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Amdoparvoviruses infect various carnivores, including mustelids, canids and felids et al. Aleutian mink disease virus (AMDV), Carnivore amdoparvovirus 1, infects mink and other Mustelidae and causes subclinical diseases or fatal chronic immune stimulation and immune complex diseases. Here, we identified a nearly complete genome (4265nt) of a novel amdoparvovirus from farmed Asian badgers (Meles meles) and tentatively named Meles meles amdoparvovirus (MMAV). Furthermore, we constructed phylogenetic trees based on the NS1 and VP2 proteins, respectively, as well as the amdoparvoviral VP2 nucleotide hypervariable region. MMAV NS1 protein shared 62.6%-88.8% sequence identities with other known amdoparvoviruses and phylogenetically clustered into a unique clade, while VP2 protein shared 76.7%-97.2% identities and clustered into two clades, of which one was more closely related to raccoon dog and arctic fox amdoparvovirus (RFAV), and the other clade is independent of other known amdoparvoviruses. According to the NS1 genetic distance-based criteria, MMAV is classified as a novel species of Amdoparvovirus. the positive rate for amdoparvovirus by PCR was 32.8% (47/143) in 143 clinical samples collected from 134 individual badgers. MMAV DNA was detected in feces, blood, spleen, liver, lung, and adipose tissue from Asian badgers. Eight isolates were sequenced from eight samples, with five obtaining approximate complete genomes. Six serum samples were PCR,CIEP and IAT-positive, with four having high serum antibody titers (＞512) against AMDV-G. Twenty-six of the 41 amdoparvovirus-positive badgers exhibited diseased symptoms, and some necropsy revealed organic lesions. Therefore, these lines of evidence suggested that we discovered novel and diverse amdoparvoviruses, MMAVs, that naturally infect Asian badgers of mustelid and may be cause Aleutian disease in badgers similar to that seen in mink. Furthermore, our findings supported that mustelid species are susceptible to diverse amdoparvoviruses and the molecular characteristics of amdoparvorviruses divergence between hosts species.

Crop plants, such as watermelon, suffer from severe Aluminum (Al3+)-toxicity in acidic soils with their primary root elongation being first arrested. However, the significance of apoplastic or symplastic Al3+-toxicity in watermelon root is scarcely reported. In this work, we identified a medium fruit type (ZJ) and a small fruit type (NBT) as Al+3-tolerant and sensitive based on their differential primary root elongation rate respectively, and used them to show the effects of symplastic besides apoplastic Al distribution in the watermelon's root. Although the Al content was higher in the root of NBT than ZJ, Al+3 allocated in their apoplast, vacuole and plastid fractions were not significantly different between the two cultivars. Thus, only a few proportion of Al+3 differentially distributed in the nucleus and mitochondria corresponded to interesting differential morphological and physiological disorders recorded in the root under Al+3-stress. The symplastic amount of Al+3 substantially induced the energy efficient catalase pathway in ZJ, and the energy consuming ascorbate peroxidase pathway in NBT. These findings coincided with obvious starch granule visibility in the root ultra-structure of ZJ than NBT, suggesting a differential energy was used in supporting the root elongation and nutrient uptake for Al+3-tolerance in the two cultivars. This work provides clues that could be further investigated in the identification of genetic components and molecular mechanisms associated with Al+3-tolerance in watermelon.

Watermelon (Citrullus lanatus) is an important horticultural crop that is sensitive to heavy metals such as lead (Pb) in polluted water or soil. However, there are no available data regarding Pb tolerance phenotyping in watermelon. Watermelon seedlings were exposed to various Pb doses (0, 20, 40, 60, 80 µm·L–1 Pb) for 14 days, after which 20 µm Pb was identified as the optimal treatment for lead tolerance analysis in watermelon because it caused significant symptoms (leaf chlorosis, stubby and yellow roots) but little damage to seedlings. Subsequently, the Pb responses were analyzed in eight watermelon varieties (V1–V8), and membership function analysis was used to determine a single Pb tolerance index. Of the eight watermelon varieties, V4 and V7 were ranked the most Pb tolerant; V1, V2, V5, and V6 were moderately Pb tolerant; and V3 and V8 were the most Pb-sensitive varieties. Compared with most Pb-sensitive varieties (V3 and V8), the most Pb-tolerant varieties (V4 and V7) maintained high antioxidant activity, and had lower malondialdehyde (MDA) and total soluble protein (TSP) contents. In addition, carotenoid and chlorophyll (both a and b) contents were stimulated and inhibited, respectively, in leaves of high-Pb translocation varieties (V4 and V8). Principal component analysis (PCA) revealed relative root length as an indicator of Pb tolerance because it correlated significantly with shoot growth. These results provide useful insight into the mechanism of Pb tolerance in cucurbit crops, as well as information regarding the breeding of watermelon with enhanced tolerance to this heavy metal (Pb).

