A Receptor Integrin ß1 Promotes Infection of Avian Metapneumovirus Subgroup C by Recognizing a Viral Fusion Protein RSD Motif.

Avian metapneumovirus subgroup C (aMPV/C) causes respiratory diseases and egg dropping in chickens and turkeys, resulting in severe economic losses to the poultry industry worldwide. Integrin ß1 (ITGB1), a transmembrane cell adhesion molecule, is present in various cells and mediates numerous viral infections. Herein, we demonstrate that ITGB1 is essential for aMPV/C infection in cultured DF-1 cells, as evidenced by the inhibition of viral binding by EDTA blockade, Arg-Ser-Asp (RSD) peptide, monoclonal antibody against ITGB1, and ITGB1 short interfering (si) RNA knockdown in cultured DF-1 cells. Simulation of the binding process between the aMPV/C fusion (F) protein and avian-derived ITGB1 using molecular dynamics showed that ITGB1 may be a host factor benefiting aMPV/C attachment or internalization. The transient expression of avian ITGB1-rendered porcine and feline non-permissive cells (DQ cells and CRFK cells, respectively) is susceptible to aMPV/C infection. Kinetic replication of aMPV/C in siRNA-knockdown cells revealed that ITGB1 plays an important role in aMPV/C infection at the early stage (attachment and internalization). aMPV/C was also able to efficiently infect human non-small cell lung cancer (A549) cells. This may be a consequence of the similar structures of both metapneumovirus F protein-specific motifs (RSD for aMPV/C and RGD for human metapneumovirus) recognized by ITGB1. Overexpression of avian-derived ITGB1 and human-derived ITGB1 in A549 cells enhanced aMPV/C infectivity. Taken together, this study demonstrated that ITGB1 acts as an essential receptor for aMPV/C attachment and internalization into host cells, facilitating aMPV/C infection.

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Multicellular spheroids, which mimic the natural organ counterparts, allow the prospect of drug screening and regenerative medicine. However, their application is hampered by low processing efficiency or limited scale. This study introduces an efficient method to drive rapid multicellular spheroid formation by a cellulose nanofibril matrix. This matrix enables the facilitated growth of spheroids (within 48 h) through multiple cell assembly into size-controllable aggregates with well-organized physiological microstructure. The efficiency, dimension, and conformation of the as-formed spheroids depend on the concentration of extracellular nanofibrils, the number of assembled cells, and the heterogeneity of cell types. The above strategy allows the robust formation mechanism of compacted tumoroids and hepatocyte spheroids.

Inhibition of γδ T Cells Alleviates Blood-Brain Barrier in Cardiac Arrest and Cardiopulmonary Resuscitation in Mice.

Ischemia/reperfusion (I/R) injury is the leading cause of death following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR). γδT cells are suggested to aggravate blood-brain barrier (BBB) injury in various pathological processes. We herein investigate the effects of γδT cells inhibitor (UC7-13D5) against I/R injury post-CA/CPR. C57BL/6 mice were subjected to CA through injection of KCL (70 µL of 0.5 mol/L) and cessation of mechanical ventilation followed by CPR. Flow cytometry was performed to measure the proportion of CD3-positive cells after intraperitoneal injection of 200 µg UC7-13D5 at 6 h, 24 h, and 48 h post-resuscitation into mice. Neurological scores and modified neurological severity scores were assessed to examine neurological functions. Brain edema was estimated via brain water content measurements. Immunohistochemistry of caspase-3 and immunofluorescence staining of claudin-1, ZO-1 and CD31 were performed to detect neuronal apoptosis, BBB integrity and angiogenesis. Microvascular morphology in the cortical area was assessed via H&E staining. Oxidative stress was determined by measuring malondialdehyde, myeloperoxidase, xanthine oxidase, superoxide dismutase, and glutathione peroxidase activities. Western blotting was performed to measure the protein levels of Nuclear factor-E2-related factor 2 (Nrf2) and Heme oxygenase-1 (HO-1). UC7-13D5 effectively depleted γδT cells. Inhibition of γδT cells improved neurological deficits and reduced brain edema post-CA/CPR. γδT cells depletion attenuated neuronal apoptosis, BBB disruption and oxidative stress and promoted angiogenesis following CA/CPR. Inhibition of γδT cells facilitated the activation of the Nrf2/HO-1 pathway in CA/CPR-induced mice. Inhibition of γδT cells alleviates neurological deficits and cerebral edema in mice with CA/CPR by inhibiting neuronal apoptosis, BBB disruption and oxidative stress, and promoting angiogenesis via activation of the Nrf2/HO-1 pathway.

