Emergence of SARS-CoV-2 with Dual-Drug Resistant Mutations During a Long-Term Infection in a Kidney Transplant Recipient.

Introduction: Various therapeutic agents are being developed for the treatment of coronavirus disease 2019 (COVID-19). Therefore, it is crucial to accumulate information regarding the features of drug-resistant viruses to these antiviral drugs. Methods: We investigated the emergence of dual-drug resistance in a kidney transplant recipient who received sotrovimab (from day 0) and remdesivir (RDV) (from day 8 to day 17). We sequenced the whole viral genomes from nasopharyngeal swabs taken on day 0 and seven points after starting treatment (on days 12, 19, 23, 37, 43, 48, and 58). The genetic traits of the wild-type (day 0) and descendant viruses (after day 12) were determined by comparing the genomes with those of a Wuhan strain and the day 0 wild-type strain, respectively. Three viral isolates (from samples collected on days 0, 23, and 37) were investigated for their escape ability and growth kinetics in vitro. Results: The sotrovimab resistant mutation (S:E340K) and the RDV resistant mutation RdRp:V792I (nt: G15814A) emerged within 12 days (day 12) and 11 days (day 19) after the treatment, respectively. The day 23 isolate harboring S:E340K/RdRp:V791I was resistant to both sotrovimab and RDV, showing 364- and 2.73-fold higher resistance respectively, compared with the wild-type. Moreover, compared with the day 23 isolate, the day 37 isolate accumulated multiple additional mutations and had a higher level of resistance to both drugs. Conclusion: Drug-resistant variants with double mutations (S:E340K/RdRp:V791I) became dominant within 23 days after starting treatment, suggesting that even a combination therapy involving sotrovimab and RDV, dual-drug resistant viruses may emerge rapidly in immunocompromised patients. The dual-resistant variants had lower virus yields than those of the wild-type virus in vitro, suggesting that they paid a fitness cost.

Use of live attenuated recombinant Newcastle disease virus carrying avian paramyxovirus 2 HN and F protein genes to enhance immune responses against species A rotavirus VP6 protein.

Numerous infectious diseases in cattle lead to reductions in body weight, milk production, and reproductive performance. Cattle are primarily vaccinated using inactivated vaccines due to their increased safety. However, inactivated vaccines generally result in weaker immunity compared with live attenuated vaccines, which may be insufficient in certain cases. Over the last few decades, there has been extensive research on the use of the Newcastle disease virus (NDV) as a live vaccine vector for economically significant livestock diseases. A single vaccination dose of NDV can sufficiently induce immunity; therefore, a booster vaccination dose is expected to yield limited induction of further immune response. We previously developed recombinant chimeric NDV (rNDV-2F2HN), in which its hemagglutinin-neuraminidase (HN) and fusion (F) proteins were replaced with those of avian paramyxovirus 2 (APMV-2). In vitro analysis revealed that rNDV-2F2HN expressing human interferon-gamma had potential as a cancer therapeutic tool, particularly for immunized individuals. In the present study, we constructed rNDV-2F2HN expressing the bovine rotavirus antigen VP6 (rNDV-2F2HN-VP6) and evaluated its immune response in mice previously immunized with NDV. Mice primarily inoculated with recombinant wild-type NDV expressing VP6 (rNDV-WT-VP6), followed by a booster inoculation of rNDV-2F2HN-VP6, showed a significantly stronger immune response than that in mice that received rNDV-WT-VP6 as both primary and booster inoculations. Therefore, our findings suggest that robust immunity could be obtained from the effects of chimeric rNDV-2F2HN expressing the same or a different antigen of a particular pathogen as a live attenuated vaccine vector.

A Successfully Treated Case of Posterior Ischemic Optic Neuropathy That Developed during Antihypertensive Therapy for Hypertensive Emergency.

A 33-year-old woman developed hypertensive emergency (268/168 mmHg) with renal failure and hypertensive retinopathy. Four hours after the initiation of antihypertensive therapy with the continuous infusion of nicardipine, her blood pressure (BP) decreased to 168/84 mmHg; however, the patient developed blindness. She was diagnosed with posterior ischemic optic neuropathy (PION). Her BP was maintained at approximately 175/90 mmHg until her vision improved. Olmesartan was initiated on day 13, and her BP decreased to approximately 135/95 mmHg without the re-exacerbation of vision loss. Although the prognosis of PION is poor, its early diagnosis and gradual antihypertensive therapy may help preserve the patient's vision.

