A set of RT-PCR assays for detection of all known avian paramyxoviruses and application in surveillance of avian paramyxoviruses in China.

BACKGROUND: Avian paramyxoviruses (APMVs), also termed avian avulaviruses, are of a vast diversity and great significance in poultry. Detection of all known APMVs is challenging, and distribution of APMVs have not been well investigated. METHODS: A set of reverse transcription polymerase chain reaction (RT-PCR) assays for detection of all known APMVs were established using degenerate primers targeting the viral polymerase L gene. The assays were preliminarily evaluated using in-vitro transcribed double-stranded RNA controls and 24 known viruses, and then they were employed to detect 4,346 avian samples collected from 11 provinces. RESULTS: The assays could detect 20-200 copies of the double-stranded RNA controls, and detected correctly the 24 known viruses. Of the 4,346 avian samples detected using the assays, 72 samples were found positive. Of the 72 positives, 70 were confirmed through sequencing, indicating the assays were specific for APMVs. The 4,346 samples were also detected using a reported RT-PCR assay, and the results showed this RT-PCR assay was less sensitive than the assays reported here. Of the 70 confirmed positives, 40 were class I Newcastle disease virus (NDV or APMV-1) and 27 were class II NDV from poultry including chickens, ducks, geese, and pigeons, and three were APMV-2 from parrots. The surveillance identified APMV-2 in parrots for the first time, and revealed that prevalence of NDVs in live poultry markets was higher than that in poultry farms. The surveillance also suggested that class I NDVs in chickens could be as prevalent as in ducks, and class II NDVs in ducks could be more prevalent than in chickens, and class II NDVs could be more prevalent than class I NDVs in ducks. Altogether, we developed a set of specific and sensitive RT-PCR assays for detection of all known APMVs, and conducted a large-scale surveillance using the assays which shed novel insights into APMV epidemiology.

Sequence variants in FSHR and CYP19A1 genes and the ovarian response to controlled ovarian stimulation.

OBJECTIVE: To examine whether sequence variants within the FSHR and CYP19A1 genes are related to the ovarian response to controlled ovarian stimulation (COS). DESIGN: Genetic association study using both single-gene and combined analyses of women with sequence variants undergoing in vitro fertilization treatment. SETTING: Academic research institute hospital. PATIENT(S): Seven hundred and five women undergoing ovarian stimulation with recombinant follicle-stimulating hormone (FSH). INTERVENTION(S): Peripheral blood extraction, DNA purification, and FSHR c.919G>A (rs6165, p.Thr307Ala) and CYP19A1 c.*19C>T (rs10046) sequence variants analyses. MAIN OUTCOME MEASURE(S): Single-gene statistical analysis and combined statistical analysis with the SPSS17.0 software; FSHR c.919G>A and CYP19A1 c.*19C>T sequence variant genotypes and clinical parameters related to the COS response as oocyte retrieval and hormone levels, doses of exogenous FSH. RESULT(S): Women with genotype Ala/Ala at FSHR position 307 had higher basal levels of FSH and were more likely to have a low ovarian response compared with other genotypes. Women with genotype TT at CYP19A1 yielded fewer oocytes after ovarian stimulation. The combined analysis of these two sequence variants revealed that these two single-nucleotide variants have a synergistic effect in conferring the risk of a low ovarian response. CONCLUSION(S): Our results support an association of sequence variants in the genes that participate in estrogen synthesis, notably the FSHR and CYP19A1 genes, with the outcome of COS.

Genome sequence characterization of canine parvoviruses prevalent in the Sichuan province of China.

Canine parvovirus 2 (CPV-2) infection is responsible for large numbers of animal deaths worldwide and is one of the most dangerous infectious diseases in young puppies. Twenty-four rectal swabs were collected from dogs with clinical signs of vomiting and haemorrhagic diarrhoea and were initially verified to be infected with CPV-2 using colloidal gold test strips. From the 24 CPV-positive samples, complete genome of 5050-5054 nucleotides was sequenced with a next-generation sequencing platform. Characteristics of the Open Reading Frames from different CPV-2 strains detected in this study were analyzed. Several VP2 point mutations were discovered, and demonstrated the co-circulation of new CPV-2a, new CPV-2b and CPV-2c in Sichuan province of China. The analysis results of the Chinese CPV-2 retrieved from the NCBI nucleotide, showed that new CPV-2a has become the predominant variant in some provinces of China. Phylogenetic analysis of global VP2 and NS1 nucleotide sequences revealed certain correlations among geographical regions, types and circulating time, which lays the foundation for further research concerning the epidemiology, genetic variation, vaccination and molecular evolutionary relationships of the CPV-2 identified at different times and from different regions.

