Prenatal exposure to bisphenols, immune responses in cord blood and infantile eczema: A nested prospective cohort study in China.

Increasing evidence shows that human exposure to bisphenols can increase the risk of allergic disease, such as child asthma. However, the mechanism by which exposure to bisphenols causes allergic disease is unclear. In addition, the effects of exposure to bisphenols during pregnancy on infantile eczema have been poorly studied. The aim of our study was to investigate the effect of bisphenols (BPA, BPF and BPS) exposure during pregnancy on immune cells in cord blood, and on the occurrence of infantile eczema. 111 mother-child pairs with urine samples from pregnant women and cord blood were recruited from a birth cohort established in February 2019 in Shenyang, China. The levels of urinary bisphenols and Th1-, Th2-, Treg- and Th17-related genes, and cytokines in cord blood, as well as the incidence of infantile eczema at 6 and 12 months follow up were determined. Our results show that BPA, BPF and BPS were detected in 100%, 63.1% and 46.8% of the urine samples, respectively. The median concentration of urine specific gravity adjusted BPA (SG-BPA) was 7.46 ng/mL. High SG-BPA levels during pregnancy was independently associated with increased risk of infantile eczema (adjusted OR = 2.731, 95%CI: 1.064-7.012, P = 0.037). Higher levels of FOXP3 gene in cord blood had a significantly lower risk of developing eczema in infants (adjusted OR=0.430, 95%CI: 0.190-0.972, P = 0.042). However, BPS and BPF levels were not associated with infantile eczema. FOXP3 gene levels in cord blood mediated the relationship between SG-BPA levels during pregnancy and infantile eczema (indirect effect: ß = 0.350 [CI:0.011,1.077]). Our findings indicate that high levels of BPA exposure during pregnancy increase the risk of infantile eczema, which may be associated with down-regulation of FOXP3 gene expression in cord blood.

A porcine alveolar macrophage cell line stably expressing CD163 demonstrates virus replication and cytokine secretion characteristics similar to primary alveolar macrophages following PRRSV infection.

The in vitro investigation of cytokine secretion induced by porcine reproductive and respiratory syndrome virus (PRRSV) requires porcine alveolar macrophages (PAMs) and their interaction with immunocytes. However, immortalized monoclonal PAMs (mPAMs) are non-permissive for PRRSV infection. The porcine CD163 receptor isolated from primary PAMs (pPAMs) confers susceptibility to PRRSV infection; thus, this approach could be used to establish a novel cell line to facilitate the exploration of PRRSV infection kinetics. Here, we amplified the coding region of the CD163 gene from pPAMs and integrated it into an mPAM line using a lentivirus expression system. After verification, the monoclonal PAM cell line stably expressing CD163 (mPAM-CD163-GFP) was infected with either the highly pathogenic PRRSV strain JXA1 or the classical PRRSV strain SD1, which produced high infectious titers of progeny virus reaching > 109 copies/mL or a 50 % tissue culture infective dose of 105.5 over at least 100 cell generations. We also investigated cytokine and Toll-like receptor expression in infected mPAM-CD163-GFP cells and pPAMs. The mPAM-CD163-GFP cell line showed similar patterns of viral replication and cytokine secretion compared with pPAMs, so it may be extremely useful for replacing primary cells for in vitro investigations of the mechanisms of cytokine secretion and interactions between PRRSV-infected PAMs and immunocytes.

Isolation and characterization of an Aves polyomavirus 1 from diseased budgerigars in China.

