LitCovid-sentences  

PMC:7115396 / 7673-19199 JSONTXT 10 Projects

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Id Subject Object Predicate Lexical cue
T43 0-24 Sentence denotes 2 Materials and methods
T44 26-50 Sentence denotes 2.1 Vaccine and viruses
T45 51-184 Sentence denotes The H120 vaccine strain is currently a widely used vaccine strain, created from 120 serial passages of the H strain isolated in 1956.
T46 185-411 Sentence denotes The ck/CH/IBYZ/2011 strain (referred to as IBYZ, GenBank KF663561.1) used in this study is classified as a QX-like IBV isolated from a flock presenting with IB symptoms by our research group in 2011 in Jiangsu Province, China.
T47 412-533 Sentence denotes Both of these strains were recovered from a full-length clone using reverse genetics, as previously described [35], [36].
T48 535-556 Sentence denotes 2.2 Eggs and animals
T49 557-869 Sentence denotes Fertilized specific pathogen free (SPF) chicken embryos were obtained from Beijing Merial Vital Laboratory Animal Technology Co., Ltd. (Beijing, China) and hatched in our lab (Lab of Chicken Infectious Disease Protection and Control, Poultry Institute, Chinese Academic of Agriculture Sciences, Yangzhou, China).
T50 870-1019 Sentence denotes For hatching, one-day-old SPF chickens from the SPF chicken embryos as described previously were incubated at 37 °C in a relative humidity of 55–65%.
T51 1020-1131 Sentence denotes All chickens were maintained in isolators under negative pressure, and food and water were provided ad libitum.
T52 1133-1177 Sentence denotes 2.3 Construction of recombinant rH120-S1/YZ
T53 1178-1289 Sentence denotes The rIBV rH120-S1/YZ strain used in this study is described in the schematic illustration presented in Fig. 1 .
T54 1290-1421 Sentence denotes The genome RNA was synthesized in vitro by T7 RNA polymerase and transfected into BHK-21 cells, as previously described [35], [36].
T55 1422-1536 Sentence denotes The cell supernatants were harvested 48 h following transfection and propagated in 10-day-old SPF chicken embryos.
T56 1537-1656 Sentence denotes The allantoic fluid was harvested for RT-PCR, whole genome sequencing, and was then stored at −80 °C until further use.
T57 1657-1889 Sentence denotes Fig. 1 Schematic diagram for the construction of the chimeric S gene and production of a full-length cDNA of rH120-S1/YZ. (A) Replacement of the H120 S1 fragment by the corresponding sequence of IBYZ for construction of pYZS1H120S2.
T58 1890-2056 Sentence denotes The plasmids pIBYZS and pH120S contained the S gene of IBYZ strain and H120 strain, respectively, were constructed during the establishment of reverse genetic system.
T59 2057-2225 Sentence denotes Primers PS1F and PS1R were used to amplify the S1 fragment of IBYZ and vector fragment, while primers PS2F and PS2R were used to amplify the S2 fragment of H120 strain.
T60 2226-2389 Sentence denotes By overlapping PCR, the pYZS1H120S2 was constructed which contained a chimeric S gene. (B) Strategy for the construction of full-length cDNA clones of rH120-S1/YZ.
T61 2390-2475 Sentence denotes Ten cDNA fragments covering the entire genome of H120 strain was amplified by RT-PCR.
T62 2476-2678 Sentence denotes Unique Bsa I sites were inserted at the junctions between each clone, a unique T7 start site was inserted at the 5′ end of clone TM1, and a 28-nucleotide T tail was inserted at the 3′ end of clone TM10.
T63 2679-2763 Sentence denotes The S fragment in the pMDTM8 plasmid of H120 strain was replaced by chimeric S gene.
T64 2764-2920 Sentence denotes By using appropriate ligation strategy, the genomic cDNA of rH120-S1/YZ was assembled by in vitro ligation using appropriate restriction sites as indicated.
T65 2922-2953 Sentence denotes 2.4 Chicken embryo dwarf assay
T66 2954-3205 Sentence denotes To determine the recombinants’ pathogenicity in chicken embryos, 0.2 mL of rH120, rIBYZ, or rH120-S1/YZ virus (diluted to 1:100 in normal saline) was inoculated into the allantoic cavities of five 10-old-day embryonated SPF chicken eggs, respectively.
T67 3206-3288 Sentence denotes Chick embryo lesions were examined for lesions at 144 h post-inoculation at 37 °C.
T68 3290-3329 Sentence denotes 2.5 Growth kinetics in chicken embryos
T69 3330-3492 Sentence denotes To examine the viral growth ability in chicken embryos, a real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) method was established.
T70 3493-3604 Sentence denotes According to the sequences of IBV from GenBank, primers were designed based on conservative area in the 5′-UTR.
T71 3605-3715 Sentence denotes The upstream primer was 5′-CCGTTGCTTGGGCTACCTAGT-3′, and the downstream primer was 5′-CGCCTACCGCTAGATGAACC-3′.