WHO grade I meningioma subtypes: MRI features and pathological analysis.

AIMS: WHO grade I meningiomas include several subtypes that differ in terms of surgical planning and prognosis. We aim to analyze the relationship between magnetic resonance imaging (MRI) features and pathological parameters for each WHO grade I meningioma subtype to improve diagnostic value. MATERIALS AND METHODS: Patients with WHO grade I meningiomas underwent pathology pathological examination and surgery at our hospital. MRI findings included signal intensity on T1WI and T2WI and, the enhancement degree in contrast-enhanced, and the degree of peritumoral edema in patients were reviewed. 1H-MRS was performed for the different meningioma subtypes. The correlation between MRI features and pathology was determined using a Kruskal-Wallis H test (P < 0.05). KEY FINDINGS: Angiomatous meningiomas mainly showed a low T1WI signal, a high T2WI signal, a high occurrence rate of peritumoral edema, mainly with moderate or severe peritumoral edema, and homogeneous enhancement. Meningothelial meningiomas mainly showed T1WI and T2WI iso-intense signals, mainly moderate or severe peritumoral edema, and moderate enhancement. Mixed, fibroblastic, and psammomatous meningiomas showed mixed signals, much overlap of the T1WI and T2WI signals, mainly mild or moderate peritumoral edema, and mild or moderate enhancement that could be inhomogeneous. A significant difference was noted in apparent diffusion coefficient (ADC) values and in some 1H-MRS indicators among different meningioma subtypes. SIGNIFICANCE: Angiomatous meningiomas were the most easily identified subtype, followed by meningothelial meningiomas. No obvious difference was observed among the mixed, fibroblastic, and psammomatous meningiomas, but differences were observed between angiomatous and meningothelial meningiomas.

MicroRNA expression profiling in exosomes derived from gastric cancer stem-like cells.

Cancer stem-like cells (CSCs) have been identified as the initial cell in formation of cancer. Quiescent CSCs can "hide out" from traditional cancer therapy which may produce an initial response but are often unsuccessful in curing patients. Thus, levels of CSC in patients may be used as an indicator to measure the chance of recurrence of cancer after therapy. The goals of our work are to develop specific exosomal miRNA clusters for gastric CSCs that can potentially predict which patients are at high risk for developing gastric cancer (GC) in order to diagnose GC at an early stage. Here, upon sorting gastric CSCs, we initially isolated and characterized exosomes secreted by both gastric CSCs and their differentiated cells (DCs). By deep sequencing of each exosomal miRNA library, 11 typical differentially expressed miRNAs were identified as signature miRNAs for CSC. Gene target prediction, GO annotation and KEGG pathway enrichment analysis showed possible functions associated with these signature miRNAs. Hence, upon research of exosomal miRNAs that would influence behavior of tumor cells and their microenvironment, this study shows that a specific miRNA signature is present in CSCs, and implies that a potential miRNA biomarker reflecting the stage of gastric cancer progression and metastasis could be developed in the foreseeable future.

Involvement of Polo-like kinase 1 (Plk1) in quiescence regulation of cancer stem-like cells of the gastric cancer cell lines.

Cancer stem cells (CSCs) have been hypothesized to initiate tumor growth and be resistant to chemoradiotherapy, and these processes appear to be closely related to CSC quiescence. Here, a CSC-like cell population with a high level of CD44 expression was obtained from the human gastric cancer cell lines MKN45 and MKN74. Using a PKH26-labeling retention assay, quiescent CSC-like cells with low levels of Ki67 and PCNA expression were found in spheres formed in serum-free medium, and exhibited resistance to drug and radiation treatments. Polo-like kinase 1 (Plk1) and ribosomal S6 kinase 1 (RSK1) were silenced in the quiescent CSC-like cells. The Plk1-specific inhibitors inhibited the activation of RSK1 and induced quiescence in the CSC-like cells, but increased RSK1 activity and resulted in apoptosis in non-CSCs. Furthermore, RSK1 silencing by inhibitors activated Plk1 and had no effect on the growth of spheres in the CSC-like cells, but did not affect phosphorylation of Plk1 and led to decreased proliferation in non-CSCs. Our results showed that Plk1 and RSK1 play important roles in the conversion of CSCs between active and quiescent states.

Comparison of three media for transport and storage of the samples collected for detection of avian influenza virus.

