Clostridium lamae sp. nov., a novel bacterium isolated from the fresh feces of alpaca.

A novel Gram-positive, anaerobic, nonspore-forming, rod-shaped bacterium, designated strain NGMCC 1.200840 T, was isolated from the alpacas fresh feces. The taxonomic position of the novel strain was determined using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed strain NGMCC 1.200840 T was a member of the genus Clostridium and closely related to Clostridium tertium DSM 2485 T (98.16% sequence similarity). Between strains NGMCC 1.200840 T and C. tertium DSM 2485 T, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were 79.91% and 23.50%, respectively. Genomic DNA G + C content is 28.44 mol%. The strain can utilise D-glucose, D-mannitol, D-lactose, D-saccharose, D-maltose, D-xylose, L-arabinose, D-cellobiose, D-mannose, D-melezitose, D-raffinose, D-sorbitol, L-rhamnose, D-trehalose, D-galactose and Arbutin to produce acid. The optimal growth pH was 7, the temperature was 37 °C, and the salt concentration was 0-0.5% (w/v). The major cellular fatty acids (> 10%) included iso-C15:0, anteiso-C15:0 and iso-C17:0 3-OH. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids and two unidentified aminolipids. Based on phenotypic, phylogenetic and chemotaxonomic characteristics, NGMCC 1.200840 T represents a novel species within the genus Clostridium, for which the named Clostridium lamae sp. nov. is proposed. The type strain is NGMCC 1.200840 T (= CGMCC 1.18014 T = JCM 35704 T).

Bacteroides rhinocerotis sp. nov., isolated from the fresh feces of rhinoceros in Beijing Zoo.

A Gram-negative strain, anaerobic, non-motile, non-spore-forming, rod-shaped bacterial strain named as NGMCC 1.200684 T was isolated from the fresh feces of rhinoceros in Beijing Zoo. Based on 16S rRNA gene sequences, phylogenetic analysis indicated that strain NGMCC 1.200684 T belonged to the genus Bacteroides and was most strongly related to the type strain of Bacteroides uniformis ATCC 8492 T (96.88%). The G + C content of the genomic DNA was determined to be 46.62%. Between strains NGMCC 1.200684 T and B. uniformis ATCC 8492 T, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were 93.89 and 67.60%, respectively. Strain NGMCC 1.200684 T can produce acid from fermentation of several substrates, including glucose, mannitol, lactose, saccharose, maltose, salicin, xylose, cellobiose, mannose, raffinose, sorbitol, trehalose, D-galactose, and maltotriose. The major cellular fatty acids (> 10%) were identified as anteiso-C15:0, iso-C15:0, iso-C14:0, and iso-C17:0 3-OH. The polar lipid profiles of strain NGMCC 1.200684 T were determined to contain diphosphatidyl glycerol, phosphatidylglycerol, phosphatidylethanolamine, three unknown phospholipids, and two unknown amino-phospholipids. Based on phenotypic, phylogenetic, and chemotaxonomic characteristics, a novel species of the genus Bacteroides, Bacteroides rhinocerotis sp. nov. is proposed. The type strain is NGMCC 1.200684 T (= CGMCC 1.18013 T = JCM 35702 T).

Susceptibility and Attenuated Transmissibility of SARS-CoV-2 in Domestic Cats.

Domestic cats, an important companion animal, can be infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This has aroused concern regarding the ability of domestic cats to spread the virus that causes coronavirus disease 2019. We systematically demonstrated the pathogenesis and transmissibility of SARS-CoV-2 in cats. Serial passaging of the virus between cats dramatically attenuated the viral transmissibility, likely owing to variations of the amino acids in the receptor-binding domain sites of angiotensin-converting enzyme 2 between humans and cats. These findings provide insight into the transmissibility of SARS-CoV-2 in cats and information for protecting the health of humans and cats.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 via Close Contact and Respiratory Droplets Among Human Angiotensin-Converting Enzyme 2 Mice.

We simulated 3 transmission modes, including close-contact, respiratory droplets and aerosol routes, in the laboratory. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be highly transmitted among naive human angiotensin-converting enzyme 2 (hACE2) mice via close contact because 7 of 13 naive hACE2 mice were SARS-CoV-2 antibody seropositive 14 days after being introduced into the same cage with 3 infected-hACE2 mice. For respiratory droplets, SARS-CoV-2 antibodies from 3 of 10 naive hACE2 mice showed seropositivity 14 days after introduction into the same cage with 3 infected-hACE2 mice, separated by grids. In addition, hACE2 mice cannot be experimentally infected via aerosol inoculation until continued up to 25 minutes with high viral concentrations.

