesses, and necrotizing pneumonia), including influenza prodrome cases and athlete infections, similar to USA300. It spread to even public transport and Hong Kong through a Japanese family. Regarding genetic diversity, ST8 CA-MRSA/J included ST and spa variants and was classified into at least three pulsed-field gel electrophoresis types, ST8 Jα to γ. Of those, ST8 Jß was associated with severe invasive infections. As for genomics, ST8 CA-MRSA/J showed high similarities to USA300, but with marked diversity in accessory genes; e.g., ST8 CA-MRSA/J possessed enhanced cytolytic peptide genes of CA-MRSA, but lacked the Panton-Valentine leukocidin phage and arginine catabolic mobile element, unlike USA300. The unique features of ST8 CA-MRSA/J included a novel mosaic SaPI (designated SaPIj50) carrying the toxic shock syndrome toxin-1 gene with high expression; the evolution included salvage (through recombination) of hospital-acquired MRSA virulence. The data suggest that ST8 CA-MRSA/J has become a successful native clone in Japan, in association with not only SSTIs but also severe invasive infections (posing a threat), requiring attention.

A new local variant (ST764) of the globally disseminated ST5 lineage of hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) carrying the virulence determinants of community-associated MRSA.

The ST5 lineage of methicillin-resistant Staphylococcus aureus (MRSA) is one of the most globally disseminated hospital-associated MRSA (HA-MRSA) lineages. We isolated a new local variant (designated ST764) over at least 5 years that causes invasive infections, including necrotizing fasciitis, and is carried by medical students, as well as household members. Analysis of the genome sequence of one isolate compared to that of the reference ST5 strain revealed that ST764 had acquired virulence traits similar to those of community-associated MRSA (CA-MRSA) through the acquisition of two new mobile genetic elements, ACMEII and SaPInn54, which carried ACME arcA and the staphylococcal enterotoxin B gene (seb), respectively, and through enhanced expression of cytolytic peptide genes, although ST764 was negative for Panton-Valentine leukocidin. Other differences between ST764 and ST5 included the acquisition of an ACMEII-related cassette (cJR1), prophage φ2NN54, and streptococcal Tn5251 and decreased numbers of copies of Tn554. As for superantigen genes, although the two possessed seg, sei, sem, sen, and seo, ST764 lacked tst, sec, sel, and sep. The data suggest that ST764 MRSA is a novel hybrid variant of ST5 HA-MRSA with the characteristics of CA-MRSA and that the evolution of ST764 includes multiple steps, e.g., acquisition of novel or nonstaphylococcal mobile elements.

Unique features of the motility and structures in the flagellate polar region of Campylobacter jejuni and other species: an electron microscopic study.

Similarly to Helicobacter pylori but unlike Vibrio cholerae O1/O139, Campylobacter jejuni is non-motile at 20°C but highly motile at ≥37°C. The bacterium C. jejuni has one of the highest swimming speeds reported (>100 µm/s), especially at 42°C. Straight and spiral bacterial shapes share the same motility. C. jejuni has a unique structure in the flagellate polar region, which is characterized by a cup-like structure (beneath the inner membrane), a funnel shape (opening onto the polar surface) and less dense space (cytoplasm). Other Campylobacter species (coli, fetus, and lari) have similar motility and flagellate polar structures, albeit with slight differences. This is especially true for Campylobacter fetus, which has a flagellum only at one pole and a cup-like structure composed of two membranes.

Virulence gene and expression analysis of community-associated methicillin-resistant Staphylococcus aureus causing iliopsoas abscess and discitis with thrombocytopenia.

Iliopsoas abscesses (IPAs) from methicillin-resistant Staphylococcus aureus (MRSA) are rare; however, IPAs from community-associated MRSA (CA-MRSA) may be increasing. In Japan, we previously described an adolescent athlete case of Panton-Valentine leukocidin (PVL)-positive ST30 CA-MRSA (strain NN12). In this study, we describe an IPA and discitis case from a variant of the successful PVL-negative CA-MRSA clone (ST8 CA-MRSA/J) in Japan. The patient was a 62-year-old man with intractable eczema, who had been diagnosed with IPAs and discitis (L1-L2). CA-MRSA (strain NN55) was isolated from blood, pus, and joint fluid. The invasive infections seemed to have originated in his intractable eczema, and the characteristics of this case, systemic myalgia and marked thrombocytopenia, seemed to have been caused by an exotoxin. Molecular genetic analysis revealed that NN55 possessed genotype ST8/spa606(t1767)/agr1/CoaIII and SCCmecIV of a novel subtype (encoding new cell-wall-anchored surface protein/J [CWASP/J]), exhibited enhanced expression of the cytolytic peptide genes, psmα and hld, and was resistant to gentamicin (caused by aacA-aphD), similar to ST8 CA-MRSA/J; however, NN55 lacked pathogenicity island SaPIj50 [carrying tst, encoding toxic shock syndrome toxin-1 (TSST-1)] of ST8 CA-MRSA/J, suggesting a variant (ST8 CA-MRSA/Jv). Strains NN12 and NN55 both caused bacteremia, IPAs, and adjacent musculoskeletal infections, preceded by intractable skin infections, and possessed high potential for adherence and enhanced expression of psmα and hld. The data suggest the role of a combination of CA-MRSA adhesin/cytolytic peptides (not PVL or TSST-1) in the pathogenesis of IPAs (and perhaps of systemic myalgia and marked thrombocytopenia).

Electron microscopic structures, serum resistance, and plasmid restructuring of New Delhi metallo-ß-lactamase-1 (NDM-1)-producing ST42 Klebsiella pneumoniae emerging in Japan.

Enterobacteriaceae, carrying the New Delhi metallo-ß-lactamase-1 (NDM-1) gene (bla (NDM-1)), have emerged and posed a threat since 2006. In Japan, bla (NDM-1)-carrying Escherichia coli was first described in 2010. In this study, we characterized NDM-1-positive Klebsiella pneumoniae strain 419 in Japan, which was isolated from the urine of a 90-year-old Japanese patient who had never been to the Indian subcontinent. K. pneumoniae 419 belonged to ST42. It possessed a surface capsule (with untypeable capsular PCR types) and was resistant to serum killing. K. pneumoniae 419 cells were occasionally flagellated or piliated and autoaggregated. K. pneumoniae 419 was resistant to ß-lactams (including carbapenems), aminoglycosides, and fluoroquinolones, and was susceptible to imipenem (or biapenem), aztreonam, polymixin B, and colistin. It possessed at least eight plasmids; of those, a 74-kb plasmid (pKPJ1) of the replicon FIIA carried bla (NDM-1) and was conjugally transferred to E. coli strains, with a 71-kb transferable azithromycin-resistant (mphA (+)) plasmid of the replicon F (pKPJ2), as a large (145-kb) plasmid (pKPJF100) through a transposition event. In addition to bla (NDM-1), pKPJ1 carried arr-2, pKPJ2 carried mphA, and pKPJF100 carried both. They were negative for the 16S rRNA methylase gene, e.g., which is frequently associated with bla (NDM-1). The data demonstrate that K. pneumoniae 419 possessed virulence- and fitness-associated surface structures, was resistant to serum killing, and possessed a unique (or rare) genetic background in terms of ST type and bla (NDM-1)-carrying plasmid.