Detection of avian influenza viruses (AIVs) is important for diagnosis, surveillance and control of avian influenza which is of great economic and public health significance. Proper transport and storage of samples is critical for the detection when the samples cannot be detected immediately. As recommended by some international or national authoritative entities and some publications, phosphate buffered saline (PBS), PBS-glycerol and brain heart infusion broth (BHIB) are frequently used for transport and storage of the samples collected for detection of AIVs worldwide. In this study, we compared these three media for transport and storage of simulated and authentic swab and feces samples collected for detection of AIVs using virus isolation and reverse transcription-PCR. The results suggest that PBS-glycerol is superior to PBS and BHIB as the sample transport and storage media. The results also suggest that the samples collected for detection of AIVs should be detected as soon as possible because the virus concentration of the samples may decline rapidly during storage within days at 4 or -20°C.

Genomic Analysis and Surveillance of the Coronavirus Dominant in Ducks in China.

The genetic diversity, evolution, distribution, and taxonomy of some coronaviruses dominant in birds other than chickens remain enigmatic. In this study we sequenced the genome of a newly identified coronavirus dominant in ducks (DdCoV), and performed a large-scale surveillance of coronaviruses in chickens and ducks using a conserved RT-PCR assay. The viral genome harbors a tandem repeat which is rare in vertebrate RNA viruses. The repeat is homologous to some proteins of various cellular organisms, but its origin remains unknown. Many substitutions, insertions, deletions, and some frameshifts and recombination events have occurred in the genome of the DdCoV, as compared with the coronavirus dominant in chickens (CdCoV). The distances between DdCoV and CdCoV are large enough to separate them into different species within the genus Gammacoronavirus. Our surveillance demonstrated that DdCoVs and CdCoVs belong to different lineages and occupy different ecological niches, further supporting that they should be classified into different species. Our surveillance also demonstrated that DdCoVs and CdCoVs are prevalent in live poultry markets in some regions of China. In conclusion, this study shed novel insight into the genetic diversity, evolution, distribution, and taxonomy of the coronaviruses circulating in chickens and ducks.

Optimal cutoff scores for dementia and mild cognitive impairment of the Montreal Cognitive Assessment among elderly and oldest-old Chinese population.

BACKGROUND: All versions of the Montreal Cognitive Assessment (MoCA) lack population-based data of 80-plus individuals. The norms and cut-off scores for mild cognitive impairment (MCI) and dementia of the MoCA are different among five Chinese versions. OBJECTIVE: To provide the cut-off scores in detecting MCI and dementia of the Peking Medical Union College Hospital version of the MoCA (MoCA-P). METHODS: In a cross-sectional survey, Chinese veterans aged ≥60 years completed the MoCA-P and the Mini-Mental State Examination (MMSE). RESULTS: Among 7,445 elderly veterans, 5,085 (68.30%) were aged ≥80 years old, 2,621 (35.20%) had 6 years of education or less, 6,847 (91.97%) were male, and 2,311 (31.04%) and 984 (13.22%) veterans were diagnosed as having MCI and dementia, respectively. Adding two points and one point to the MoCA scores for the primary and middle school groups, respectively, can fully adjust for the notable impact of education but cannot compensate for the effect of age. In the three age groups (60-79, 80-89, and ≥90 years old), the optimal MoCA-P cut-off scores for detecting MCI were ≤25, ≤24, and ≤23, respectively, and for detecting dementia were ≤24, ≤21, and ≤19, respectively, which demonstrated relatively high sensitivities and specificities. The areas under the curves for the MoCA-P for detecting MCI and dementia (0.937 and 0.908, respectively) were greater than those for the MMSE (0.848 and 0.892, respectively). CONCLUSION: Compared with the MMSE, the MoCA-P is significantly better for detecting MCI in the elderly, particularly in the oldest old population, and it also displays more effectiveness in detecting dementia.

Evaluation of avian influenza virus isolated from ducks as a potential live vaccine candidate against novel H7N9 viruses.

Recent outbreaks of a novel H7N9 avian influenza virus in humans in China raise pandemic concerns and underscore an urgent need to develop effective vaccines. Theoretically, live influenza vaccines are of multiple advantages over traditional inactivated influenza vaccines to be used in a pandemic, because they can be produced rapidly, safely, and inexpensively. However, studies on live vaccines against the novel H7N9 virus are limited. In this study, we evaluated a potential live influenza vaccine candidate using an H7N3 avian influenza virus isolated from ducks with controls of two recombinant viruses generated through reverse genetics. The potential candidate could be produced efficiently using chicken embryonated eggs, and is homogenous to the novel H7N9 virus in their viral hemagglutinin genes. The potential candidate is likely low pathogenic to birds and mammals, and likely sensitive to oseltamivir and amantadine, as suggested by its genomic sequences. Its low pathogenicity was further supported through inoculation in mice, chicken embryonated eggs and chickens. Specific antibodies elicited in mice were detectable at least during the period between day 14 and day 56 after intranasal administration of the candidate for one time. Titers of the specific antibodies increased significantly with a boost intranasal administration or a higher inoculation dose. The induced specific antibodies were of substantial cross-reactivity with the novel H7N9 virus. These primary but promising evaluation data suggest that the duck influenza virus could be used as a potential live vaccine candidate, favorably through a prime-boost route, to mitigate the severity of the possible pandemic caused by the newly emerging H7N9 virus, and is valuable to be further evaluated.