Although mink enteritis virus (MEV) is an acute, virulent, and highly contagious pathogen in minks, there is currently a lack of a quick diagnostic method. By conjugating colloidal gold nanoparticles with the MEV-specific monoclonal antibody, monoclonal antibody (MAb) 14, we developed a single-step competitive immunochromatographic strip (ICS) assay for simple determination of MEV. The optimal concentrations of the colloidal gold-coupled MAb 14 (coating antibody), the capture protein (MEV VP2 protein), and the goat anti-mouse antibody were 1.0, 0.8, and 1.0 mg/ml, respectively. The limit of detection was approximately 512 hemagglutination units/100 μl of MEV B strain. Other common viruses of mink were tested to evaluate the specificity of the ICS, and the results showed no cross-reactivity for other pathogens. In comparison with the Anigen Rapid canine parvovirus (CPV) Ag Test Kit (BioNote, Korea) in testing 289 samples, the percentage of agreement and relative sensitivity and specificity of the MEV ICS assay were 94.1, 93.2, and 97.1%, respectively. The ICS test was found to be a sufficiently sensitive and specific detection method for the convenient and rapid detection of MEV.

Human bocavirus 1 (HBoV1), an autonomous human parvovirus, causes acute respiratory tract infections in young children. HBoV1 infects well-differentiated (polarized) human airway epithelium cultured at an air-liquid interface (HAE-ALI). HBoV1 expresses a large nonstructural protein, NS1, that is essential for viral DNA replication. HBoV1 infection of polarized human airway epithelial cells induces a DNA damage response (DDR) that is critical to viral DNA replication involving DNA repair with error-free Y-family DNA polymerases. HBoV1 NS1 or the isoform NS1-70

Canine distemper virus (CDV) is currently circulating in domestic and wild animals. The host range of CDV comprises all families within the order Carnivora, and it has recently expanded to nonhuman primates, moreover the host range still has the potential for further expansion. In this research, a CDV strain named BT was isolated from a vaccinated domestic dog in Changchun, Northeast of China, and identified by Electron microscope (EM) and Immunofluorescence Assay (IFA). The whole genome sequence of the virus was obtained by RT-PCR and Sanger sequencing. Phylogenetic analysis was performed based on the Coding sequence (CDS) of hemagglutinin (H) protein and the complete genome sequence of the virus respectively, the results showed that the CDV-BT strain still classified into the Asia-1 lineage. This research shows that CDV-BT strain’s antigenicity of the epitope 444GDKYPIHFNDER455 in nucleocapsid (N) protein and the epitope 178ARGDIFPPY186 in H protein were significantly different from vaccine strain by amino acid substitutions, and suggests that the characterization of genetic diversity among the circulating CDVs is essential for future CDV’s research and disease monitoring.

ObjectiveThis study aimed to develop a comprehensive but easy to apply histologic grading system to score osteoarthritic changes in the whole knee joint for both spontaneous and posttraumatic osteoarthritis (OA) mouse models.MethodsThe new OA grading scheme was developed based on extensive literature review and the authors’ experience in mouse OA models with relatively long periods of observation (up to 24 months of age or 24-week post-surgery). Semi-quantitative assessments of the histopathologic OA changes were applied to all four quadrants of the knee. Grading elements per quadrant were defined as follows: cartilage (0-7) including three new grading elements for early- and late-stage OA, respectively; osteophyte (0-2) covering chondro-osteophytes in both outer and inner joint margins; subchondral bone (0-2) containing subchondral bone thickening and destruction; synovitis (0-2) comprised of both synovial plica and intercondylar notch; and peri-articular tissues (0-2) highlighting ectopic chondrogenesis and ossification in the knee capsule, ligament, and musculature.ResultsStatistical analyses showed that the new grading system had high intra- and inter-observer reproducibility (Pearson’s correlation coefficients r >0.9) for both experienced and novice scorers. Sensitivity and reliability analyses confirmed the ability of the new system to detect minimal OA progression between two timepoints with a two-week interval and to accurately identify tissue-specific OA severity within the knee joint.ConclusionsThe comprehensive histologic grading system presented here covers all-stage osteoarthritic changes in all major knee joint tissues of mice, which enable us to score OA severity for the whole joint reproducibly and accurately without software-assisted time-consuming measurements.

BACKGROUND This retrospective study was conducted at a single center in China and aimed to compare rocuronium with succinylcholine for rapid sequence induction intubation in the Emergency Department of a hospital. MATERIAL AND METHODS An orotracheal intubation procedure was performed in a total of 267 patients by direct laryngoscopy using an intravenous bolus injection of 1 mg/kg of succinylcholine (n=141; SY group) or 1.2 mg/kg of rocuronium (n=126; RM group) for a rapid sequence induction in the emergency department. The success of orotracheal intubation was evaluated by a capnography curve. The modified Cormack-Lehane score was used to grade the direct laryngoscopy. RESULTS There was no statistically significant difference in numbers of patients with successful first-attempt orotracheal intubation between the groups (112 vs. 87, P=0.067). Fewer intubation failures under direct laryngoscopy were reported in the SY group than in the RM group (23 [16%] vs. 34 [27%], P=0.037). The number of intubation attempts was higher in the RM group than in the SY group (1.52±0.87 per patient vs. 1.27±0.60 per patient, P=0.032). CONCLUSIONS The findings from this study support results from previous studies, showing that even in the Emergency Department setting, rocuronium was equivalent to succinylcholine in achieving rapid sequence induction intubation, when the dose was appropriate. However, as current clinical guidelines highlight, succinylcholine has more contraindications and adverse effects, including hyperkalemia, which should be monitored, and rocuronium has a longer duration of action.