Analysis of the susceptibility of lung cancer patients to SARS-CoV-2 infection.

Recent studies have reported that COVID-19 patients with lung cancer have a higher risk of severe events than patients without cancer. In this study, we investigated the gene expression of angiotensin I-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) with prognosis in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Lung cancer patients in each age stage, subtype, and pathological stage are susceptible to SARS-CoV-2 infection, except for the primitive subtype of LUSC. LUAD patients are more susceptible to SARS-CoV-2 infection than LUSC patients. The findings are unanimous on tissue expression in gene and protein levels.

Correction to: The mouse and ferret models for studying the novel avian-origin human influenza A (H7N9) virus.

An amendment to this paper has been published and can be accessed via the original article.

Novel avian-origin human influenza A(H7N9) can be transmitted between ferrets via respiratory droplets.

The outbreak of human infections caused by novel avian-origin influenza A(H7N9) in China since March 2013 underscores the need to better understand the pathogenicity and transmissibility of these viruses in mammals. In a ferret model, the pathogenicity of influenza A(H7N9) was found to be less than that of an influenza A(H5N1) strain but comparable to that of 2009 pandemic influenza A(H1N1), based on the clinical signs, mortality, virus dissemination, and results of histopathologic analyses. Influenza A(H7N9) could replicate in the upper and lower respiratory tract, the heart, the liver, and the olfactory bulb. It is worth noting that influenza A(H7N9) exhibited a low level of transmission between ferrets via respiratory droplets. There were 4 mutations in the virus isolated from the contact ferret: D678Y in the gene encoding PB2, R157K in the gene encoding hemagglutinin (H3 numbering), I109T in the gene encoding nucleoprotein, and T10I in the gene encoding neuraminidase. These data emphasized that avian-origin influenza A(H7N9) can be transmitted between mammals, highlighting its potential for human-to-human transmissibility.

The transcriptome of MHV-infected RAW264.7 cells offers an alternative model for macrophage innate immunity research.

BACKGROUND: Macrophages are the primary innate immune cells encountered by the invading coronaviruses, and their abilities to initiate inflammatory reactions, to maintain the immunity homeostasis by differential polarization, to train the innate immune system by epigenic modification have been reported in laboratory animal research. METHODS: In the current in vitro research, murine macrophage RAW 264.7 cell were infected by mouse hepatitis virus, a coronavirus existed in mouse. At 3-, 6-, 12-, 24-, and 48-h post infection (hpi.), the attached cells were washed with PBS and harvested in Trizol reagent. Then The harvest is subjected to transcriptome sequencing. RESULTS: The transcriptome analysis showed the immediate (3 hpi.) up regulation of DEGs related to inflammation, like Il1b and Il6. DEGs related to M2 differential polarization, like Irf4 showed up regulation at 24 hpi., the late term after viral infection. In addition, DEGs related to metabolism and histone modification, like Ezh2 were detected, which might correlate with the trained immunity of macrophages. CONCLUSIONS: The current in vitro viral infection study showed the key innated immunity character of macrophages, which suggested the replacement value of viral infection cells model, to reduce the animal usage in preclinical research.

Flu-CED: A comparative transcriptomics database of influenza virus-infected human and animal models.

BACKGROUND: The continuing emergence of influenza virus has highlighted the value of public databases and related bioinformatic analysis tools in investigating transcriptomic change caused by different influenza virus infections in human and animal models. METHODS: We collected a large amount of transcriptome research data related to influenza virus-infected human and animal models in public databases (GEO and ArrayExpress), and extracted and integrated array and metadata. The gene expression matrix was generated through strictly quality control, balance, standardization, batch correction, and gene annotation. We then analyzed gene expression in different species, virus, cells/tissues or after antibody/vaccine treatment and imported sample metadata and gene expression datasets into the database. RESULTS: Overall, maintaining careful processing and quality control, we collected 8064 samples from 103 independent datasets, and constructed a comparative transcriptomics database of influenza virus named the Flu-CED database (Influenza comparative expression database, https://flu.com-med.org.cn/). Using integrated and processed transcriptomic data, we established a user-friendly website for realizing the integration, online retrieval, visualization, and exploration of gene expression of influenza virus infection in different species and the biological functions involved in differential genes. Flu-CED can quickly query single and multi-gene expression profiles, combining different experimental conditions for comparative transcriptome analysis, identifying differentially expressed genes (DEGs) between comparison groups, and conveniently finding DEGs. CONCLUSION: Flu-CED provides data resources and tools for analyzing gene expression in human and animal models infected with influenza virus that can deepen our understanding of the mechanisms underlying disease occurrence and development, and enable prediction of key genes or therapeutic targets that can be used for medical research.