Identification of a potential novel type of influenza virus in Bovine in China.

Bovine influenza virus was first identified in the USA in 2013, and the virus represents a potential novel type of influenza viruses. However, the distribution and evolution of the virus remain unknown. We conducted a pilot survey of bovine influenza virus in China, and identified three bovine influenza viruses which are highly homogenous to the ones identified in the USA, suggesting that the bovine influenza virus likely circulates widely and evolves slowly in the world.

Identification and survey of a novel avian coronavirus in ducks.

The rapid discovery of novel viruses using next generation sequencing (NGS) technologies including DNA-Seq and RNA-Seq, has greatly expanded our understanding of viral diversity in recent years. The timely identification of novel viruses using NGS technologies is also important for us to control emerging infectious diseases caused by novel viruses. In this study, we identified a novel duck coronavirus (CoV), distinct with chicken infectious bronchitis virus (IBV), using RNA-Seq. The novel duck-specific CoV was a potential novel species within the genus Gammacoronavirus, as indicated by sequences of three regions in the viral 1b gene. We also performed a survey of CoVs in domestic fowls in China using reverse-transcription polymerase chain reaction (RT-PCR), targeting the viral nucleocapsid (N) gene. A total of 102 CoV positives were identified through the survey. Phylogenetic analysis of the viral N sequences suggested that CoVs in domestic fowls have diverged into several region-specific or host-specific clades or subclades in the world, and IBVs can infect ducks, geese and pigeons, although they mainly circulate in chickens. Moreover, this study provided novel data supporting the notion that some host-specific CoVs other than IBVs circulate in ducks, geese and pigeons, and indicated that the novel duck-specific CoV identified through RNA-Seq in this study is genetically closer to some CoVs circulating in wild water fowls. Taken together, this study shed new insight into the diversity, distribution, evolution and control of avian CoVs.

Complete genome sequence of a hemagglutination-negative avian paramyxovirus type 4 isolated from china.

We report here the complete genome sequence of a nonpathogenic and hemagglutination-negative avian paramyxovirus type 4 isolated from a duck in southern China. Phylogenetic analysis of the genome sequence indicated that the waterfowl virus possibly has evolved into the Eastern and Western Hemisphere lineages.

Panorama phylogenetic diversity and distribution of type A influenza viruses based on their six internal gene sequences.

BACKGROUND: Type A influenza viruses are important pathogens of humans, birds, pigs, horses and some marine mammals. The viruses have evolved into multiple complicated subtypes, lineages and sublineages. Recently, the phylogenetic diversity of type A influenza viruses from a whole view has been described based on the viral external HA and NA gene sequences, but remains unclear in terms of their six internal genes (PB2, PB1, PA, NP, MP and NS). METHODS: In this report, 2798 representative sequences of the six viral internal genes were selected from GenBank using the web servers in NCBI Influenza Virus Resource. Then, the phylogenetic relationships among the representative sequences were calculated using the software tools MEGA 4.1 and RAxML 7.0.4. Lineages and sublineages were classified mainly according to topology of the phylogenetic trees and distribution of the viruses in hosts, regions and time. RESULTS: The panorama phylogenetic trees of the six internal genes of type A influenza viruses were constructed. Lineages and sublineages within the type based on the six internal genes were classified and designated by a tentative universal numerical nomenclature system. The diversity of influenza viruses circulating in different regions, periods, and hosts based on the panorama trees was analyzed. CONCLUSION: This study presents the first whole views to the phylogenetic diversity and distribution of type A influenza viruses based on their six internal genes. It also proposes a tentative universal nomenclature system for the viral lineages and sublineages. These can be a candidate framework to generalize the history and explore the future of the viruses, and will facilitate future scientific communications on the phylogenetic diversity and evolution of the viruses. In addition, it provides a novel phylogenetic view (i.e. the whole view) to recognize the viruses including the origin of the pandemic A(H1N1) influenza viruses.

The host responses in sheep artificially infected with Theileria sp. (China).

Studies on host responses in sheep artificially infected with Theileria sp. (China) were discussed and summarized mainly on typical high fever periods, merozoits and schizoon observation, antibody response.