Bourbon virus (BRBV) was first isolated from a patient hospitalized at the University of Kansas Hospital in 2014. Since then, several deaths have been reported to be caused by BRBV infection in the Midwest and Southern United States. BRBV is a tick-borne virus that is widely carried by lone star ticks. It belongs to genus Thogotovirus of the Orthomyxoviridae family. Currently, there are no treatments or vaccines available for BRBV or thogotovirus infection caused diseases. In this study, we reconstituted a replicon reporter system, composed of plasmids expressing the RNA-dependent RNA polymerase (RdRP) complex (PA, PB1, and PB2), nucleocapsid (NP) protein, and a reporter gene flanked by the 3' and 5' untranslated region (UTR) of the envelope glycoprotein (GP) genome segment. By using the luciferase reporter, we screened a few small molecule compounds of anti-endonuclease that inhibited the nicking activity by parvovirus B19 (B19V) NS1, as well as FDA-approved drugs targeting the RdRP of influenza virus. Our results demonstrated that myricetin, an anti-B19V NS1 nicking inhibitor, efficiently inhibited the RdRP activity of BRBV and virus replication. The IC50 and EC50 of myricetin are 2.22 and 4.6 μM, respectively, in cells. Myricetin had minimal cytotoxicity in cells, and therefore the therapeutic index of the compound is high. In conclusion, the BRBV replicon system is a useful tool to study viral RNA replication and to develop antivirals, and myricetin may hold promise in treatment of BRBV infected patients.

Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders., During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5′-GUA AAG CUA CGG GAC GGU-3′), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [KD] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.

Broad coverage of mink enteritis virus (MEV) vaccination program in northeast of China has provided effective protection from mink viral enteritis. Nevertheless, MEV vaccine failures were reported due to continually evolving and changing virulence of field variants or wild-type MEV. In this study, a combined loop-mediated isothermal amplification (LAMP) and single nucleotide polymorphism (SNP) method, named LAMP-SNP assay, was developed for detection and differentiation of wild-type and vaccine strains of MEV. Four primers in MEV-VP2-LAMP were used to detect both wild-type and vaccine strains of MEV in our previous publication, and other four primers in LAMP-SNP were designed to amplify the NS1 gene in wild-type MEV and only used to detect wild-type viruses. The LAMP-SNP assay was performed in a water bath held at a constant temperature of 65 °C for 60 min. LAMP-SNP amplification can be judged by both electrophoresis and visual assessment with the unaided eyes. In comparison with virus isolation as the gold standard in testing 171 mink samples, the percentage of agreement and relative sensitivity and specificity of the LAMP-SNP assay were 97.1, 100%, and 94.0%, respectively. There were no cross-reactions with other mink viruses. The LAMP-SNP assay was found to be a rapid, reliable and low-cost method to differentiate MEV vaccine and field variant strains.

PEDV remains one of the most important swine diseases that infects pigs of all ages. It causes devastating viral enteric disease in piglets with a high mortality rate, leading to significant threats and huge economic loss to the pork industry. In this study, a transcriptomic shotgun sequencing (RNA-Seq) procedure was used to study gene responses against PEDV infection. Genome-wide analysis of differentially expressed genes (DEGs) was performed in Vero E6 cells post-PEDV infection. mTOR signaling pathway activator-MHY1485, and inhibitor-PP242 were used to study the antiviral function. Results revealed that the IRF3 was significantly up-regulated post-PEDV infection. Although most of the IFN-regulatory and –related genes evaluated in this study were either down-regulated or remained unchanged, IL11 behaved significantly up-regulated, with the peak at 16 hpi. Nearly 90% of PEDV infections were suppressed in the PP242 pretreated cells whereas the reverse effect was observed in the MYH1485 pretreated cells. Results indicated that the mTOR signaling pathway played a vital role in the PEDV antiviral regulation in the Vero E6 cells. Future studies will contribute to better understand the cellular antiviral mechanism against PEDV., Highlights • RNA-Seq was used to study gene responses against PEDV infection. • Genome-wide analysis of DEGs was performed in Vero E6 cells post-PEDV infection. • The mTOR signaling pathway activator and inhibitor can affect the PEDV infection rate.