The mouse and ferret models for studying the novel avian-origin human influenza A (H7N9) virus.

BACKGROUND: The current study was conducted to establish animal models (including mouse and ferret) for the novel avian-origin H7N9 influenza virus. FINDINGS: A/Anhui/1/2013 (H7N9) virus was administered by intranasal instillation to groups of mice and ferrets, and animals developed typical clinical signs including body weight loss (mice and ferrets), ruffled fur (mice), sneezing (ferrets), and death (mice). Peak virus shedding from respiratory tract was observed on 2 days post inoculation (d.p.i.) for mice and 3-5 d.p.i. for ferrets. Virus could also be detected in brain, liver, spleen, kidney, and intestine from inoculated mice, and in heart, liver, and olfactory bulb from inoculated ferrets. The inoculation of H7N9 could elicit seroconversion titers up to 1280 in ferrets and 160 in mice. Leukopenia, significantly reduced lymphocytes but increased neutrophils were also observed in mouse and ferret models. CONCLUSIONS: The mouse and ferret model enables detailed studies of the pathogenesis of this illness and lay the foundation for drug or vaccine evaluation.

Characteristics of gut microbiota in representative mice strains: Implications for biological research.

BACKGROUND: Experimental animals are used to study physiological phenomena, pathological mechanisms, and disease prevention. The gut microbiome is known as a potential confounding factor for inconsistent data from preclinical studies. Although many gut microbiome studies have been conducted in recent decades, few have focused on gut microbiota fluctuation among representative mouse strains. METHODS: A range of frequently used mouse strains were selected from 34 isolation packages representing disease-related animal (DRA), immunity defect animal (IDA), or gene-editing animal (GEA) from the BALB/c and C57BL/6J backgrounds together with normal mice, and their microbial genomic DNA were isolated from mouse feces to sequence for the exploration of gut microbiota. RESULTS: Mouse background strain, classification, introduced source, introduced year, and reproduction type significantly affected the gut microbiota structure (p < 0.001 for all parameters), with background strain contributing the greatest influence (R2  = 0.237). In normal groups, distinct gut microbiota types existed in different mouse strains. Sixty-four core operational taxonomic units were obtained from normal mice, and 12 belonged to Lactobacillus. Interestingly, the gut microbiota in C57BL/6J was more stable than that in BALB/c mice. Furthermore, the gut microbiota in the IDA, GEA, and DRA groups significantly differed from that in normal groups (p < 0.001 for all). Compared with the normal group, there was a significantly higher Chao1 and Shannon index (p < 0.001 for all) in the IDA, GEA, and DRA groups. Markedly changed classes occurred with Firmicutes and Bacteroidetes. The abundances of Helicobacter, Blautia, Enterobacter, Bacillus, Clostridioides, Paenibacillus, and Clostridiales all significantly decreased in the IDA, GEA, and DRA groups, whereas those of Saccharimonas, Rikenella, and Odoribacter all significantly increased.

Safety and protective capability of an inactivated SARS-CoV-2 vaccine on pregnancy, lactation and the growth of offspring in hACE2 mice.

The mass inoculation of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine to induce herd immunity is one of the most effective measures to fight COVID-19. The vaccination of pregnant women cannot only avoid or reduce the probability of infectious diseases, but also offers the most effective and direct protection for neonates by means of passive immunization. However, there is no randomized clinical data to ascertain whether the inactivated vaccination of pregnant women or women of childbearing age can affect conception and the fetus. We found that human angiotensin-converting enzyme 2 (hACE2) mice that were vaccinated with two doses of CoronaVac (an inactivated SARS-CoV-2 vaccine) before and during pregnancy exhibited normal weight changes and reproductive performance indices; the physical development of their offspring was also normal. Following intranasal inoculation with SARS-CoV-2, pregnant mice in the immunization group all survived; reproductive performance indices and the physical development of offspring were all normal. In contrast, mice in the non-immunization group all died before delivery. Analyses showed that inoculation of CoronaVac was safe and did not exert any significant effects on pregnancy, lactation, or the growth of offspring in hACE2 mice. Vaccination effectively protected the pregnant mice against SARS-CoV-2 infection and had no adverse effects on the growth and development of the offspring, thus suggesting that inoculation with an inactivated SARS-CoV-2 vaccine may be an effective strategy to prevent infection in pregnant women.