A case of malignant hypertension as a presentation of atypical hemolytic uremic syndrome.

INTRODUCTION: Malignant hypertension (mHTN) damages multiple target organs, including the kidneys. mHTN has been regarded as one of the causes of secondary thrombotic microangiopathy (TMA); however, a high prevalence of complement gene abnormalities was recently reported in cohorts of mHTN. CASE REPORT: We herein describe a 47-year-old male who presented with severe hypertension, renal failure (serum creatinine (sCr): 11.6 mg/dL), heart failure, retinal hemorrhage, hemolytic anemia, and thrombocytopenia. Renal biopsy findings were consistent with acute hypertensive nephrosclerosis. The patient was diagnosed with secondary TMA associated with mHTN. However, his previous medical history of TMA of unknown origin and family history of atypical hemolytic uremic syndrome (aHUS) suggested as aHUS presenting mHTN, and genetic testing revealed a pathogenic C3 mutation (p.I1157T). The patient required plasma exchange and hemodialysis for 2 weeks and was able to withdraw from dialysis by antihypertensive therapy without eculizumab. Renal function gradually improved to a sCr level of 2.7 mg/dL under antihypertensive therapy for 2 years after the event. There was no recurrence, and renal function was preserved throughout a 3-year follow-up. DISCUSSION: mHTN is a common presentation of aHUS. In cases of mHTN, abnormalities in complement-related genes may be involved in the development of the disease.

Chimeric Newcastle Disease Virus Vectors Expressing Human IFN-γ Mediate Target Immune Responses and Enable Multifaceted Treatments.

The therapeutic potential of Newcastle disease virus (NDV) has been reported as both an oncolytic agent and a vaccine vector against many antigens. However, in the individuals already immunized with NDVs, second and subsequent administration does not provide substantial benefits. In this study, two types of recombinant chimeric NDVs using APMV-2 F and HN genes were generated. In rNDV-2HN, the wild-type NDV HN gene was replaced with the APMV-2 HN gene, and in rNDV-2F/2HN, both wild-type F and HN genes were replaced with APMV-2 F and HN genes, respectively. We enhanced the immune responses of these chimeric viruses by inserting the human IFN-γ gene. To examine the escape from NDV antiserum, each virus was treated with diluted NDV antiserum, and HEp-2 cells were infected with these virus particles. The two constructed chimeric viruses indicated notably lower virus-neutralizing titer compared to wild-type NDV and escaped the action of NDV antiserum. These two chimeric viruses infected both respiratory and colon cancer cell lines, indicating their potential as a cancer treatment tool. Chimeric viruses with enhanced immune responses can be considered a novel therapeutic strategy in cancer treatment that can be administered multiple times and used to enhance immune cells interaction.

Downregulation of calcium-regulated heat stable protein 1 expression by low-temperature stimulation causes reduction of interferon-ß expression and sensitivity to influenza viral infection.

Influenza is prevalent in temperate countries during winter when the environment is dry and cold; however, in tropical and subtropical countries, it is prevalent during the hot, humid rainy season. Thus, temperature and humidity conditions affect influenza outbreaks in different climates. Although the reason for this may be related to host conditions and the conditions under which the virus can survive, it is difficult to analyze changes in host viral responses owing to environmental changes at the cellular level. In the current study, to find candidate genes related with temperature, we analyzed the effects of low-temperature stimulation on influenza virus infection using immortalized respiratory cell lines with the same genetic background established in our laboratory. Although two cell lines with different immune response strengths exhibited enhancement of influenza virus replication following low-temperature stimulation, the mechanisms and degrees were different. In cell lines that showed greater changes, promotion of viral replication was found to involve genes related to temperature, including TRPM2 and CARHSP1. In particular, CARHSP1 expression was decreased by low-temperature stimulation in several respiratory cell lines. In knockdown experiments, because reduction of interferon-ß production and sensitivity were observed, the decline may create an environment in which the initial infection cannot be controlled. This procedure may be effective for identifying candidate genes related to the host/viral responses to changes in temperature, and these results can help elucidate the relationships of temperature, humidity, and host responses with viral infection.