Canine parvovirus (CPV) is an important pathogen in domestic dogs, and the original antigenic types CPV-2 and its variants, CPV-2a, 2b and 2c, are prevalent worldwide. A multiplex TaqMan real-time PCR method was developed for the detection and differentiation of four antigenic types of CPV. A set of primers and probes, CPV-305F/CPV-305R and CPV-2-305P (for CPV-2)/CPV-2a-305P (for CPV-2a, 2b and 2c), was able to differentiate CPV-2 and its variants (CPV-2a, 2b and 2c). Another set of primers and probes, CPV-426F/CPV-426R and CPV-2-426P (for CPV-2 and 2a)/CPV-2b-426P (for CPV-2b)/CPV-2c-426P (for CPV-2c), was able to differentiate CPV-2a (2), CPV-2b, and CPV-2c. With these primers and probes, the multiplex TaqMan real-time PCR assay detected effectively and differentiated CPV-2, 2a, 2b and 2c by two separate real-time PCRs. No cross reactivity was observed with canine distemper virus, canine adenovirus, and canine coronavirus. The detection limit of the assay is 101 genome copies/μL for CPV-2, CPV-2a, CPV-2b, and 102 copies/μL for CPV-2c. The multiplex real-time PCR has 100% agreement with DNA sequencing. We provide a sensitive assay that simultaneously detects and differentiate four antigenic types of CPV and the method was also used for quantification of CPVs viral genome., Highlights • The Multiplex TaqMan real-time PCR can simultaneously detect and differentiate four antigenic types of CPV. • The method is suit for using in detection of CPVs in China. • The method showed a high specificity and sensitivity. • The method was also used for quantification of CPVs viral genome.

In this study, the complete chloroplast (cp) genome of Sophora tonkinensis Gagnep. was determined through Illumina sequencing method. The complete chloroplast genome of S. tonkinensis was 155,640 b...

Mink enteritis virus (MEV), an autonomous parvovirus, causes acute hemorrhagic enteritis in minks. The molecular pathogenesis of MEV infection has not been fully understood. In this study, we observed significantly increased apoptosis in the esophagus, small intestine, mesenteric lymph nodes, and kidney in minks experimentally infected with strain MEVB. In vitro infection of feline F81 cells with MEVB decreased cell viability and induced cell cycle arrest at G(1) phase and apoptosis. By screening MEV nonstructural proteins (NS1 and NS2) and structural proteins (VP1 and VP2), we demonstrated that the MEV NS1 induced apoptosis in both F81 and human embryonic kidney 293T (HEK293T) cells, similar to that induced during MEV infection in minks. We found that the NS1 protein-induced apoptosis in HEK293T cells was mediated not by the death receptor but by the mitochondrial pathway, as demonstrated by mitochondrial depolarization, opening of mitochondrial transition pore, release of cytochrome c, and activation of caspase-9 and -3. Moreover, in NS1-transfected cells, we observed an increase of Bax expression and its translocation to the mitochondria, as well as an increased ratio of the Bax/Bcl-2, reactive oxygen species (ROS) production, and activated p38 mitogen-activated protein kinase (MAPK) and p53. Taken together, our results demonstrated that MEV induces apoptosis through activation of p38 MAPK and the p53-mediated mitochondrial apoptotic pathway induced by NS1 protein, which sheds light on the molecular pathogenesis of MEV infection. IMPORTANCE MEV causes fatal hemorrhagic enteritis in minks. Apoptosis is a cellular mechanism that effectively sacrifices virus-infected cells to maintain homeostasis between the virus and host. In this study, we demonstrated that MEV induces apoptosis both in vivo and in vitro. Mechanistically, the viral large nonstructural protein NS1 activates p38 MAPK, which leads p53 phosphorylation to mediate the mitochondrial apoptotic pathway but not the death receptor-mediated apoptotic pathway. This is the first report to uncover the mechanism underlying MEV-induced apoptosis.

Human bocavirus 1 (HBoV1), which belongs to the genus Bocaparvovirus of the Parvoviridae family, causes acute respiratory tract infections in young children. In vitro, HBoV1 infects polarized primary human airway epithelium (HAE) cultured at an air-liquid interface (HAE-ALI). HBoV1 encodes a small nonstructural protein, nuclear protein 1 (NP1), that plays an essential role in the maturation of capsid protein (VP)-encoding mRNAs and viral DNA replication. In this study, we determined the broad interactome of NP1 using the proximity-dependent biotin identification (BioID) assay combined with mass spectrometry (MS). We confirmed that two host mRNA processing factors, DEAH-box helicase 15 (DHX15) and cleavage and polyadenylation specificity factor 6 (CPSF6; also known as CFIm68), a subunit of the cleavage factor Im complex (CFIm), interact with HBoV1 NP1 independently of any DNA or mRNAs. Knockdown of CPSF6 significantly decreased the expression of capsid protein but not that of DHX15. We further demonstrated that NP1 directly interacts with CPSF6 in vitro and colocalizes within the virus replication centers. Importantly, we revealed a novel role of CPSF6 in the nuclear import of NP1, in addition to the critical role of CPSF6 in NP1-facilitated maturation of VP-encoding mRNAs. Thus, our study suggests that CPSF6 interacts with NP1 to escort NP1 imported into the nucleus for its function in the modulation of viral mRNA processing and viral DNA replication. IMPORTANCE Human bocavirus 1 (HBoV1) is one of the significant pathogens causing acute respiratory tract infections in young children worldwide. HBoV1 encodes a small nonstructural protein (NP1) that plays an important role in the maturation of viral mRNAs encoding capsid proteins as well as in viral DNA replication. Here, we identified a critical host factor, CPSF6, that directly interacts with NP1, mediates the nuclear import of NP1, and plays a role in the maturation of capsid protein-encoding mRNAs in the nucleus. The identification of the direct interaction between viral NP1 and host CPSF6 provides new insights into the mechanism by which a viral small nonstructural protein facilitates the multiple regulation of viral gene expression and replication and reveals a novel target for potent antiviral drug development.