T72 3716-3837 Sentence denotes The amplification product was cloned to pMD18-T vector (Takara) as a positive plasmid and its concentration was measured.
T73 3838-3945 Sentence denotes A gradient dilution of 5 × 102–5 × 108 copies/μL of the plasmid was used as template for quantitation test.
T74 3946-4058 Sentence denotes By plotting the cycle threshold (CT) values against the copies of the plasmid, the standard curve was generated.
T75 4059-4250 Sentence denotes 18 10-day-old embryonated SPF chicken eggs (six eggs/group) were inoculated with rH120, rIBYZ, and rH120-S1/YZ at a dose of 107 viral RNA copies/100 μL, and used for growth curve experiments.
T76 4251-4412 Sentence denotes The allantoic fluids were collected separately by syringe from the six inoculated embryonated eggs of each group at 12, 18, 24, 36, 48, 72, 96 h per inoculation.
T77 4413-4681 Sentence denotes RNA was extracted from the allantoic fluids using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. cDNA was obtained by reverse transcription using a PrimeScript RT Master Mix Perfect Real Time Kit (TaKaRa, Otsu, Shiga, Japan).
T78 4682-4898 Sentence denotes The viral copies were measured by absolute quantitative method of Real-time PCR, which was performed using SYBR® Premix Ex Taq™ II (TaKaRa, Otsu, Shiga, Japan) on an Applied Biosystems 7500 Fast Real-time PCR System.
T79 4899-4974 Sentence denotes The standard curve was plotted against the log of the template copy number.
T80 4975-5097 Sentence denotes All of the assays were run in triplicate and the copy number of each virus was calculated according to the standard curve.
T81 5099-5133 Sentence denotes 2.6 Titration of rIBVs in embryos
T82 5134-5377 Sentence denotes Serial 10-fold dilutions from 105 copies to 109 copies per 0.1 mL of virus of the recombinant strain, rH120-S1/YZ, and its parental strains (rH120 and rIBYZ) were inoculated into the allantoic cavity of 10-day-old embryonated SPF chicken eggs.
T83 5378-5493 Sentence denotes For each dilution, 0.1 mL of the virus solution was injected into each egg and 10 eggs were used for each dilution.
T84 5494-5529 Sentence denotes PBS was used as a negative control.
T85 5530-5621 Sentence denotes After a 24 h incubation, the dead embryos were considered nonspecific deaths and discarded.
T86 5622-5730 Sentence denotes The embryos were examined for the presence of specific lesions caused by the virus after a 144-h incubation.
T87 5731-5862 Sentence denotes Dead and live embryos that displayed IBV infectious signs (e.g., dwarfing, curling, and stunting) were considered positive samples.
T88 5863-5966 Sentence denotes The 50% embryo infectious dose (EID50) of these rIBVs was calculated using the Reed-Muench method [37].
T89 5968-5987 Sentence denotes 2.7 Safety testing
T90 5989-6051 Sentence denotes 2.7.1 Assessment of pathogenicity in one-day-old SPF chickens
T91 6052-6173 Sentence denotes The chickens were separately housed in isolators under consistent conditions, and food and water was provided ad libitum.
T92 6174-6272 Sentence denotes A total of 120 one-day-old SPF chickens were randomly divided into four groups (n = 30 per group).
T93 6273-6445 Sentence denotes The chickens in the experimental groups were intranasally inoculated with 200 μL allantoic fluid per chick containing 105EID50 of the rH120, rIBYZ, and rH120-S1/YZ strains.
T94 6446-6531 Sentence denotes The control group (n = 30) was inoculated with 200 μL sterile PBS via the same route.
T95 6532-6588 Sentence denotes The morbidity and mortality was followed-up for 14 days.
T96 6589-6760 Sentence denotes All experimental groups were monitored daily for clinical signs related to IB infection for 14 days post-infection (dpi), including coughing, sneezing, and tracheal rales.
T97 6761-6834 Sentence denotes Dead chickens were examined for gross tracheal, lung, and kidney lesions.
T98 6836-6890 Sentence denotes 2.7.2 Tissue tropism of early viral infection in vivo
T99 6891-6977 Sentence denotes A total of 100 1-day-old SPF chickens were assigned to four groups (n = 25 per group).
T100 6978-7240 Sentence denotes The birds were inoculated with 200 μL allantoic fluid containing 105 EID50 of the rH120, rIBYZ, or rH120-S1/YZ strains, respectively via eye drop and the intranasal route and 200 μL PBS per chick was administered to the control group (n = 25) via the same route.
T101 7241-7426 Sentence denotes The tracheas, lungs, kidneys, and bursa from the 5 inoculated birds per group were harvested at 1, 3, 5, and 7 dpi, weighed, and collected into 1 mL PBS per sample and frozen at −80 °C.
T102 7427-7633 Sentence denotes After grinding the samples, the viral RNA was extracted using TRIzol, and the cDNA was obtained by reverse transcription using a PrimeScript RT Master Mix Perfect Real Time Kit (TaKaRa, Otsu, Shiga, Japan).
