A Single-Step Method for Harvesting Influenza Viral Particles from MDCK Cell Culture Supernatant with High Yield and Effective Impurity Removal.

Influenza vaccines, which are recommended by the World Health Organization (WHO), are the most effective preventive measure against influenza virus infection. Madin-Darby canine kidney (MDCK) cell culture is an emerging technology used to produce influenza vaccines. One challenge when purifying influenza vaccines using this cell culture system is to efficiently remove impurities, especially host cell double-stranded DNA (dsDNA) and host cell proteins (HCPs), for safety assurance. In this study, we optimized ion-exchange chromatography methods to harvest influenza viruses from an MDCK cell culture broth, the first step in influenza vaccine purification. Bind/elute was chosen as the mode of operation for simplicity. The anion-exchange Q chromatography method was able to efficiently remove dsDNA and HCPs, but the recovery rate for influenza viruses was low. However, the cation-exchange SP process was able to simultaneously achieve high dsDNA and HCP removal and high influenza virus recovery. For the SP process to work, the clarified cell culture broth needed to be diluted to reduce its ionic strength, and the optimal dilution rate was determined to be 1:2 with purified water. The SP process yielded a virus recovery rate exceeding 90%, as measured using a hemagglutination units (HAUs) assay, with removal efficiencies over 97% for HCPs and over 99% for dsDNA. Furthermore, the general applicability of the SP chromatography method was demonstrated with seven strains of influenza viruses recommended for seasonal influenza vaccine production, including H1N1, H3N2, B (Victoria), and B (Yamagata) strains, indicating that the SP process could be utilized as a platform process. The SP process developed in this study showed four advantages: (1) simple operation, (2) a high recovery rate for influenza viruses, (3) a high removal rate for major impurities, and (4) general applicability.

Evaluation of the Genetic Diversity, Population Structure and Selection Signatures of Three Native Chinese Pig Populations.

Indigenous pig populations in Hainan Province live in tropical climate conditions and a relatively closed geographical environment, which has contributed to the formation of some excellent characteristics, such as heat tolerance, strong disease resistance and excellent meat quality. Over the past few decades, the number of these pig populations has decreased sharply, largely due to a decrease in growth rate and poor lean meat percentage. For effective conservation of these genetic resources (such as heat tolerance, meat quality and disease resistance), the whole-genome sequencing data of 78 individuals from 3 native Chinese pig populations, including Wuzhishan (WZS), Tunchang (TC) and Dingan (DA), were obtained using a 150 bp paired-end platform, and 25 individuals from two foreign breeds, including Landrace (LR) and Large White (LW), were downloaded from a public database. A total of 28,384,282 SNPs were identified, of which 27,134,233 SNPs were identified in native Chinese pig populations. Both genetic diversity statistics and linkage disequilibrium (LD) analysis indicated that indigenous pig populations displayed high genetic diversity. The result of population structure implied the uniqueness of each native Chinese pig population. The selection signatures were detected between indigenous pig populations and foreign breeds by using the population differentiation index (FST) method. A total of 359 candidate genes were identified, and some genes may affect characteristics such as immunity (IL-2, IL-21 and ZFYVE16), adaptability (APBA1), reproduction (FGF2, RNF17, ADAD1 and HIPK4), meat quality (ABCA1, ADIG, TLE4 and IRX5), and heat tolerance (VPS13A, HSPA4). Overall, the findings of this study will provide some valuable insights for the future breeding, conservation and utilization of these three Chinese indigenous pig populations.

Identification of Runs of Homozygosity Islands and Functional Variants in Wenchang Chicken.

Wenchang chickens, a native breed in the Hainan province of China, are famous for their meat quality and adaptability to tropical conditions. For effective management and conservation, in the present study, we systematically investigated the characteristics of genetic variations and runs of homozygosity (ROH) along the genome using re-sequenced whole-genome sequencing data from 235 Wenchang chickens. A total of 16,511,769 single nucleotide polymorphisms (SNPs) and 53,506 ROH segments were identified in all individuals, and the ROH of Wenchang chicken were mainly composed of short segments (0-1 megabases (Mb)). On average, 5.664% of the genome was located in ROH segments across the Wenchang chicken samples. According to several parameters, the genetic diversity of the Wenchang chicken was relatively high. The average inbreeding coefficient of Wenchang chickens based on FHOM, FGRM, and FROH was 0.060 ± 0.014, 0.561 ± 0.020, and 0.0566 ± 0.01, respectively. A total of 19 ROH islands containing 393 genes were detected on 9 different autosomes. Some of these genes were putatively associated with growth performance (AMY1a), stress resistance (THEMIS2, PIK3C2B), meat traits (MBTPS1, DLK1, and EPS8L2), and fat deposition (LANCL2, PPARγ). These findings provide a better understanding of the degree of inbreeding in Wenchang chickens and the hereditary basis of the characteristics shaped under selection. These results are valuable for the future breeding, conservation, and utilization of Wenchang and other chicken breeds.