Crop plants, such as watermelon, suffer from severe Aluminum (Al

Background Canine parvovirus (CPV) and feline parvovirus (FPV) are causative agents of diarrhea in dogs and cats, which manifests as depression, vomiting, fever, loss of appetite, leucopenia, and diarrhea in young animals. CPV and FPV can single or mixed infect cats and cause disease. To diagnose sick animals effectively, an effective virus diagnostic and genome typing method with high sensitivity and specificity is required. Results In this study, a conserved segment containing one SNP A4408C of parvovirus was used for real-time PCR amplification. Subsequently, data were auto-analyzed and plotted using Applied Biosystems® High Resolution Melt Software v3.1. Results showed that CPV and FPV can be detected simultaneously in a single PCR reaction. No cross-reactions were observed with canine adenovirus, canine coronavirus, and canine distemper virus. The assay had a detection limit of 4.2 genome copies of CPV and FPV. A total of 80 clinical samples were subjected to this assay, as well as to conventional PCR-sequence assay and virus isolation. Results showed that the percentage of agreement of the assay and other methods are high. Conclusions In short, we have developed a diagnostic test for the accurate detection and differentiation of CPV and FPV in fecal samples, which is also cost effective.

Coupling between G-protein-coupled receptors (GPCRs) and the G proteins is a key step in cellular signaling. Despite extensive experimental and computational studies, the mechanism of specific GPCR-G protein coupling remains poorly understood. This has greatly hindered effective drug design of GPCRs that are primary targets of ~1/3 of currently marketed drugs. Here, we have employed all-atom molecular simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method to decipher the mechanism of the GPCR-G protein interactions. Adenosine receptors (ARs) were used as model systems based on very recently determined cryo-EM structures of the A1AR and A2AAR coupled with the Gi and Gs proteins, respectively. Changing the Gi protein to the Gs led to increased fluctuations in the A1AR and agonist adenosine (ADO), while agonist 5’-N-ethylcarboxamidoadenosine (NECA) binding in the A2AAR could be still stabilized upon changing the Gs protein to the Gi. Free energy calculations identified one stable low-energy conformation for each of the ADO-A1AR-Gi and NECA-A2AAR-Gs complexes as in the cryo-EM structures, similarly for the NECA-A2AAR-Gi complex. In contrast, the ADO agonist and Gs protein sampled multiple conformations in the ADO-A1AR-Gs system. GaMD simulations thus indicated that the ADO-bound A1AR preferred to couple with the Gi protein to the Gs, while the A2AAR could couple with both the Gs and Gi proteins, being highly consistent with experimental findings of the ARs. More importantly, detailed analysis of the atomic simulations showed that the specific AR-G protein coupling resulted from remarkably complementary residue interactions at the protein interface, involving mainly the receptor transmembrane 6 helix and the Gα α5 helix and α4-β6 loop. In summary, the GaMD simulations have provided unprecedented insights into the dynamic mechanism of specific GPCR-G protein interactions at an atomistic level, which is expected to facilitate future drug design efforts of the GPCRs.

Toll-like receptors (TLRs) can recognize conserved molecular patterns and initiate a wide range of innate and adaptive immune responses against invading infectious agents. The aim of this study was to assess the transcript profile of mink TLRs (mTLRs) in mink peripheral blood mononuclear cells (PBMCs) and a range of tissues, and to explore the potential role of mTLRs in the antiviral immune response process. The results indicated that the mTLR partial nucleotide sequences had a high degree of nucleotide identity with ferret sequences (95-98%). Phylogenetic analysis showed that mammalian TLRs grouped into five TLR families, with a closer relationship of the mTLRs with those of ferret than the other mammalian sequences. Moreover, all the mTLRs were ubiquitously expressed in lymphoid organs (spleen and lymph nodes) and PBMCs. Interestingly, the mTLR expression patterns in lung, uterus, and heart showed quite a lot of similarity. Another remarkable observation was the wide expression of mTLR1-3 mRNAs in all tissues. Among the analyzed tissues, skeletal muscle was revealed to being the lowest repertoire of mTLR expression. Additionally, mink PBMCs exposed to the canine distemper virus revealed significant upregulation of mTLR2, mTLR4, mTLR7, and mTLR8 mRNAs, indicating that mTLRs have a role in innate immunity in the mink. Collectively, our results are the first to establish the basic expression patterns of mTLRs and the relationship between mTLRs and a virus, which will contribute to better understanding of the evolution and the functions of mTLRs in the innate immune system in minks.

