LitCovid-sentences  

PMC:7115396 / 2182-43411 JSONTXT 12 Projects

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T16 0-15 Sentence denotes 1 Introduction
T17 16-267 Sentence denotes Infectious bronchitis (IB) was first described as a respiratory disease affecting chicks in the US in 1931 as an acute and highly contagious viral disease, and continues to cause major economic loss within the poultry industry worldwide [1], [2], [3].
T18 268-442 Sentence denotes As a result of the damage to the respiratory system, the clinical signs of this disease include depression, coughing, head-shaking, as well as nasal and ocular discharge [1].
T19 443-629 Sentence denotes Since the virus causing IB replicates in many non-respiratory epithelial surfaces (e.g., kidney and gonads), nephritis, reduced egg production, and egg quality is also observed [2], [3].
T20 630-850 Sentence denotes The causative agent of IB is infectious bronchitis virus (IBV), a non-segmented, positive-sense, single-stranded RNA virus, which belongs to the genus Gammacoronavirus, family Coronaviridae, in the order Nidovirales [4].
T21 851-1057 Sentence denotes The full length RNA genome of IBV is 27.6 kb in length and encodes non-structural proteins, four accessory proteins, and four structural proteins (spike [S], matrix [M], nucleocapsid [N], and envelope [E]).
T22 1058-1141 Sentence denotes The S protein is cleaved into S1 and S2 as two subunits at the S1/S2 cleavage site.
T23 1142-1294 Sentence denotes The S1 protein contains the receptor-binding domain and mediates viral attachment to host cells, whereas S2 is responsible for membrane fusion [5], [6].
T24 1295-1534 Sentence denotes Errors generated during the genomic replication of IBV and the selective pressure following the use of live attenuated vaccines or multiple infections with different IBV serotypes has led to the emergence of numerous IBV variants [7], [8].
T25 1535-1699 Sentence denotes In particular, small change as little as 5% in the amino acid composition of S1 gene may lead to alteration in cross protection among closely related serotypes [9].
T26 1700-1811 Sentence denotes In China, IBV was first observed during the early 1980s, and outbreaks have since been frequently reported [7].
T27 1812-2048 Sentence denotes The QX-like genotype is thought to have originated during the mid-1990s and continues to be the predominant strain, whereas the LDT3, 4/91, and Taiwan subtypes have also recently been frequently isolated in China [10], [11], [12], [13].
T28 2049-2248 Sentence denotes Severe nephritis, false layer syndrome, and high mortality are observed in chickens infected with this viral genotype, which represents a major problem in the poultry industry, particularly in China.
T29 2249-2393 Sentence denotes Using a live attenuated or inactivated vaccine is typically considered to be the most cost-effective means of controlling viral infections [14].
T30 2394-2744 Sentence denotes Unfortunately, inactivated IB vaccines are not effective if used alone, which could induce little or no protection against egg loss [15], [16], as well as impaired ciliary activity in the trachea [17]; thus, birds must continue to be given one or a series of vaccinations with live attenuated IB vaccines to provide broad heterologous protection [1].
T31 2745-3104 Sentence denotes Commercial attenuated live vaccines used against IBV in China include the H120, LDT3, and 4/91 strains [7]; however, phylogenetic analysis indicates that the QX-like genotype is genetically distant from the strains described above, which may explain the poor cross-protectivity against infection in chickens immunized with these classical vaccines [18], [19].
T32 3105-3210 Sentence denotes Recently, there has been increasing research on live attenuated vaccines against the QX-like IBV strains.
T33 3211-3469 Sentence denotes Different strains of QX-like IBVs were serially passaged in embryonated eggs or primary chicken kidney cells (CK) to obtain vaccines that are less virulent and exhibit high immunogenicity against the QX-like epidemic IBV strains [20], [21], [22], [23], [24].
T34 3470-3747 Sentence denotes However, in addition to causing tissue damage or secondary bacterial infections in young vaccinated chicks [25], [26], live attenuated vaccines of RNA viruses may not be genetically stable or they may be associated with the tendency to revert back to a virulent form [9], [14].
T35 3748-3879 Sentence denotes Moreover, the potential for recombination between vaccine and virulent strains can lead to the creation of new virulent virus [27].
T36 3880-4128 Sentence denotes Furthermore, it takes a substantial amount of time to prepare a extensively passaged attenuated vaccine strain, which may not be able to effectively solved the loss problem caused by the rapid variation exhibited by prevalent strains on the flocks.
T37 4129-4341 Sentence denotes In considering the limitations of the live attenuated and inactivated IBV vaccines, reverse genetic IBV vaccines have been developed in recent years as they display increased safety and efficacy [28], [29], [30].
T38 4342-4653 Sentence denotes As reverse genetic technology is primarily based on the H120 and Beaudette strains [31], [32], [33], both of which are passage-generated attenuated strains, the mechanism of virus virulence attenuation remains unclear, thereby posing difficulties in the development of attenuated vaccines using this technology.