Development of genus-specific universal primers for the detection of flaviviruses.

BACKGROUND: Flaviviruses are representative arboviruses carried by arthropods and/or vertebrates; these viruses can pose a public health concern in many countries. By contrast, it is known that a novel virus group called insect-specific flaviviruses (ISFs) also infects arthropods, although no such virus has yet been isolated from vertebrates. The characteristics of ISFs, which affect replication of human-pathogenic flaviviruses within co-infected mosquito cells or mosquitoes without affecting the mosquitoes themselves, mean that we should pay attention to both ISFs and human-pathogenic flaviviruses, despite the fact that ISFs appear not to be directly hazardous to human health. To assess the risk of diseases caused by flaviviruses, and to better understand their ecology, it is necessary to know the extent to which flaviviruses are harbored by arthropods. METHODS: We developed a novel universal primer for use in a PCR-based system to detect a broad range of flaviviruses. We then evaluated its performance. The utility of the novel primer pair was evaluated in a PCR assay using artificially synthesized oligonucleotides derived from a template viral genome sequence. The utility of the primer pair was also examined by reverse transcription PCR (RT-PCR) using cDNA templates prepared from virus-infected cells or crude supernatants prepared from virus-containing mosquito homogenates. RESULTS: The novel primer pair amplified the flavivirus NS5 sequence (artificially synthesized) in all samples tested (six species of flavivirus that can cause infectious diseases in humans, and flaviviruses harbored by insects). In addition, the novel primer pair detected viral genomes in cDNA templates prepared from mosquito cells infected with live flavivirus under different infectious conditions. Finally, the viral genome was detected with high sensitivity in crude supernatants prepared from pooled mosquito homogenates. CONCLUSION: This PCR system based on a novel primer pair makes it possible to detect arthropod-borne flaviviruses worldwide (the primer pair even detected viruses belonging to different genetic subgroups). As such, an assay based on this primer pair may help to improve public health and safety, as well as increase our understanding of flavivirus ecology.

CG223, a novel BET inhibitor, exerts TGF-ß1-mediated antifibrotic effects in a murine model of bleomycin-induced pulmonary fibrosis.

Pulmonary fibrosis is a progressive disease with poor prognosis and limited therapeutic options. In this study, we evaluated the potential therapeutic effects of CG223, a novel inhibitor of bromodomain and extra-terminal motif (BET) proteins, on pulmonary fibrosis by focusing on the transforming growth factor-ß1 (TGF-ß1) pathway. In a murine model of bleomycin-induced pulmonary fibrosis, CG223 attenuated fibrosis while reducing the infiltration of inflammatory cells into the lungs. Fibroblasts expressing BRD4, a member of the BET protein family, were enriched in the tissue regions corresponding to bleomycin-induced fibrotic lesions. Additionally, pulmonary fibroblasts isolated from bleomycin-instilled mice showed a significantly increased association of BRD4 with the promoters of two pro-fibrotic genes linked to the entry into the TGF-ß1 autocrine/paracrine loop, thrombospondin 1 (Thbs1) and integrin ß3 (Itgb3), as well as with the promoter of a myofibroblast marker gene, actin alpha 2 (Acta2). Subsequent in vitro studies with murine primary lung fibroblasts showed that the mRNA induction of Thbs1, Itgb3, and Acta2 by TGF-ß1 can be inhibited by CG223 in a dose-dependent manner. Taken together, CG223-induced BRD4 inhibition suppressed lung fibrogenesis by affecting multiple genes, including those involved in the triggering of the TGF-ß1 autocrine/paracrine loop.

Proteins produced by Streptococcus species in the lower respiratory tract can modify antiviral responses against influenza virus in respiratory epithelial cells.