TLR8 is an important sensor of single-stranded RNA (ssRNA) from the viral genome and plays an essential role in innate antiviral responses via the recognition of conserved viral molecular patterns. In this report, TLR8 in the Chinese raccoon dog was characterized and analyzed for the first time. The full-length sequence of raccoon dog TLR8 (RdTLR8) cDNA was cloned by rapid amplification of cDNA ends (RACE) and is 3191bp with a 3117-bp open reading frame (ORF) encoding 1038 amino acids. The putative protein exhibits typical features of the TLR families, with 19 leucine-rich repeats (LRRs) in the extracellular domain and a cytoplasmic TIR domain. Comparative analyses of the RdTLR8 amino acid sequence indicated a 73.6-99.4% sequence identity with dog, horse, pig, sheep, cattle, human and mouse TLR8. Phylogenetic analysis grouped 71 mammalian TLR proteins into five sub-families, wherein RdTLR8 was clustered into a monophyletic TLR8 clade in the TLR9 family, which was completely coincident with the evolutionary relationship among mammals. Quantitative real-time PCR analysis revealed extensive expression of RdTLR8 in tissues from healthy Chinese raccoon dogs with the highest expression in the peripheral blood mononuclear cells (PBMCs) and the lowest expression in the skeletal muscle. HEK293 cells cotransfected with a RdTLR8 expression plasmid and an NF-κB-luciferase reporter plasmid significantly responded to the agonist 3M-002, indicating a functional TLR8 homolog. In addition, raccoon dog PBMCs exposed to the canine distemper virus (CDV) wild strain CDV-PS and the TLR8 agonist 3M-002 showed significant upregulation of RdTLR8 mRNA and proinflammatory cytokines TNF-α and IFN-α, suggesting that RdTLR8 might play an important role in the immune response to viral infections in the Chinese raccoon dog.

Canine distemper virus (CDV) is a member of the genus Morbillivirus within the family Paramyxoviridae and has caused severe economic losses in China. Nucleocapsid protein (N) is the major structural viral protein and can be used to diagnose CDV and other morbilliviruses. In this study, a specific monoclonal antibody, 1N8, was produced against the CDV N protein (amino acids 277-471). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that (350)LNFGRSYFDPA(360) was the minimal linear epitope that could be recognized by mAb 1N8. ELISA assays revealed that mouse anti-CDV sera could also recognize the minimal linear epitope. Alignment analysis of the amino acid sequences indicated that the epitope was highly conserved among CDV strains. Furthermore, the epitope was conserved among other morbilliviruses, which was confirmed with PRRV using western blotting. Taken together, the results of this study may have potential applications in the development of suitable diagnostic techniques for CDV or other morbilliviruses.

Paddy-upland rotation, as a popular cultivation mode in many Asian countries, could largely increase land use efficiency and its productivity. In recent years, watermelon production is badly impaired by unknown factors that result in watermelon malformed roots in rice-watermelon rotation area. In this study, we examined the effects of several herbicides, which are often used for the weed control in rice season, on the growth of succeeding culture of watermelon. The effects of four different herbicides (penoxsulam, bensulfuron-methyl, chylapof-butyl and fhiroxypyr) with different concentrations at 1, 10, and 100 ppb, were investigated on three cucurbitaceae crops of watermelon, squash, and bottle gourd. All species were severely affected in plant growth by penoxsulam and bensulfuron-methyl herbicides at as low as 10 ppb after one week, whereas there was no obvious toxic effect of chylapof-butyl and fhiroxypyr treatments on plant growth even after two weeks. Among the three cucurbitaceae crops, watermelon showed the highest sensitivity to herbicide exposure, and the root tip ultrastructure micrograph further conformed the sensitivity of watermelon to herbicides. Moreover, the expression of auxin and cytokinin response factors suggested that plant hormones, auxin and cytokinin, play important roles in root growth and development under herbicide stress. In conclusion, we postulated that the residues of herbicides from application in rice production is one of the major barriers for successful production of watermelon crop. The present study provides new insights for avoiding the detrimental effects of herbicide residues in soil and improving watermelon production in sustainable rice-watermelon rotation cultivation mode.

The highly contagious canine distemper virus (CDV) is a non-segmented single-stranded negative-sense RNA virus, which belongs to the Morbillivirus genus of the Paramyxoviridae family. The phosphoprotein (P) of CDV plays the important role in the virus replication and pathogenesis. In this study, we characterized four monoclonal antibodies (MAbs), designated as Pc7, Pc8, Pc11 and Pc25 MAbs against the P protein of CDV-PS strain. A series of overlapping P protein-derived peptides representing the CDV-PS phosphoprotein (aa232-507) were screened to identify linear peptide epitopes recognized by each MAb. Finally, four epitopes, 238SHGMGIVAGSTN249 (E2-9), 264GPSVSAENVRQ274 (E6-2), 390INPELRPIIGR400 (E27-2) and 252TQSALKSTG260 (E4-9), are minimal linear epitopes recognized by the Pc7, Pc8, Pc11 and Pc25 MAbs, respectively. Each identified B-cell epitope was able to be recognized by CDV positive dog serum. Alignment analysis of the amino acid sequences indicated that the linear B-cell epitope of the Pc11 MAb is relatively conserved among different CDV strains, but the linear B-cell epitopes recognized by Pc7, Pc8 and Pc25 MAbs are not conserved among CDV strains. Our results revealed that the E27-2 peptide might be a common B-cell binding epitope of CDV antibodies. These findings may provide a useful basis for the development of new diagnostic assays for CDV.