T39 4654-4817 Sentence denotes In addition to the function of host invasion and increasing genetic diversity, the IBV S1 subunit can induce virus neutralizing and cross-reactive antibodies [34].
T40 4818-5062 Sentence denotes Therefore, some researchers have created an attenuated IBV vaccine by replacing the S1 or S genes from other virulent strains using reverse genetic technology to generate protection against strains of the corresponding serotype [5], [28], [30].
T41 5063-5345 Sentence denotes In the present study, we constructed a recombinant strain, using the reverse genetic system. rH120-S1/YZ is based on the backbone of the vaccine strain H120, and the S1 gene was replaced with that of the QX-like nephropathogenic strain, ck/CH/IBYZ/2011, which was isolated in China.
T42 5346-5489 Sentence denotes Our results demonstrate that this recombinant strain is safe and provides effective protection in young chickens against QX-like IBV challenge.
T43 5491-5515 Sentence denotes 2 Materials and methods
T44 5517-5541 Sentence denotes 2.1 Vaccine and viruses
T45 5542-5675 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 5676-5902 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 5903-6024 Sentence denotes Both of these strains were recovered from a full-length clone using reverse genetics, as previously described [35], [36].
T48 6026-6047 Sentence denotes 2.2 Eggs and animals
T49 6048-6360 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 6361-6510 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 6511-6622 Sentence denotes All chickens were maintained in isolators under negative pressure, and food and water were provided ad libitum.
T52 6624-6668 Sentence denotes 2.3 Construction of recombinant rH120-S1/YZ
T53 6669-6780 Sentence denotes The rIBV rH120-S1/YZ strain used in this study is described in the schematic illustration presented in Fig. 1 .
T54 6781-6912 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 6913-7027 Sentence denotes The cell supernatants were harvested 48 h following transfection and propagated in 10-day-old SPF chicken embryos.
T56 7028-7147 Sentence denotes The allantoic fluid was harvested for RT-PCR, whole genome sequencing, and was then stored at −80 °C until further use.
T57 7148-7380 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 7381-7547 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 7548-7716 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 7717-7880 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 7881-7966 Sentence denotes Ten cDNA fragments covering the entire genome of H120 strain was amplified by RT-PCR.
T62 7967-8169 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 8170-8254 Sentence denotes The S fragment in the pMDTM8 plasmid of H120 strain was replaced by chimeric S gene.
T64 8255-8411 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 8413-8444 Sentence denotes 2.4 Chicken embryo dwarf assay
T66 8445-8696 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 8697-8779 Sentence denotes Chick embryo lesions were examined for lesions at 144 h post-inoculation at 37 °C.
T68 8781-8820 Sentence denotes 2.5 Growth kinetics in chicken embryos
T69 8821-8983 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 8984-9095 Sentence denotes According to the sequences of IBV from GenBank, primers were designed based on conservative area in the 5′-UTR.
T71 9096-9206 Sentence denotes The upstream primer was 5′-CCGTTGCTTGGGCTACCTAGT-3′, and the downstream primer was 5′-CGCCTACCGCTAGATGAACC-3′.
T72 9207-9328 Sentence denotes The amplification product was cloned to pMD18-T vector (Takara) as a positive plasmid and its concentration was measured.
T73 9329-9436 Sentence denotes A gradient dilution of 5 × 102–5 × 108 copies/μL of the plasmid was used as template for quantitation test.
T74 9437-9549 Sentence denotes By plotting the cycle threshold (CT) values against the copies of the plasmid, the standard curve was generated.
T75 9550-9741 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 9742-9903 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 9904-10172 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 10173-10389 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 10390-10465 Sentence denotes The standard curve was plotted against the log of the template copy number.
T80 10466-10588 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 10590-10624 Sentence denotes 2.6 Titration of rIBVs in embryos
T82 10625-10868 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 10869-10984 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 10985-11020 Sentence denotes PBS was used as a negative control.
T85 11021-11112 Sentence denotes After a 24 h incubation, the dead embryos were considered nonspecific deaths and discarded.
T86 11113-11221 Sentence denotes The embryos were examined for the presence of specific lesions caused by the virus after a 144-h incubation.
T87 11222-11353 Sentence denotes Dead and live embryos that displayed IBV infectious signs (e.g., dwarfing, curling, and stunting) were considered positive samples.
T88 11354-11457 Sentence denotes The 50% embryo infectious dose (EID50) of these rIBVs was calculated using the Reed-Muench method [37].
T89 11459-11478 Sentence denotes 2.7 Safety testing
T90 11480-11542 Sentence denotes 2.7.1 Assessment of pathogenicity in one-day-old SPF chickens
T91 11543-11664 Sentence denotes The chickens were separately housed in isolators under consistent conditions, and food and water was provided ad libitum.
T92 11665-11763 Sentence denotes A total of 120 one-day-old SPF chickens were randomly divided into four groups (n = 30 per group).