Seasonal influenza spreads during winter in temperate countries. Primary viral pneumoniae resulting from aggravation triggers acute respiratory distress syndrome, which is a serious respiratory disorder. We have identified a unique pattern of lung microbiota in patients with the syndrome. In this study, we hypothesized that the unique microbiota was also associated with primary influenza viral pneumoniae. Bacterial culture supernatants of Streptococcus oralis and Streptococcus mitis detected from the patients significantly increased viral replication (maximum 10-fold increase) in lung epithelial cells. Our results suggest that the lung environment microbiota is significantly involved in viral replication.

Unique patterns of lower respiratory tract microbiota are associated with inflammation and hospital mortality in acute respiratory distress syndrome.

BACKGROUND: The lung microbiome maintains the homeostasis of the immune system within the lungs. In acute respiratory distress syndrome (ARDS), the lung microbiome is enriched with gut-derived bacteria; however, the specific microbiome associated with morbidity and mortality in patients with ARDS remains unclear. This study investigated the specific patterns of the lung microbiome that are correlated with mortality in ARDS patients. METHODS: We analyzed the lung microbiome from the bronchoalveolar lavage fluid (BALF) of patients with ARDS and control subjects. We measured the copy numbers of 16S rRNA and the serum and BALF cytokines (interleukin [IL]-6, IL-8, receptor for advanced glycation end products, and angiopoietin-2). RESULTS: We analyzed 47 mechanically ventilated patients diagnosed with (n = 40) or without (n = 7; control) ARDS. The alpha diversity was significantly decreased in ARDS patients compared with that of the controls (6.24 vs. 8.07, P = 0.03). The 16S rRNA gene copy numbers tended to be increased in the ARDS group compared with the controls (3.83 × 106 vs. 1.01 × 105 copies/mL, P = 0.06). ARDS patients were subdivided into the hospital survivor (n = 24) and non-survivor groups (n = 16). Serum IL-6 levels were significantly higher in the non-survivors than in the survivors (567 vs. 214 pg/mL, P = 0.027). The 16S rRNA copy number was significantly correlated with serum IL-6 levels in non-survivors (r = 0.615, P < 0.05). The copy numbers and relative abundance of betaproteobacteria were significantly lower in the non-survivors than in the survivors (713 vs. 7812, P = 0.012; 1.22% vs. 0.08%, P = 0.02, respectively). Conversely, the copy numbers of Staphylococcus, Streptococcus and Enterobacteriaceae were significantly correlated with serum IL-6 levels in the non-survivors (r = 0.579, P < 0.05; r = 0.604, P < 0.05; r = 0.588, P < 0.05, respectively). CONCLUSIONS: The lung bacterial burden tended to be increased, and the alpha diversity was significantly decreased in ARDS patients. The decreased Betaproteobacteria and increased Staphylococcus, Streptococcus and Enterobacteriaceae might represent a unique microbial community structure correlated with increased serum IL-6 and hospital mortality. TRIAL REGISTRATION: The institutional review boards of Hiroshima University (Trial registration: E-447-4, registered 16 October 2019) and Kyoto Prefectural University of Medicine (Trial registration: ERB-C-973, registered 19 October 2017) approved an opt-out method of informed consent.

Demethylation around the transcriptional start site of the IFN-ß gene induces IFN-ß production and protection against influenza virus infection.

Seasonal influenza is related to lifestyle-associated risk factors and it has been suggested that the epigenetic state of the individual plays an important role in the severity of infection. It is well known that epigenetics stringently regulate gene expression in each tissue and that aberrant epigenetic states can influence disease development. Despite some studies, limited information is available on changes in epigenetic states before and after influenza virus infection; in particular, it is unknown whether the epigenetic state at specific sites affects subsequent infection. Here, we analyzed CpG methylation states in clones derived from human primary small airway epithelial cells with the same genetic background but different viral replication rates. Our study revealed that demethylating CpGs downstream of the IFN-ß transcription start site using a CRISPR/dCas9 system suppressed viral replication during subsequent influenza virus infection. Thus, our observations suggest that epigenome editing might provide adequate protection against the influenza virus.

Molecular detection of microbial colonization in cervical mucus of women with and without endometriosis.