T103 7634-7740 Sentence denotes The viral RNA copies from each of the different samples were detected by real-time PCR as described above.
T104 7741-7857 Sentence denotes All assays were run in triplicate and the copy number for each virus was calculated according to the standard curve.
T105 7859-7880 Sentence denotes 2.7.3 Histopathology
T106 7881-8133 Sentence denotes After inoculation with 105 EID50 dose of the rH120, rIBYZ, and rH120-S1/YZ strains, three infected chickens per group were randomly selected to be euthanized by bleeding, and the tissue pathology was examined in the different groups at 3, 5, 7, 10 dpi.
T107 8134-8358 Sentence denotes The tracheas, lungs, kidneys, and bursas of three dead or other randomly-selected chickens from the experimental and control groups (inoculated with PBS) were collected at 5 or 7 dpi and further processed for histopathology.
T108 8359-8480 Sentence denotes The samples were fixed in formalin, embedded in paraffin, cut into 5 μm sections, and stained with hematoxylin and eosin.
T109 8481-8557 Sentence denotes The slides were examined under light microscopy for the presence of lesions.
T110 8559-8580 Sentence denotes 2.7.4 Viral shedding
T111 8581-8795 Sentence denotes To determine the level of viral shedding of the different recombinants, 10 birds per group were inoculated with 200 μL allantoic fluid containing 105 EID50 of the rH120, rIBYZ, or rH120-S1/YZ strains, respectively.
T112 8796-8895 Sentence denotes 200 μL PBS per chick was administered to the control group via eye drop and the intranasal route. .
T113 8896-9017 Sentence denotes Oral and cloacal swabs were collected from each bird into 1 mL PBS at 7, 14, 18, 22, 26, and 30 dpi and stored at −80 °C.
T114 9018-9279 Sentence denotes After freezing and thawing three times and centrifuging at 8,000 × g for 5 min at 4 °C, relative amount of virus present in 200 μL of supernatants of each sample was quantified by RNA extraction, reverse transcription, and real-time PCR as described previously.
T115 9281-9321 Sentence denotes 2.8 Efficacy of rH120-S1/YZ vaccination
T116 9323-9345 Sentence denotes 2.8.1 Viral challenge
T117 9346-9430 Sentence denotes In total, 30 one-day-old SPF chickens were divided into three groups of 10 chickens.
T118 9431-9582 Sentence denotes The three groups were intranasally vaccinated with the rH120 and rH120-S1/YZ strains at a dose of 104 EID50/200 μL, and the control group received PBS.
T119 9583-9744 Sentence denotes Two weeks post-vaccination, the chickens in each group were challenged with the QX-like strain, rIBYZ, at 106 EID50/200 μL via eye drop and the intranasal route.
T120 9745-9844 Sentence denotes Any clinical signs, as well as the percentage of morbidity and mortality were recorded for 14 days.
T121 9845-9927 Sentence denotes The dead chickens were also examined for gross tracheal, lung, and kidney lesions.
T122 9929-10010 Sentence denotes 2.8.2 Antibody response measured by an enzyme-linked immunosorbent assay (ELISA)
T123 10011-10087 Sentence denotes 45 1-day-old SPF chickens were divided into three groups (n = 15 per group).
T124 10088-10347 Sentence denotes After vaccinating with the rH120 and rH120-S1/YZ strains at a dose of 104 EID50/200 μL, the sera of 10 birds from each group were randomly collected and antibodies were tested using an ELISA created in our lab at 7, 14, 21, and 28 days post-vaccination (dpv).
T125 10348-10521 Sentence denotes ELISA plates were coated with antigen of culture supernatants of inactivated IBV rMJ , a vero cells adaption strain domesticated from its parental strain of IBYZ in our lab.
T126 10522-10657 Sentence denotes Individual chicken sera collected from different groups diluted to 1:200 were loaded and the plates were incubated at 37 °C for 60 min.
T127 10658-10800 Sentence denotes IBV-specific IgG was detected with anti-chicken IgY (IgG) (whole molecule)-peroxidase antibodies produced in rabbits (Sigma-Aldrich, Germany).
T128 10801-11012 Sentence denotes After incubated with 3,3′,5,5′-Tetramethylbenzidine(TMB) substrate for 15 min and terminated with 2 mol/L H2SO4 solution, the absorbance at 490 nm was measured using a Model 680 microplate reader (Bio-Rad, USA).
T129 11014-11039 Sentence denotes 2.9 Statistical analysis
T130 11040-11148 Sentence denotes GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA, USA) was used for the statistical analyses.
T131 11149-11370 Sentence denotes For the antibody test, growth kinetics, tissue tropism, and viral shedding test, the collected data were analyzed using a two-way ANOVA to determine whether there was a significant difference between the different groups.
T132 11371-11526 Sentence denotes The significance was considered as follows: significant at P ≤ 0.05 (*); highly significant at P ≤ 0.01 (**); and extremely significant at P ≤ 0.001 (***).