Aves polyomavirus 1 (APV) causes inflammatory disease in psittacine birds, especially in young budgerigar. In this study, an APV virus (SD18 strain) was isolated from a diseased psittacine birds breeding facility. The full genome (4981 bp) of SD18 was determined and analyzed. Phylogenetic analysis of full genome sequences indicated all the APV strains form two groups. The SD18 strain showed close relationship with APV isolated from Poland, however, the other Chinese strains are located in group II, which suggested different genotypes APVs are co-circulating in China. Compared with the consensus sequence of APV full genome, the SD18 strain contains 13 nucleotide mutations, and 2 unique amino acid substitutions (R179M and Q382K) located in VP2/3 and Large T proteins. To explore the pathogenicity of the virus, the SD18 strain was used to challenge 2-week-old budgerigars. All infected birds died no later than 5 days post infection, and virus was detected in multiple organs including brain, heart, ingluvies, liver, and intestine, which indicated that SD18 is fatal and causes systemic infection in young budgerigar. In vitro studies showed that SD18 replicated efficiently in CEF cells and reached the highest viral titers at 9 days post infection. Notably, replication of SD18 stimulated IFN-ß response in CEF cells and overexpression of the VP4 or VP4Delta proteins significantly inhibited IFN-ß promoter activation, which could be the strategy of APV to escape from the host innate immunity.

Identification and characterization of novel genotypes of psittacine beak and feather disease virus from budgerigar in China.

Psittacine beak and feather disease (PBFD) is a common disease in psittacine bird that caused by beak and feather disease virus (BFDV). BFDV is widely spread and threatening psittacine birds worldwide. However, the BFDV infection in China remains largely unknown. In this study, a surveillance study of BFDV was conducted in three budgerigar breeding facilities, which showed that 66.6% of collected faeces samples were positive for BFDV. Full genomes of nine BFDV circulating in the three budgerigar breeding facilities (three for each facility) were determined and analysed. The full genomes shared 75.9% to 87.5% identity with the known genotype BFDV. Phylogenetic analysis of the full genome indicated that the BFDV circulating in China formed a separated group, and the nine isolates fell into three subgroups, suggesting that different unique BFDV genotypes are circulating in China. Notably, the Cap genes of three strains (SD3, SD5 and SD9) showed low identity (67.9% to 70%) to all the known genotypes of BFDV. Phylogenetic analysis showed that these three Cap genes formed a unique lineage that is different from all known genotypes, which suggested that the SD3, SD5 and SD9 strains identified in this study belong to a novel genotype that has not been reported. However, the origin of this genotype remains unclear. All the data indicated that the different unique genotypes of BFDV are co-circulating in China, and active surveillance of BFDV is warranted.

Establishment of rapid detection method and surveillance of budgerigar fledgling disease virus using a TaqMan Real-Time PCR.

Budgerigar fledgling disease virus (BFDV) infection causes sudden death, abdominal distention, and feather abnormality in psittacine birds. In this study, we developed a TaqMan Real-time PCR assay to detect BFDV by targeting a conserved region in VP1 gene. The detection limit of the assay was 30 DNA gene copies, 1000 times more sensitive than conventional PCR. The coefficients of variation were less than 1.09% in either intra- or inter-assays, indicating high reproducibility. By using this method, the prevalence of BFDV in China was evaluated. 56 feces samples were collected from four psittacine birds breeding facilities in China. The results showed 28 out of 56 samples were positive for BFDV in Real-Time PCR assay, while only 19 samples were positive in PCR assay. Three facilities were positive for BFDV with positive rates from 60% to 87.5%. Further sequence analysis of VP1 genes from the positive samples indicated that VP1 genes fell into two different lineages in phylogenetic tree, suggesting that different genotypes BFDV are co-circulating in China.

Profile and source apportionment of volatile organic compounds from a complex industrial park.

Industrial emissions, mainly from industrial parks, are important sources of ambient volatile organic compounds (VOCs). Identification of the major sources of VOCs from industrial parks has practical significance in emission reduction. In this study, the major species of VOCs from a residential area located downwind of a complex industrial park were sampled with Tenax absorption tubes and analyzed by thermal desorption coupled with gas chromatography/mass spectrometry (TD-GC/MS). Receptor models of factor analysis with nonnegative constraints (FA-NNC) and positive matrix factorization (PMF) were employed to recognize the potential emission sources, which suggested an association with the production processes in the nearby industrial park. In order to validate the sources, the profiles of VOC emissions of related workshops under actual manufacturing processes were acquired. It was found that xylenes & amines, phenols and esters were the major species of VOCs for the workshops of foundry, refractory materials and printing, respectively. Similarity analysis indicated that the detected profiles of VOC emissions from the dominant industrial types had good correlations with the identified factors from receptor models. Source contributions to VOCs in the receptor region exhibited that foundry production was the primary contributor (56-64%), followed by refractory material production (22-26%) and printing (14-18%). This study provides a strategy for source apportionment of VOCs from a local complex industrial park, which is helpful in the development of targeted control strategies.