A novel base editor SpRY-ABE8eF148A mediates efficient A-to-G base editing with a reduced off-target effect.

Adenine base editors (ABEs) can mediate two transition mutations, A-to-G and T-to-C, which are suitable for repairing G·C-to-T·A pathogenic variants, the most significant human pathogenic variant. By combining the protospacer adjacent motif (PAM)less SpRY nuclease with F148A-mutated TadA∗8e deaminase, we developed a new editor, SpRY-ABE8eF148A, in this study, which has narrowed the editing range and enhanced A-to-G editing efficiency in most sites with NR/YN PAMs. Furthermore, compared with SpRY-ABE8e, SpRY-ABE8eF148A significantly decreased the RNA off-target effect. Therefore, this engineered base editor, SpRY-ABE8eF148A, expanded the editing scope and improved the editing precision for G·C-to-T·A pathogenic variants. Besides, we established a bioinformatics tool, adenine base-repairing sgRNA database of pathogenic variant (ARDPM), to facilitate the development of precise editors.

Inhibition of Γδ T Cells Alleviates Brain Ischemic Injury in Cardiopulmonary-Cerebral Resuscitation Mice.

BACKGROUND: A half-million people in the United States suffer from cardiac arrest (CA) requiring cardiopulmonary resuscitation (CPR). An inflammatory mechanism is associated with neuronal injury in the presence of cerebral ischemia. T lymphocytes are identified as crucial regulators of inflammation. Therefore, we investigated the relationship between CA/CPR-induced ischemia injury and T lymphocytes. METHODS: C57BL/6 mice were subjected to CA through injection of KCl (30 µL of 0.5 mol/L) and cessation of mechanical ventilation followed by CPR. The survival rate and neurologic deficit scores were assessed. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was carried out to detect neuronal death. Histologic changes were observed by hematoxylin-eosin staining. The levels of Trgv4, Trgv5 and Trgv7 were quantified by RT-qPCR. Inflammatory responses were identified by measurement of IL-1ß, IL-6 and IL-17. RESULTS: Downregulated γδ T cells improved survival and neurologic outcomes and inhibits neuronal apoptosis. γδ T inhibition protected brains from CA/CPR-mediated tissue damage. UC7-13D5 treatment inhibited the levels of γδ T markers. Knockdown of γδ T cells ameliorated neuroinflammation. CONCLUSIONS: Inhibition of γδ T cells ameliorates ischemic injury in mice with CA/CPR by attenuating inflammation and neuronal apoptosis.

Establishment of hydrolysis probe system real-time PCR assay for rapid detection of canine circovirus.

In the study, we established a hydrolysis probe-based real-time polymerase chain reaction (PCR) assay to rapidly detect Canine circovirus (CanineCV) DNA in faecal samples. We designed a pair of specific primers and one probe targeting Rep in CanineCV, and sensitivity, specificity, and repeatability tests were performed to evaluate the efficacy of the assay. The assay showed high sensitivity and a minimum detection limit of 8.42 × 101 copies/µL, which is 1000-fold more sensitive compared to traditional PCR. The method was also highly specific, without cross-reaction with other common canine viruses. Moreover, the assay showed high repeatability, and the mean intra-assay and inter-assay coefficients of variation were 0.26 and 0.36%, respectively. The results of the detection of clinical samples showed that the positive detection rate of CanineCV was 14.04% (8/57). Notably, 8% of clinical samples were co-infected with other canine pathogens. In conclusion, the establishment of a hydrolysis probe-based real-time PCR method provides a fast, sensitive, specific, reliable, and repeatable method for CanineCV detection. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03031-z.

Establishment of Duplex SYBR Green I-based Real-time PCR Assay for Simultaneous Detection of Duck Hepatitis A Virus-1 and Duck Astrovirus-3.