Canine distemper virus (CDV) is a major pathogen not only in raccoon dogs but also in a variety of carnivorous animals, including domesticated animals, particularly if they have not been vaccinated. In this study, a wild-type strain of CDV was isolated from lung tissue from a raccoon dog kept at a fur farm in Jilin Province, China. Cytopathic effects typical of CDV infection were observed after three blind passages in Vero cells, yielding a virus titer of 10(4.6) TCID50/mL. Virus identification was carried out by RT-PCR, immunofluorescence, electron microscopy, and genome sequencing. The results showed that the isolated virus, termed the SY strain, corresponded to the Asia-1 genotype of CDV and has a genome of 15,690 nucleotides. This represents the first complete nucleotide sequence of a CDV strain circulating in raccoon dogs in China.

Canine distemper virus (CDV), a paramyxovirus, causes a severe highly contagious lethal disease in carnivores, such as mink. Mink lung epithelial cells (Mv.1.Lu cells) are sensitive to CDV infection and are homologous to the natural host system of mink. The current study analyzed the response of Mv.1.Lu cells to CDV infection by iTRAQ combined with LC-MS/MS. In total, 151 and 369 differentially expressed proteins (DEPs) were markedly up-regulated or down-regulated, respectively. Thirteen DEPs were validated via real-time RT-PCR or western blot analysis. Network and KEGG pathway analyses revealed several regulated proteins associated with the NF-κB signaling pathway. Further validation was performed by western blot analysis and immunofluorescence assay, which demonstrated that different CDV strains induced NF-κB P65 phosphorylation and nuclear translocation. Moreover, the results provided interesting information that some identified DEPs possibly associated with the pathogenesis and the immune response upon CDV infection. This study is the first overview of the responses to CDV infection in Mv.1.Lu cells, and the findings will help to analyze further aspects of the molecular mechanisms involved in viral pathogenesis and the immune responses upon CDV infection.

Hemorrhagic pneumonia in mink is a fatal disease caused by Pseudomonas aeruginosa. Very little is known about P. aeruginosa in relation to genotype and the mechanisms underlying antimicrobial resistance in mink. A total of 110 P. aeruginosa samples were collected from mink from Chinese mink farms between 2007 and 2015. Samples underwent molecular genotyping using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST), antimicrobial susceptibility and its mechanism were investigated at the molecular level. The PFGE identified 73 unique types and 15 clusters, while MLST identified 43 (7 new) sequence types (ST) and 12 sequence type clonal complexes (STCC). Sequence types and PFGE showed persistence of endemic clones in cities Wendeng (Shandong, China) and Dalian (Liaoning, China), even in different timelines. The MLST also revealed the gene correlation of the mink P. aeruginosa across different time and place. The ST1058 (n = 14), ST882 (n = 11), and ST2442 (n = 10) were the predominant types, among which ST1058 was the only one found both in Shandong province and Dalian (Liaoning, China). The MLST for P. aeruginosa infection in mink was highly associated with that in humans and other animals, implying possible transmission events. A small proportion of mink exhibited drug resistance to P. aeruginosa (9/69, 13%) with resistance predominantly to fluoroquinolone, aminoglycoside, and β-lactamase. Eight strains had mutations in the quinolone-resistance determining regions (QRDR). High proportions (65%; 72/110) of the fosA gene and 2 types of glpt deletion for fosmycin were detected. Furthermore, in the whole genome sequence of one multidrug resistant strain, we identified 27 genes that conferred resistance to 14 types of drugs.

Background The Nucleoprotein (NP) is the most abundant and highly immunogenic protein in canine distemper virus (CDV), playing an important role in CDV viral replication and assembly. Results In this study, a specific monoclonal antibody, named C8, was produced against the NP protein C terminal (amino acids 401–523). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis.The results indicated that 444GDKYPIHFNDER455 was the minimal linear epitope that could be recognized by mAb C8. Sequence alignments demonstrated that this linear epitope is less conserved among three CDV genotypes. We next analyzed the level of conservation of the defined epitope in19 Chinese CDV clinical isolates, and it has one site variation in amino acid among these CDV isolations. 2 isolates have the amino acid mutations F451L, while one has P448Ssubstitution.Phylogenetic analysis showed the two isolates with F451Lsubstitution had a closer relationship in a virulent strain ZJ-7, so the epitope may be a significant tag associated with virus virulence. Conclusion This collection of mAb along with defined linear epitope may provide useful reagents for investigations of NP protein function and the development of CDV specific diagnostics.