Characterization of a moderately pathogenic pseudorabies virus variant isolated in China, 2014.

In this study, we reported a moderately pathogenic pseudorabies virus (PRV) variant isolated from one Bartha-K61-vaccinated pig farm in Weifang, Shandong Province, China, 2014. The sick piglets in the farm were characterized by anorexia, weight loss and neurologic symptoms but did not die. Sequence alignment of the gE gene indicated that it belonged to a new mutated PRV strain and about 15% amino acid sites had mutations, deficiencies and insertions compared to the other PRV strains. The gD gene had two amino acid insertions and ten amino acid mutations in comparison with the Bartha-K61 vaccine strain. The TK and gM genes were the same as one highly pathogenic PRV TJ strain. Evidence from virus isolation, laboratory challenge, serological detection and histopathologic examination confirmed that the etiological agent of the disease is PRV SD1404, which is a moderately pathogenic strain and causes piglets to be sick but not to die. PRV SD1404 strain is different from other reports and should be paid more attention to avoid economic losses.

Genetic characterization and mutation analysis of Qihe547 Aujeszky's disease virus in China.

BACKGROUND: Aujeszky's disease virus (ADV) can cause neurologic disease in young pigs, respiratory disease in older pigs and abortion or birth of mummified fetuses or stillborn neonates. The re-emergence of Aujeszky's disease (AD) in pig farms vaccinated with live vaccine (Bartha-K61) caused substantial economic losses to Chinese pig industry since late 2011. A field ADV, named Qihe547, was isolated from pigs that exhibited suspected AD clinical symptoms. To better understand the genetic characteristics and mutations of Qihe547 ADV, the whole genome was sequenced and analyzed. RESULTS: The genomic length of Qihe547 ADV was 143,404 bp, with 73.59% G + C contents. Phylogenetic analysis based on the whole genome of ADV strains revealed that Chinese ADV strains were located to one group with three subgroups. Qihe547 ADV was closely related to these novel ADV strains isolated in China since 2012. Qihe547 presented numerous hypervariable regions compared with oversea ADV strains. In 34 genes of Qihe547 ADV, amino acid (AA) insertion or deletion were observed. In addition, numerous AA mutations were found in the main protective antigen genes (gB, gC and gD genes). The differences of potential antigenic peptides in the main protective antigens between Qihe547 ADV and ADV Bartha were discovered in the dominant antigenic regions of gB (AA59-AA126, AA507-AA734),the extracellular region of gC and gD. CONCLUSION: High diversity was observed between Qihe547 and foreign ADV isolates. The AA variations and the differences of potential antigenic peptides in the important functional regions of the main protective antigen (gB, gC and gD) of ADV Qihe547 may contribute to immune evasion of the virus and may be partial reason that the virus escapes from the vaccination of Bartha-K61 vaccine. In a word, the effect of the variations obviously requires further research.

Establishment and Characterization of a High and Stable Porcine CD163-Expressing MARC-145 Cell Line.

Isolation and identification of diverse porcine reproductive and respiratory syndrome viruses (PRRSVs) play a fundamental role in PRRSV research and disease management. However, PRRSV has a restricted cell tropism for infection. MARC-145 cells are routinely used for North American genotype PRRSV isolation and vaccine production. But MARC-145 cells have some limitations such as low virus yield. CD163 is a cellular receptor that mediates productive infection of PRRSV in various nonpermissive cell lines. In this study, we established a high and stable porcine CD163- (pCD163-) expressing MARC-145 cell line toward increasing its susceptibility to PRRSV infection. Indirect immunofluorescence assay (IFA) and Western blotting assays showed that pCD163 was expressed higher in pCD163-MARC cell line than MARC-145 cells. Furthermore, the ability of pCD163-MARC cell line to propagate PRRSV was significantly increased as compared with MARC-145 cells. Finally, we found that pCD163-MARC cell line had a higher isolation rate of clinical PRRSV samples and propagated live attenuated PRRS vaccine strains more efficiently than MARC-145 cells. This pCD163-MARC cell line will be a valuable tool for propagation and research of PRRSV.