Ginsenoside-Rb1 attenuates dilated cardiomyopathy in cTnT(R141W) transgenic mouse.

Familial dilated cardiomyopathy (FDCM) is caused by defective genes and specific medicines are not currently available to treat this. Ginsenoside-Rb1 provides cardioprotection in the experimental models of myocardial ischemia-reperfusion injury. Here we investigate Rb1's effect on DCM in cTnT(R141W) transgenic mouse. The transgene-positive mice aged 2 months were randomized into the model group and Rb1 [70 mg/(kg.day)] group; transgene-negative mice were used as a control. After 4-month treatment, cardiac function was assessed by echocardiography; cardiac tissues were prepared for histology and electron microscopy. Expression levels of molecular markers of cardiac hypertrophy, fibrosis, and intercalated disc proteins were detected by RT-PCR. Rb1 significantly decreased mortality, chamber dilation, and contractile dysfunction in cTnT(R141W) mice. Rb1 attenuated cardiac hypertrophy, interstitial fibrosis, ultrastructural degeneration, and intercalated disc remodeling in DCM hearts. Western blotting showed that Rb1 significantly decreased heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression and signal transduction and activators of transcription 3 (STAT3) activation, which were gradually increased in DCM hearts. Our results showed that Rb1 clearly alleviated cardiac dysfunction and remodeling in the cTnT(R141W) transgenic mouse, indicating its potential utility in the treatment of FDCM.

Bacterial community analysis of floor dust and HEPA filters in air purifiers used in office rooms in ILAS, Beijing.

Air purifiers with high-efficiency particulate air (HEPA) filters remove not only particulate matter but also airborne microorganisms in indoor environments. We investigated the bacterial community in HEPA filters (used for 1 year) and that in the floor dust of 12 office rooms in Beijing. We found that the viable bacteria proportion in the filter was significantly higher than that in the floor dust (p < 0.001). The Non-Metric Multi-Dimensional Scaling analysis showed that the bacterial communities in the filters and dust were significantly different (p = 0.001). The Chao1, Shannon-Wiener and phylogenetic diversity values in the filter were significantly higher than those in the dust (p < 0.001). The predominant bacterial classes in the filter were Alphaproteobacteria and Actinobacteria, whereas those in the dust were Bacteroidia, Clostridia and Bacilli. Human occupancy contributed more to the bacterial community in the filter than that in the dust. Klebsiella and Alloprevotella in the dust and filters positively correlated with the occupancy density. Soil bacteria contributed to a significantly higher proportion of the bacteria in the HEPA filter (p < 0.001). In contrast, human oral, indoor air and outdoor haze contributed to a higher proportion of the bacteria in the dust samples (p < 0.001, p < 0.01 and p < 0.05, respectively). As HEPA filters serve as an ecological niche for indoor bacteria, they should be carefully investigated during the assessment of indoor environmental health.

Author Correction: Bacterial community analysis of floor dust and HEPA filters in air purifiers used in office rooms in ILAS, Beijing.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Different immunization methods lead to altered gut flora and varied responses to Mycobacterium tuberculosis infection in mice.

INTRODUCTION: Vaccination is an essential means for prevention of tuberculosis infection, but the effects of various vaccines on the intestinal flora of mice and their response to Mycobacterium tuberculosis (Mtb) infection remain poorly understood. METHODOLOGY: In this study, two different vaccinations - ESAT6 and ESAT6 + TLR8 agonists - were administered to mice transgenic for human TLR8 to investigate gut microbiota characteristics following vaccination. Gut microbiota was investigated by next generation sequencing in the MiSeq Sequencing System. Adonis analysis was used to evaluate the effect of variables on gut bacterial community stucture. Chao1, Shannon index, and phylogenetic diversity index were used to explore the gut bacterial diversity. RESULTS: The results showed that different vaccines have significant influence on mice intestinal bacteria (adonis analysis, p < 0.01), with gut bacterial diversity within the ESAT6 + TLR8 agonists group being significantly decreased compared to the ESAT6 treatment group (p < 0.01). Following infection with Mtb via tail vein injection, the bacterial community structure within the control versus vaccinated groups altered significantly (adonis analysis, p < 0.01), and the altered changed genera were markedly different between the groups. Following infection, Bifidobacteria differed between the groups, indicated that they play a vital role in the response to infection. CONCLUSIONS: Our results indicated that different vaccines might have distinct influences on intestinal flora, and their role should not be ignored.