PROBLEM: Intrauterine microbial colonization and its association with the pathogenesis of endometriosis via an innate immune cascade have been reported. As a potential source of microbial transmission, information on microbial colonization in cervical mucus is unknown. We investigated pattern of microbiota in the cervical mucus collected from women with and without endometriosis using next-generation sequencing (NGS) technology. METHOD OF STUDY: Cervical mucus samples were collected from women with (n = 30) and without (n = 39) endometriosis. The communities of microbiota in cervical mucus in the endometriosis group and the control group were examined by Gram staining and NGS targeting the V5-V6 region of 16S ribosomal RNA gene. Copy number of some target bacteria was detected by real-time PCR. RESULTS: We confirmed visual presence of bacteria in cervical mucus by Gram staining. NGS analysis showed that distribution of microbiota was similar in cervical mucus of women with and without endometriosis regardless of the phases of the menstrual cycle. In addition to predominant Lactobacilli spp., the populations of Corynebacterium, Enterobacteriaceae, Flavobacterium, Pseudomonas, and Streptococcus were increased in the endometriosis group. Of them, Enterobacteriaceae and Streptococcus were identified as the more significant candidates in the endometriosis group than in controls by real-time PCR (P < 0.05 for each). CONCLUSION: Our NGS analysis of cervical mucus indicated that among a variable microbiota, two candidates (Enterobacteriaceae and Streptococcus) were more frequently detected in women with endometriosis. Further investigation is needed to elucidate a mechanistic link of these bacteria in the pathophysiology of endometriosis.

Relationship between periodontal disease and butyric acid produced by periodontopathic bacteria.

BACKGROUND: Periodontopathic bacteria such as Porphyromonas gingivalis produce a large amount of butyric acid as a metabolite. Though butyric acid has been reported to have an anti-inflammatory effect on inflammatory diseases in the gastrointestinal tract, it has been suggested to contribute to the progression of periodontal disease in the oral cavity. The concentration of butyric acid in periodontal tissue of patients with periodontitis patients is reported to increase with the progress of the periodontal disease state. However, the influence of butyric acid on periodontal disease progression is not well known. MAIN TEXT: In this review, we have considered the relationship between butyric acid and periodontal disease with respect to the findings reported till date and the knowledge we newly obtained [Shirasugi M et al. Biochem Biophys Res Commun, 2017]. We have studied the relationship between butyric acid and periodontal disease by analyzing the effect of butyric acid on normal human gingival fibroblasts, which are a major component of periodontal tissue. We observed that gingival fibroblasts underwent cytostasis and apoptosis via extrinsic and intrinsic pathways upon long-term exposure to butyric acid. In addition, we showed that TNF-α produced by gingival fibroblasts treated with butyric acid plays an important role in inducing exogenous apoptosis. CONCLUSION: Butyric acid produced by periodontopathic bacteria may promote progress of the periodontal disease state. Butyric acid is known to act as an HDAC inhibitor. Thus, we believe that advanced epigenetic analysis of the effects of butyric acid on gingival fibroblasts will help elucidate the periodontal disease pathology and facilitate discovery of new targets for periodontal disease treatment.

A Refractory Case of Secondary Membranous Nephropathy Concurrent with IgG4-related Tubulointerstitial Nephritis.

A 58-year-old man with type 1 autoimmune pancreatitis was referred to nephrologists for severe proteinuria. Laboratory data revealed a high serum IgG4 level, hypoalbuminemia, and massive proteinuria, which were compatible with nephrotic syndrome. The renal pathological findings confirmed the diagnosis of secondary membranous nephropathy concurrent with IgG4-related tubulointerstitial nephritis. Despite the improvement of interstitial markers, the proteinuria was refractory to prednisolone, requiring cyclosporine to achieve complete remission. Membranous nephropathy is a rare manifestation of IgG4-related kidney disease. This case shows that the therapeutic response to prednisolone significantly differs between glomerular lesions and interstitial lesions of IgG4-related kidney disease.

A combined human case of Dirofilaria ursi infection in dorsal subcutaneous tissue and Anisakis simplex sensu stricto (s.s.) infection in ventral subcutaneous tissue.