Source Apportionment of Polycyclic Aromatic Hydrocarbons in Sediment by the Application of Non-Negative Factor Analysis: A Case Study of Dalian Bay.

An improved method, factor analysis with non-negative constraints (FA-NNC) was adopted to apportion the sources of sediment polycyclic aromatic hydrocarbons (PAHs) in Dalian Bay, China. Cosine similarity and Monte Carlo uncertainty analysis were used to assist the FA-NNC source resolution. The results identified three sources for PAHs, which were overall traffic, diesel engine emissions and residential coal combustion. The contributions of these sources were quantified as 78 ± 4.6% from overall traffic, 12 ± 3.2% from diesel engine emissions, and 10 ± 1.9% from residential coal combustion. The results from the Monte Carlo uncertainty analysis indicated that the model was robust and convergent.

Vaccine resistant pseudorabies virus causes mink infection in China.

BACKGROUND: Pseudorabies, a highly contagious infectious disease of swine is caused by pseudorabies virus (PRV). PRV can cause fatal infection in other animal species. RESULTS: We report a deadly outbreak of pseudorabies that killed 87.2% (3522/4028) minks in a farm in 2014 in Shandong Province, China. PRV was isolated by using Vero cell culture and detected in mink samples by PCR from minks died during the outbreak. Epidemiological analysis indicated that 5.8% of minks (33/566) were PCR positive to PRV in Shandong Province. Phylogenetic analysis indicated that the PRV strains isolated from minks in this study were in the same clade with the Chinese porcine PRV isolates, which are resistant to the PRV vaccine. CONCLUSIONS: We demonstrated that pseudorabies virus caused an outbreak of minks in a farm in Shandong Province of China and the virus has a very high infection rate in minks in Shandong Province, which is a challenge for the fur industry in China.

Severe Fever with Thrombocytopenia Syndrome Virus Infection in Minks in China.

We analyzed the seroprevalence of tick-borne severe fever with thrombocytopenia syndrome virus (SFTSV) in farm-raised minks using double antigen ELISA (enzyme-linked immunosorbent assay) kit and indicated that 8.4% (15/178) of the minks had antibodies to the nucleoprotein of SFTSV and 72.7% (8/11) of mink farms had minks positive to SFTSV. The ELISA results were further confirmed by presence of neutralization to SFTSV in the mink sera. Our results suggested that minks were widely infected with SFTSV in China.

Genetic analysis of porcine circovirus type 2 from dead minks.

Circovirus infection is a growing problem in the field of veterinary and public health. It is associated with enteric diseases in both mammalian and avian hosts. In this study, we detected and isolated porcine circovirus strains in the tissue samples of minks that died from diarrhoea in Shandong Province, China. We sequenced the whole genome of two porcine strains of Circovirus, designated as MiSD-1 and MiSD-2， which had a 97.34% similarity on nucleotide sequence and were closely related to porcine circovirus type 2 (PCV2), but distantly related to mink circoviral species. Phylogenetically MiSD-1 and MiSD-2 are a part of the PCV2b genotype cluster, which is a highly prevalent genotype worldwide. The closer relationship of MiSD-1 and MiSD-2 to PCV2 from pigs than to other mink circoviral species may be evidence of cross-species transmission and considerable zoonotic potential.

Generation by reverse genetics of an effective attenuated Newcastle disease virus vaccine based on a prevalent highly virulent Chinese strain.