A glance at the gut microbiota of five experimental animal species through fecal samples.

Experimental animals including the ferret, marmoset, woodchuck, mini pig, and tree shrew have been used in biomedical research. However, their gut microbiota have not been fully investigated. In this study, the gut microbiota of these five experimental animals were analyzed with 16S rRNA sequencing. The phyla Firmicutes, Bacteroidetes, and Fusobacteria were present in the gut microbiota of all the species. Specific phyla were present in different animals: Proteobacteria in the ferret, Tenericutes in the marmoset, and Spirochaetes in the mini pig. Fusobacterium and unidentified Clostridiales were the dominant genera in the ferret, whereas Libanicoccus, Lactobacillus, Porphyromonas, and Peptoclostridium were specific to marmoset, mini pig, woodchuck, and tree shrew, respectively. A clustering analysis showed that the overall distribution of microbial species in the guts of these species mirrored their mammalian phylogeny, and the microbiota of the marmoset and tree shrew showed the closest bray_curtis distances to that of humans. PICRUSt functional prediction separated the woodchuck from the other species, which may reflect its herbivorous diet. In conclusion, both the evolutionary phylogeny and daily diet affect the gut microbiota of these experimental animals, which should not be neglected for their usage in biomedical research.

Ocular conjunctival inoculation of SARS-CoV-2 can cause mild COVID-19 in rhesus macaques.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmitted through the respiratory route, but potential extra-respiratory routes of SARS-CoV-2 transmission remain uncertain. Here we inoculated five rhesus macaques with 1 × 106 TCID50 of SARS-CoV-2 conjunctivally (CJ), intratracheally (IT), and intragastrically (IG). Nasal and throat swabs collected from CJ and IT had detectable viral RNA at 1-7 days post-inoculation (dpi). Viral RNA was detected in anal swabs from only the IT group at 1-7 dpi. Viral RNA was undetectable in tested swabs and tissues after intragastric inoculation. The CJ infected animal had a higher viral load in the nasolacrimal system than the IT infected animal but also showed mild interstitial pneumonia, suggesting distinct virus distributions. This study shows that infection via the conjunctival route is possible in non-human primates; further studies are necessary to compare the relative risk and pathogenesis of infection through these different routes in more detail.

Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques.

Coronavirus disease 2019 (COVID-19), which is caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic. It is unclear whether convalescing patients have a risk of reinfection. We generated a rhesus macaque model of SARS-CoV-2 infection that was characterized by interstitial pneumonia and systemic viral dissemination mainly in the respiratory and gastrointestinal tracts. Rhesus macaques reinfected with the identical SARS-CoV-2 strain during the early recovery phase of the initial SARS-CoV-2 infection did not show detectable viral dissemination, clinical manifestations of viral disease, or histopathological changes. Comparing the humoral and cellular immunity between primary infection and rechallenge revealed notably enhanced neutralizing antibody and immune responses. Our results suggest that primary SARS-CoV-2 exposure protects against subsequent reinfection in rhesus macaques.

Age-related rhesus macaque models of COVID-19.

BACKGROUND: Since December 2019, an outbreak of the Corona Virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2) in Wuhan, China, has become a public health emergency of international concern. The high fatality of aged cases caused by SARS-CoV-2 was a need to explore the possible age-related phenomena with non-human primate models. METHODS: Three 3-5 years old and two 15 years old rhesus macaques were intratracheally infected with SARS-CoV-2, and then analyzed by clinical signs, viral replication, chest X-ray, histopathological changes and immune response. RESULTS: Viral replication of nasopharyngeal swabs, anal swabs and lung in old monkeys was more active than that in young monkeys for 14 days after SARS-CoV-2 challenge. Monkeys developed typical interstitial pneumonia characterized by thickened alveolar septum accompanied with inflammation and edema, notably, old monkeys exhibited diffuse severe interstitial pneumonia. Viral antigens were detected mainly in alveolar epithelial cells and macrophages. CONCLUSION: SARS-CoV-2 caused more severe interstitial pneumonia in old monkeys than that in young monkeys. Rhesus macaque models infected with SARS-CoV-2 provided insight into the pathogenic mechanism and facilitated the development of vaccines and therapeutics against SARS-CoV-2 infection.