BACKGROUND: Dirofilaria ursi is a filarial nematode that parasitizes the subcutaneous tissues of the American black bear (Ursus americanus) and Japanese black bear (Ursus thiabetanus japonicus). D. ursi that has parasitized black bears has the potential to subsequently infect humans. In addition, extra-gastrointestinal anisakiasis is less common in Japan. CASE PRESENTATION: We report a case of ventral subcutaneous anisakiasis and dorsal subcutaneous dirofilariasis that was acquired in Fukushima, in the northern part of Japan. The patient was an 83-year-old Japanese female, and subcutaneous parasitic granulomas were present on her left abdomen (near the navel) and left scapula. A pathological examination of the surgically dissected tissue sections from each region demonstrated eosinophilic granulomas containing different species of parasites. To enable the morphological and molecular identification of these parasites, DNA was extracted from paraffin-embedded sections using DEXPAT reagent, and the cytochrome oxidase 2 (COX2), internal transcribed spacer 1 (ITS1), 5.8S and ITS2 regions of the Anisakis larvae, and the 5S rRNA region of the male Dirofilaria were sequenced. The PCR products were examined and compared with DNA databases. Molecular analysis of the COX2 and 5S rRNA sequences of each worm revealed that the nematode found in the ventral region belonged to Anisakis simplex sensu stricto (s.s.) and the male Dirofilaria found in the dorsal region was classified as D. ursi. CONCLUSION: The present case showed a combined human case of D. ursi and A. simplex s.s. infections in subcutaneous tissues. The results of this study will contribute to the identification of unknown parasites in histological sections.

Normal human gingival fibroblasts undergo cytostasis and apoptosis after long-term exposure to butyric acid.

The causes of periodontal disease are complex. Butyric acid, a metabolite of periodontopathic bacteria such as Porphyromonas gingivalis, acts as a histone deacetylase inhibitor that has a direct effect on mRNA expression. Butyric acid produced by Clostridium butyricum in the intestinal tract induces differentiation of regulatory T cells, thereby suppressing inflammation in the gut. Mice lacking Clostridium butyricum in the intestinal tract suffer from colitis. By contrast, butyric acid in the oral cavity worsens periodontal disease. Periodontal disease is a chronic condition in which periodontal tissue is exposed to virulence factors (such as butyric acid); however, no study has examined the effects of long-term exposure to butyric acid. The present study demonstrated that long-term exposure of human gingival fibroblasts (HGFs) to butyric acid induced cytostasis and apoptosis via the intrinsic and extrinsic pathways. Butyric acid inhibited the division of HGFs by altering expression of mRNAs encoding cyclins. Butyric acid induced apoptosis in HGFs via the intrinsic pathway, followed by activation of caspase 9; there was no DNA damage or p53 activation. Butyric acid also upregulated expression of TNF-α mRNA and protein by HGFs. Furthermore TNF-α induced apoptosis by activating caspase 8 (the extrinsic pathway) and by inducing production of pro-inflammatory cytokines. Taken together, the results show that butyric acid induced cytostasis and apoptosis in HGFs, accompanied by production of pro-inflammatory cytokines. It thus acts as a death ligand and plays a critical role as a prophlogistic substance.

Effect of Esters on the Permeation of Chemicals with Different Polarities through Synthetic Artificial Membranes Using a High-Throughput Diffusion Cell Array.

This study investigated the effects of 25 kinds of esters that are used in cosmetics on the permeation of four model compounds with different polarities (caffeine [CF], aminopyrine [AMP], benzoic acid [BA], and flurbiprofen [FP]). The amount of each model compound that permeated through two types of artificial membrane (silicone and Strat-M®) was measured and correlated with the physicochemical properties of the esters, including their solubility, viscosity, wettability, surface tension, and uptake. The amount of each model compound that permeated through the silicone membrane was not significantly correlated with the solubility of the esters but was significantly correlated with all other measured physical properties of the esters. Similar correlations were observed for the amounts of AMP, BA, and FP that passed through the Strat-M® membrane. However, the amount of CF that permeated through the Strat-M® membrane also correlated with the solubility of the esters. There was a highly significant correlation between the amount permeating through the silicone and Strat-M® membranes because the model compounds had high lipophilicity. These findings demonstrated that to control the permeation of various chemicals through artificial membranes, it is important to consider the uptake of the esters and that the solubility of the esters is also an important consideration when using a more complex membrane.