Duck viral hepatitis (DVH) mainly affects ducklings under 1 month of age, causes liver necrosis, enlargement, and hemorrhage, and is highly lethal, seriously jeopardizing the duck industry. The prevalence of duck hepatitis A virus (DHAV-1) and duck astrovirus type 3 (DAstV-3) is increasing, and coinfection is common. Moreover, the similar clinical characteristics of the DHAV-1 and DAstV-3 infections and the high frequency of coinfection make diagnosis difficult. In this study, to establish a method for the rapid, simultaneous detection of DHAV-1 and DAstV-3, two pairs of specific primers were designed according to their conserved gene regions. An SYBR® Green I-based qPCR assay was successfully established that can quickly and differentially detect the two viruses. Moreover, the assay is highly specific and does not show cross-reaction with other common viruses. The detection limit of the method is 7.34 × 101 copies/µl and 3.78 × 101 copies/µl for DHAV-1 and DAstV-3, respectively, indicating high sensitivity. A total of 34 clinical samples were tested using the established method; the positive rates for DHAV-1 and DAstV-3 were 14.71% and 8.82%, respectively, and that for coinfection was 2.94% (1/34), which was better than that obtained with conventional PCR. In summary, the SYBR Green I-based qPCR assay established in this study has high specificity, good sensitivity and accuracy, high feasibility, and is rapid. Thus, it can be a powerful tool for the coinfection detection of DHAV-1 and DAstV-3 and for future epidemiologic studies.

Artículo regular­Establecimiento de un ensayo dúplex de PCR en tiempo real basado en SYBR Green I para la detección simultánea del virus de la hepatitis A del pato-1 y del astrovirus del pato tipo 3. La hepatitis viral del pato (DVH) afecta principalmente a los patitos menores de 1 mes de edad, causa necrosis hepática, agrandamiento y hemorragia, y es altamente letal, lo que pone en grave peligro la industria del pato. La prevalencia del virus de la hepatitis A del pato (DHAV-1) y del astrovirus del pato tipo 3 (DAstV-3) está aumentando y la coinfección es común. Además, las características clínicas similares de las infecciones por el virus de la hepatitis A del pato y el astrovirus del pato tipo 3 así como la alta frecuencia de coinfección dificultan el diagnóstico. En este estudio, para establecer un método para la detección rápida y simultánea por el virus de la hepatitis A del pato y el astrovirus del pato tipo 3, se diseñaron dos pares de iniciadores específicos según sus regiones génicas conservadas. Se estableció con éxito un ensayo cuantitativo de PCR basado en SYBR® Green I que pudo detectar rápida y diferencialmente los dos virus. Además, el ensayo es muy específico y no muestró reacción cruzada con otros virus comunes. El límite de detección del método fue de 7.34 × 101 copias/µl y de 3.78 × 101 copias/µl para el virus de la hepatitis A del pato y para el astrovirus del pato tipo 3, respectivamente, lo que indica una alta sensibilidad. Se analizaron un total de 34 muestras clínicas utilizando el método establecido; las tasas positivas para el virus de la hepatitis A del pato y para el astrovirus del pato tipo 3 fueron del 14.71% y 8.82%, respectivamente y la de coinfección fue del 2.94% (1/34), que fue mejor que la obtenida con el método de PCR convencional. En resumen, el ensayo cuantitativo de PCR basado en SYBR Green I establecido en este estudio tiene alta especificidad, buena sensibilidad y precisión, alta viabilidad y es rápido. Por lo tanto, puede ser una herramienta poderosa para la detección de coinfecciones con el virus de la hepatitis A del pato y astrovirus del pato tipo 3 y para futuros estudios epidemiológicos.

The spread of Tembusu virus in China from 2010 to 2019.

Tembusu virus (TMUV) is a positive-sense RNA virus that is associated with severe reduction in egg production and even death in ducks. TMUV infection shows high incidence and is a threat to the global duck industry. However, the possible origin, genotype, and codon usage bias of TMUV are not very clear. Here, we addressed these questions by analyzing the available genomic sequences from China. The results showed that the ancestor of avian TMUV was most likely a mosquito TMUV. Moreover, three TMUV clades were identified by three different phylogenetic analysis methods. The TMUV genome exhibits a stronger mutation pressure than natural selection pressure. Our findings provide important insights that reveal the ongoing TMUV spread in China and can aid in future prevention and control.

Genomic characterization and phylogenetic analysis of a new canine picornavirus variant in the mainland of China.