Toll-like receptors (TLRs) can recognize conserved molecular patterns and initiate a wide range of innate and adaptive immune responses against invading infectious agents. The aim of this study was to assess the transcript profile of mink TLRs (mTLRs) in mink peripheral blood mononuclear cells (PBMCs) and a range of tissues, and to explore the potential role of mTLRs in the antiviral immune response process. The results indicated that the mTLR partial nucleotide sequences had a high degree of nucleotide identity with ferret sequences (95-98%). Phylogenetic analysis showed that mammalian TLRs grouped into five TLR families, with a closer relationship of the mTLRs with those of ferret than the other mammalian sequences. Moreover, all the mTLRs were ubiquitously expressed in lymphoid organs (spleen and lymph nodes) and PBMCs. Interestingly, the mTLR expression patterns in lung, uterus, and heart showed quite a lot of similarity. Another remarkable observation was the wide expression of mTLR1-3 mRNAs in all tissues. Among the analyzed tissues, skeletal muscle was revealed to being the lowest repertoire of mTLR expression. Additionally, mink PBMCs exposed to the canine distemper virus revealed significant upregulation of mTLR2, mTLR4, mTLR7, and mTLR8 mRNAs, indicating that mTLRs have a role in innate immunity in the mink. Collectively, our results are the first to establish the basic expression patterns of mTLRs and the relationship between mTLRs and a virus, which will contribute to better understanding of the evolution and the functions of mTLRs in the innate immune system in minks.

Canine parvovirus (CPV) reproduces by co-opting the resources of host cells, inevitably causing cytotoxic effects to the host cells. Feline kidney F81 cells are sensitive to CPV infection and show disparate growing statuses at different time points post-infection. This study analysed the response of F81 cells to CPV infection at successive infection time points by iTRAQ-based quantitative proteomics. Differentially expressed proteins (DEPs) during 60 h of infection and at selected time points post-infection were identified by an analysis of variance test and a two-tailed unpaired t test, respectively. DEPs with similar quantitative changes were clustered by hierarchical clustering and analysed by gene ontology enrichment, revealing that 12 h and 60 h post-infection were the optimal times to analyse the autonomous parvovirus replication and apoptosis processes, respectively. Using the MetacoreTM database, 29 DEPs were enriched in a network involved in p53 regulation. Besides, a significantly enriched pathway suggests that the CPV-induced cytopathic effect was probably due to the deficiency of functional CFTR caused by CPV infection. This study uncovered the systemic changes in key cellular factors involved in CPV infection and help to understand the molecular mechanisms of the anti-cancer activity of CPV and the cytopathic effects induced by CPV infection.

Canine parvovirus (CPV) type 2c is a new antigenic variant of CPV-2. Since the year 2000 it has spread to several countries, causing severe hemorrhagic enteritis in dogs. In 2014 and 2015, 58 fecal samples were collected from dogs in Beijing with suspected CPV infection. Regardless of the vaccination status of the dogs, 43 samples were found positive for CPV according to PCR results; i.e., 18, 7, and 18 respectively belonged to antigenic types new CPV-2a, new CPV-2b, and CPV-2c. A phylogenetic tree based on their VP2 gene sequences indicated that the Chinese CPV-2c strains form a separate cluster. In addition to synonymous mutations, the CPV-2c strains also contain a unique coding mutation in VP2 that leads to glycine at residue 5, instead of the highly conserved alanine at this position in all other CPV-2c strains sequenced to date. Using F81 cells, several novel isolates of CPV-2c, each with the Ala5Gly mutation, were obtained. One of these was used to infect experimentally beagle dogs, which subsequently developed the typical clinical symptoms of CPV infection. Hence, it appears that CPV-2c is still evolving in China, a finding that warrants continuous surveying and the eventual adaptation of current vaccines.

Background Mink enteritis virus (MEV) causes mink viral enteritis, an acute and highly contagious disease whose symptoms include violent diarrhea, and which is characterized by high morbidity and mortality. Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a recently developed technique for the rapid detection of bacterial and viral DNA. Here we describe a novel nanoPCR assay for the clinical detection and epidemiological characterization of MEV. Results This assay is based upon primers specific for the conserved region of the MEV NS1 gene, which encodes nonstructural protein 1. Under optimized conditions, the MEV nanoPCR assay had a detection limit of 8.75 × 101 copies recombinant plasmids per reaction, compared with 8.75 × 103 copies for conventional PCR analysis. Moreover, of 246 clinical mink samples collected from five provinces in North-Eastern China, 50.8% were scored MEV positive by our nanoPCR assay, compared with 32.5% for conventional PCR. Furthermore no cross reactivity was observed for the nanoPCR assay with respect to related viruses, including canine distemper virus (CDV) and Aleutian mink disease parvovirus (AMDV). Phylogenetic analysis of four Chinese wild type MEV isolates using the nanoPCR assay indicated that they belonged to a small MEV clade, named “China type”, in the MEV/FPLV cluster, and were closely clustered in the same location. Conclusions Our results indicate that the MEV China type clade is currently circulating in domestic minks in China. We anticipate that the nanoPCR assay we have described here will be useful for the detection and epidemiological and pathological characterization of MEV. Electronic supplementary material The online version of this article (doi:10.1186/s12917-014-0312-6) contains supplementary material, which is available to authorized users.