Genome-wide re-sequencing data reveals the genetic diversity and population structure of Wenchang chicken in China.

Wenchang (WC) chicken, the only indigenous chicken breed listed in Chinese genetic resources in Hainan province, is well known for its excellent meat quality and is sold all over southeast Asia. In recent years, the number of WC has decreased sharply with considerable variability in the quality at market. To explore the genetic diversity and population structure of WC chickens, the whole-genome data of 235 WC individuals from three conservation farms were obtained using the Illumina 150 bp paired-end platform and used in conjunction with the sequencing data from 123 individuals from other chicken breeds (including eight Chinese indigenous chicken breeds and three foreign or commercial breeds) downloaded from a public database. A total of 12 111 532 SNPs were identified, of which 11 541 878 SNPs were identified in WC. The results of gene enrichment analyses revealed that the SNPs harbored in WC genomes are mainly related to environmental adaptation, disease resistance and meat quality traits. Genetic diversity statistics, quantified by expected heterozygosity, observed heterozygosity, linkage disequilibrium, nucleotide diversity and fixation statistics, indicated that WC displays high genetic diversity compared with other Chinese indigenous chicken breeds. Genetic structure analyses showed that each population displayed great differentiation between WC and the other breeds, indicating the uniqueness of WC. In conclusion, the results of our study provide the first genomic overview of genetic variants, genetic diversity and population structure of WC from three conservation farms. This information will be valuable for the future breeding and conservation of WC and other surveyed populations.

Evaluation of the soil profile quality of subsided land in a coal mining area backfilled with river sediment based on monitoring wheat growth biomass with UAV systems.

Underground coal mining leads to land subsidence, and the situation is particularly serious in the Coal-Grain Complex in eastern China, causing the crop production to be reduced or to be taken out. Backfilling with Yellow River sediment is one of the effective methods to solve the land subsidence in this area, but a key issue is how to select the optimal soil reconstruction profile so that the crop yield after backfilling and reclamation is unaffected. The main purpose of this study is to verify the feasibility of selecting the optimal soil reconstruction profile by rapid monitoring of crop growth and judging soil quality with the aid of unmanned aerial vehicle systems (UAVs). A control treatment and 13 experimental treatments were established for the study area. The control treatment consisted of using 30 cm topsoil and 90 cm subsoil and the topsoil is a proxy for native (undisturbed) soil from the study sites. All other treatments consisted of using varying combinations of subsoil and sediment overlain by 30 cm of topsoil. The vegetation indices from the UAV multispectral images, and the plant height and vegetation coverage from the UAV RGB images were used for estimation of the winter wheat biomass in a random forest regression. The results showed that the random forest regression model yielded accurate estimation of the aboveground biomass. Furthermore, knowledge of plant height and vegetation coverage improved the accuracy of prediction such that crop growth was well characterized. The optimal soil profile consisted of 0.3 m topsoil + 0.2 m subsoil + 0.2 m sediment + 0.2 m subsoil + 0.3 m sediment. A fast and effective airborne monitoring method for soil quality was established, thus providing greatly improved monitoring efficiency.

Comparison of N-linked glycosylation on hemagglutinins derived from chicken embryos and MDCK cells: a case of the production of a trivalent seasonal influenza vaccine.

N-linked glycosylation plays critical roles in folding, receptor binding, and immunomodulating of hemagglutinin (HA), the main antigen in influenza vaccines. Chicken embryos are the predominant production host for influenza vaccines, but Madin-Darby canine kidney (MDCK) cells have emerged as an important alternative host. In this study, we compared glycosylation patterns, including the occupancy of potential glycosylation sites and the distribution of different glycans, on the HAs of three strains of influenza viruses for the production a trivalent seasonal flu vaccine for the 2015-2016 Northern Hemisphere season (i.e., A/California/7/2009 (H1N1) X179A, A/Switzerland/9715293/2013 (H3N2) NIB-88, and B/Brisbane/60/2008 NYMC BX-35###). Of the 8, 12, and 11 potential glycosylation sites on the HAs of H1N1, H3N2, and B strains, respectively, most were highly occupied. For the H3N2 and B strains, MDCK-derived HAs contained more sites being partially occupied (<95%) than embryo-derived HAs. A highly sensitive glycan assay was developed where 50 different glycans were identified, which was more than what has been reported previously, and their relative abundance was quantified. In general, MDCK-derived HAs contain more glycans of higher molecular weight. High-mannose species account for the most abundant group of glycans, but at a lower level as compared to those reported in previous studies, presumably due to that lower abundance, complex structure glycans were accounted for in this study. The different glycosylation patterns between MDCK- and chicken embryo-derived HAs may help elucidate the role of glycosylation on the function of influenza vaccines. KEY POINTS: • For the H3N2 and B strains, MDCK-derived HAs contained more partially (<95%) occupied glycosylation sites. • MDCK-derived HAs contained more glycans of higher molecular weight. • A systematic comparison of glycosylation on HAs used for trivalent seasonal flu vaccines was conducted.