A new canine picornavirus (CanPV) variant, designated as dog/SH1901/CHN/2019, was detected in a pool of various canine fecal samples in the mainland of China using a viral metagenomic analysis, and its nearly complete genome sequence was determined and analyzed. Sequence analyses revealed that it had a standard picornavirus genome organization, a type I internal ribosome entry site (IRES) in the 5'UTR. However, dog/SH1901/CHN/2019 has generated a serial of unique aa mutations and 7aa insertion when compared with the closely related CanPVs. Phylogenetic analysis and pairwise sequence comparisons based on the P1, 2C, 3C, and 3D protein sequences showed that dog/SH1901/ CHN/2019 was closely related to CanPV strains 244 F, 325 F and 6D, which clustered into an independent evolutionary clade and distantly related to CanPV strain A128thr of the genus Mischivirus, which indicated the unclassified CanPV strains may belong to a novel species or genus in the family Picornaviridae. This study extends our knowledge on the evolution and genetic diversity of CanPVs.

Duplex SYBR Green I-based real-time PCR assay for the rapid detection of canine kobuvirus and canine astrovirus.

A duplex SYBR Green I-based real-time PCR assay was established for the simultaneous detection of canine kobuvirus (CaKoV) and canine astrovirus (CaAstV). This assay can easily distinguish the two viruses according to their different melting temperatures (Tm) of 80 °C for CaKoV and 86.5 °C for CaAstV; other canine enteroviruses used as controls showed no specific melting peaks. The detection limit of this assay was determined to be 101 copies/µL for both viruses. This method exhibited high repeatability and reproducibility, with a coefficient of variation less than 1.5 %. A total of 48 fecal samples were collected for clinical testing by real-time PCR and confirmed by sequencing. Real-time PCR assay showed a 10.4 % CaKoV-positive rate and a 4.2 % CaAstV-positive rate, and the positive rate of co-infection of the two viruses was 2.1 %, which was consistent with the sequencing results. This assay has many advantages over conventional PCR: it is rapid, sensitive, specific, and reliable for detecting these two viruses in one sample, and it can be used as a tool to detect CaKoV and CaAstV infection or co-infection in clinical settings.

Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus.

The similar clinical characteristics of canine circovirus (CaCV) and canine astrovirus (CaAstV) infections and high frequency of co-infection make diagnosis difficult. In this study, a duplex SYBR Green I-based real-time polymerase chain reaction (PCR) assay was established for the rapid, simultaneous detection of CaCV and CaAstV. Two pairs of specific primers were designed based on the Rep gene of CaCV and the Cap gene of CaAstV. By using the real-time PCR assay method, the two viruses can be distinguished by the difference in melting temperatures, 79 °C and 86 °C for CaCV and CaAstV, respectively. This assay had high specificity, showing no cross-reaction with other common canine viruses, as well as high sensitivity, with minimum detection limits of 9.25 × 101 copies/µL and 6.15 × 101 copies/µL for CaCV and CaAstV, respectively. Based on the mean coefficient of variation, the method had good reproducibility and reliability. In a clinical test of 57 fecal samples, the rates of positive detection by real-time PCR were 14.04% (8/57) and 12.28% (7/57) for CaCV and CaAstV, respectively, and the rate of co-infection was 8.77% (5/57). In conclusion, the newly established duplex SYBR Green I-based real-time PCR assay is sensitive, specific, reliable, and rapid and is an effective tool for the detection of co-infections with CaCV and CaAstV.

Identification and full-genome sequencing of canine kobuvirus in canine fecal samples collected from Anhui Province, eastern China.

Canine kobuvirus (CaKoV), a newly described virus, is the causative agent of gastroenteritis in dogs. In this study, 57 fecal samples from dogs with diarrhea in Anhui Province, eastern China, were collected. Among these, five samples were identified to be infected with CaKoV, by polymerase chain reaction targeting the CaKoV 3D gene. The five CaKoV strains were subjected to phylogenetic analysis. The sequences of VP1 from the five CaKoV strains were 93.6%-96.1% identical to each other and 91.75%-97.95% identical to other reported CaKoV VP1 sequences. In addition, the complete genome of one strain was successfully amplified and sequenced. The genome consisted of 8223 nucleotides and shared 94.6%-97.0% nucleotide and 93.1%-94.0% amino acid sequence identity with other CaKoV isolates. Phylogenetic analysis revealed that the CaKoV strain from Anhui Province was similar to other Chinese strains, and it was more closely related to feline and mouse kobuviruses than to sheep and bovine kobuviruses. Interestingly, all of the CaKoV-positive samples were coinfected with canine parvovirus. The finding of CaKoV infection in dogs with diarrhea and coinfection with canine parvovirus are a cause for concern and highlight the need for management and preventive measures.