OBJECTIVES: To investigate whether the differences between the circulating Newcastle disease virus (NDV) isolates and the used vaccine might account for the current ND outbreaks in vaccinated poultry flocks. RESULTS: A reverse genetics system using prevalent genotype VIId isolate SG10 was constructed and a mutant virus, named aSG10, was developed by changing the virulent F protein cleavage site motif "(112)RRQKR↓F(117)" into an avirulent motif "(112)GRQGR↓L(117)". The attenuated pathogenicity of aSG10 was confirmed from the mean death time and intracerebral pathogenicity index. aSG10 and LaSota both protected vaccinated birds from death after challenge with highly virulent genotype VII NDV, strain SG10. However, aSG10 significantly reduced the challenge virus shedding from the vaccinated birds compared to LaSota vaccine. We also generated a recombinant virus, aSG10-enhanced green fluorescent protein (EGFP), which expresses EGFP. aSG10-EGFP stably expressed EGFP for at least 10 passages. CONCLUSIONS: The mutant, aSG10, can be safely used as a vaccine vector and is a potential vaccine candidate in increasing the protective efficacy for the control of current ND epidemic in China.

Characterization of two pigeon paramyxovirus type 1 isolates in China.

For over three decades, there has been a continuing panzootic caused by a virulent variant avian paramyxovirus type 1 strain, the so-called pigeon paramyxovirus type 1. It is found primarily in racing pigeons, but it has also spread to wild birds and poultry. In this study, two pigeon paramyxovirus type 1 strains, SD12 and BJ13, obtained from diseased pigeons in China, were characterized. Phylogenetic analysis based on complete sequences allowed characterization of both strains as genotype VI, class II. Further phylogenetic analysis of a 374-nucleotide section of the fusion gene showed that SD12 fell into lineage VIbii-d and BJ13 into VIbii-f. The deduced amino acid sequence of the cleavage site of the fusion protein confirmed that both isolates contained the virulent motif (112)K/RRQKR↓F(117) at the cleavage site. Nevertheless, the values of intracerebral pathogenicity indices showed the SD12 isolate to be a velogenic strain and BJ13 isolate to be a mesogenic strain. The SD12 isolate was further investigated via clinical observation, RNA detection, histopathology and viral serology in experimentally infected 3-week-old chickens. It showed a mild pathological phenotype in chickens, with viral replication restricted to a few tissues. The molecular mechanism for the SD12 isolate to have a virulent motif but low levels of virulence for chickens requires further study.

Detection and characterization of avastrovirus associated with diarrhea isolated from minks in China.

Astroviruses are becoming a growing concern in veterinary and public health. Many astrovirus species are associated with enteric diseases have been described in both mammalian and avian hosts. In the present study, 23 fecal samples from diarrheic minks were collected in Liaoning and Shandong Province, and an investigation of astrovirus was performed using biochemical methods and RT-PCR assay with specific primers. A total of four mink astroviral isolates were detected from sick minks with diarrhea problems. Further sequencing and characterization of the partial ORF1b gene and ORF2 gene segments revealed low sequence identities (20.0-85.3 and 31.8-87.2%) with known astroviral strains, indicating the emergence of a novel clade of astroviruses. Some new features of the astroviral genome have also been discovered. The phylogenetic tree revealed that all samples were distantly related to mink astrovirus and were closely related to chicken astroviruses and turkey astroviruses. MK/DL-1, MK/DL-2, MK/SD-1, and MK/SD-2 formed a new clade and were found to be more closely related to astroviruses from birds than to other mink strains, indicating past cross-species transmission and considerable zoonotic potential.

Complete genome sequence of a Vero cell-adapted isolate of porcine epidemic diarrhea virus in eastern China.

In early 2012, a widespread porcine epidemic diarrhea virus (PEDV) occurred in eastern China. A cell-adapted isolate, SD-M, was at the four-passage level of virulent field strain SD, which was isolated from a 2-day-old dead suckling piglet that had suffered from severe diarrhea in Shandong Province, China. We report here the complete genome sequence of SD-M. This sequence will promote a better understanding of the molecular pathogenesis of PEDV.

[Effects of extraneous inorganic nitrogen forms on the dynamics of soil amino sugars].

Substrate availability affects microbial growth, whereas extraneous nitrogen forms can significantly affect microbial metabolic processes. As for soil amino sugars, the stable residues in microbial cell wall, their synthesis, decomposition and turnover are closely related to the availability of extraneous carbon and nitrogen. Using isotope tracing technique to study soil amino sugars can further understand the substrate utilization profiles by soil microorganisms. In this study, two incubation tests were conducted, with glucose plus 15N-labelled NH4+ or NO3- as the substrates, respectively. The 15N enrichment in each kind of soil amino sugars was identified by gas chromatography/ mass spectrometry (GC/MS) to trace the dynamics of soil 15N-labelled and native amino sugars. During the incubation, the content of soil 15N-labelled amino sugars increased significantly, and the transformation rate from NH4+ to amino sugars was significantly higher than that from NO3-, suggesting the preferred utilization of NH4+ than NO3- by soil microorganisms. Significant changes in the amounts of soil unlabelled amino sugars were observed. The amount of unlabelled glucosamine increased with NH4+ addition, but decreased gradually with NO3- addition. The content of unlabelled muramic acid decreased gradually, especially with NO3- addition. Either the increase or the decrease of galactosamine did not exceed 20% to the original value. These compound-specific changes showed that the heterogeneous microbial residues played different roles on the turnover and stabilization of nitrogen in soil matrix. Fungal cell wall residues were easily accumulated in soil matrix, which benefited the stabilization of soil organic matter, while bacterial cell wall residues were easily degraded, playing an important role in the turnover of soil organic matter.

[Determination of phthalic acid esters in soil by gas chromatography-tandem mass spectrometry].

A method based on gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed for the determination of phthalic acid esters (PAEs) in soil. The soil sample was extracted by dichloromethane-acetone (1: 1, v/v) with an ultrasonic instrument, and the extract was cleaned up with a Florisil column. The identification and quantification of the analytes were carried out in the multiple reaction monitoring (MRM) mode after the GC separation on an HP-5MS column (30 m x 0.25 mm x 0.25 microm). The eight point calibration curves showed a good linearity for all the congeners (0.997 3 - 0.997 6). The limits of detection (LODs) ranged from 0.1 to 0.5 microg/kg. The relative standard deviations (RSDs) were not more than 14.3% and the spiked recoveries were in the range of 72.9% - 106.2%. The developed analytical method is rapid and accurate, and suitable for the determination of PAEs in soil.

Genotypic evolution and antigenic drift of H9N2 influenza viruses in China from 1994 to 2008.

H9N2 influenza viruses have been circulating in China since 1994, but a systematic investigation of H9N2 in northern China has not been undertaken since 2004. Here, using the sequences of 22 viruses we isolated from poultry and pigs in northern China during 2003-2008, in combination with sequences available in a public database, we analyzed the evolution of H9N2 influenza viruses in China from 1994 to 2008. Our findings demonstrated that the H9N2 viruses in China underwent extensive reassortment, and novel genotypes continued to emerge. Among 330 viruses, 54 genotypes were observed including 19 novel genotypes that have not been recognized before, and major genotypes were further divided into five series (BJ/94-, G1-, BG-, F/98- and Aq-series). Different epidemiological and biological features among these series were recognized. The BJ/94- and F/98-series viruses were circulating in both southern and northern China, while the other three series viruses were mainly detected in southern China. BJ/94-series influenza viruses predominated in China before 2000 and were gradually replaced by F/98-series viruses that became the predominant viruses since 2004. At least five antigenic groups could be identified over the study period, during which a significant antigenic drift likely occurred between 2002 and 2003. Animal experiments demonstrated that F/98-series viruses were able to replicate and transmit more effectively in chickens than BJ/94-series viruses. The continuing evolution of H9N2 influenza viruses in China emphasizes the importance of H9N2 influenza virus surveillance throughout this region to aid pandemic prediction and prevention.