Simultaneous detection of duck circovirus and novel goose parvovirus via SYBR green I-based duplex real-time polymerase chain reaction analysis.

Beak atrophy and dwarfism syndrome (BADS) is commonly caused by co-infection with duck circovirus (DuCV) and novel goose parvovirus (NGPV). Therefore, concurrent detection of both viruses is important for monitoring and limiting BADS, although such a diagnostic test has not been reported. In this study, we developed a duplex, SYBR Green I-based real-time polymerase chain reaction (PCR) assay to enable the simultaneous detection of DuCV and NGPV. The assay readily distinguished between the two viruses, based on their different melting temperatures (Tm), where the Tm for DuCV was 80 °C and that for NGPV was 84.5 °C. Other non-target duck viruses that were tested did not show melting peaks. The detection limit of the duplex assay was 101 copies/µL for both viruses. This method exhibited high repeatability and reproducibility, and both the inter-assay and intra-assay variation coefficients were <1.6%. Thirty-one fecal samples were collected for clinical testing using real-time PCR analysis, and the results were confirmed using sequencing. The rate of co-infection was 6.5%, which was consistent with the sequencing results. This duplex real-time PCR assay offers advantages over other tests, such as rapid, sensitive, specific, and reliable detection of both viruses in a single sample, which enables the quantitative detection of DuCV and NGPV in clinical samples. Using this test may be instrumental in reducing the incidence of BADS and the associated economic losses in the duck and goose industries.

Development of an SYBR Green I-based real-time PCR assay for the rapid detection of canine kobuvirus.

Canine kobuvirus (CaKoV) is a causative agent of gastroenteritis in dogs. Rapid detection of CaKoV is important for preventing and controlling this condition. In this study, an SYBR Green I-based quantitative real-time PCR assay was established for CaKoV detection. Specific primers targeting a highly conserved region of the CaKoV 3D gene were developed. After optimization, the method detected a minimum of 1 × 101 copies/µL with high specificity, stability, and repeatability. Moreover, the entire process only required approximately 1.5 h for completion. Our results were supported by those obtained for clinical samples, in which our developed method was successfully applied. The newly established real-time PCR is a rapid, sensitive, specific, and repeatable method for the quantitative detection of CaKoV and can, therefore, be used in epidemiological studies.

Development of a recombinase-aided amplification assay for detection of orf virus.

Orf, caused by orf virus (ORFV), is an important zoonotic disease that infects goat and sheep, leading to huge economic losses. ORFV can also cause cutaneous lesions in people who come in close contact with the diseased animals. Although accurate diagnostic methods for ORFV infection exist, there is a need for a rapid, specific, and sensitive method for easy clinical application. Here, we successfully established a recombinase-aided amplification (RAA) assay for rapid detection of ORFV. The analytical sensitivity of the assay for ORFV detection is 1 × 101 copies per reaction. Moreover, no cross-reaction was observed with other common DNA viruses. A total of 45 archived suspected ORFV infected nasal scab skin samples were examined by RAA and SYBR Green-based real-time polymerase chain reaction (PCR). Compared with the real-time PCR assay, the kappa values of the RAA assay for ORFV detection was 0.845 (p <0.001), indicating that both assay results were fully in agreement. In conclusion, this detection assay provides a rapid, sensitive, and specific method for ORFV detection and is suitable for ORFV clinical testing.

Study of molecular association in acetic acid-water binary solution by Raman spectroscopy.

Raman spectra of acetic acid-water binary solutions with different concentrations have been measured in order to study molecular association of acetic acid. We find that the symmetric and asymmetric OH stretching vibration of water (3242 and 3443 cm-1) have marked changes of Raman shift when the volume fraction of acetic acid (VAA) is 0.3 and 0.8, respectively, which demonstrates that the hydrogen bonding of the water is affected, causing association molecule (acetic acid-water structure) to undergo two phase transitions. Furthermore, the peak of the HCH bending vibration is blue-shifted at VAA = 0.8, which shows that the acetic acid-acetic acid structure undergoes a phase transition and the acetic acid side-on dimer is formed. These results also indicate that the CH vibration mode in CH⋯O is HCH bending vibration. Finally, the phase transition process of association molecules (hydrated monomer, linear dimer, acetic acid side-on dimer and water-separated dimer) has been obtained in acetic acid-water binary solutions through theoretical analysis.