T93 11764-11936 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 11937-12022 Sentence denotes The control group (n = 30) was inoculated with 200 μL sterile PBS via the same route.
T95 12023-12079 Sentence denotes The morbidity and mortality was followed-up for 14 days.
T96 12080-12251 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 12252-12325 Sentence denotes Dead chickens were examined for gross tracheal, lung, and kidney lesions.
T98 12327-12381 Sentence denotes 2.7.2 Tissue tropism of early viral infection in vivo
T99 12382-12468 Sentence denotes A total of 100 1-day-old SPF chickens were assigned to four groups (n = 25 per group).
T100 12469-12731 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 12732-12917 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 12918-13124 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 13125-13231 Sentence denotes The viral RNA copies from each of the different samples were detected by real-time PCR as described above.
T104 13232-13348 Sentence denotes All assays were run in triplicate and the copy number for each virus was calculated according to the standard curve.
T105 13350-13371 Sentence denotes 2.7.3 Histopathology
T106 13372-13624 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 13625-13849 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 13850-13971 Sentence denotes The samples were fixed in formalin, embedded in paraffin, cut into 5 μm sections, and stained with hematoxylin and eosin.
T109 13972-14048 Sentence denotes The slides were examined under light microscopy for the presence of lesions.
T110 14050-14071 Sentence denotes 2.7.4 Viral shedding
T111 14072-14286 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 14287-14386 Sentence denotes 200 μL PBS per chick was administered to the control group via eye drop and the intranasal route. .
T113 14387-14508 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 14509-14770 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 14772-14812 Sentence denotes 2.8 Efficacy of rH120-S1/YZ vaccination
T116 14814-14836 Sentence denotes 2.8.1 Viral challenge
T117 14837-14921 Sentence denotes In total, 30 one-day-old SPF chickens were divided into three groups of 10 chickens.
T118 14922-15073 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 15074-15235 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 15236-15335 Sentence denotes Any clinical signs, as well as the percentage of morbidity and mortality were recorded for 14 days.
T121 15336-15418 Sentence denotes The dead chickens were also examined for gross tracheal, lung, and kidney lesions.
T122 15420-15501 Sentence denotes 2.8.2 Antibody response measured by an enzyme-linked immunosorbent assay (ELISA)
T123 15502-15578 Sentence denotes 45 1-day-old SPF chickens were divided into three groups (n = 15 per group).
T124 15579-15838 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 15839-16012 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 16013-16148 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 16149-16291 Sentence denotes IBV-specific IgG was detected with anti-chicken IgY (IgG) (whole molecule)-peroxidase antibodies produced in rabbits (Sigma-Aldrich, Germany).
T128 16292-16503 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 16505-16530 Sentence denotes 2.9 Statistical analysis
T130 16531-16639 Sentence denotes GraphPad Prism 7 software (GraphPad Software Inc., La Jolla, CA, USA) was used for the statistical analyses.
T131 16640-16861 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 16862-17017 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 (***).
T133 17019-17029 Sentence denotes 3 Results
T134 17031-17078 Sentence denotes 3.1 Characterization of the rH120-S1/YZ strain
T135 17079-17312 Sentence denotes After being inoculated with the recombinant strains, typical IB lesions in the chick embryos from each group were observed, including amniotic membrane thickening, dwarfing, stunted growth, curling, or death of the embryo (Fig. 2 A).
T136 17313-17387 Sentence denotes The EID50 of the virus was calculated according to the Reed-Muench method.
T137 17388-17541 Sentence denotes The titers of the rH120, rIBYZ, and rH120-S1/YZ strains were determined to be 107.3 EID50/mL, 106.8 EID50/mL, and 108.4 EID50/mL, respectively (Fig. 2B).
T138 17542-17681 Sentence denotes Fig. 2 The biological characteristics of the rIBVs in SPF chicken embryos. (A) Pathogenicity of recombinant IBVs in infected chick embryos.
T139 17682-17811 Sentence denotes Five 10-old-day embryonated SPF chicken eggs per group were inoculated with 0.2 mL virus diluent of rH120, rIBYZ, or rH120-S1/YZ.
T140 17812-18103 Sentence denotes Chick embryo lesions were observed at 144 h post-inoculation at 37 °C. (B) EID50 of the recombinant IBVs, rH120, rIBYZ, and rH120-S1/YZ in SPF chicken embryonated eggs. (C) The multicycle growth kinetics of the recombinant IBVs, rH120, rIBYZ, and rH120-S1/YZ in SPF chicken embryonated eggs.
T141 18104-18165 Sentence denotes All data are presented as the mean ± standard deviation (SD).
T142 18166-18214 Sentence denotes Some of the error bars are too small to be seen.
T143 18215-18265 Sentence denotes The qRT-PCR was carried out with three replicates.
T144 18266-18553 Sentence denotes Markers of statistical difference were acquired by comparing between rH120 group and rH120-S1/YZ group. ***Indicates an extremely significant difference at P ≤ 0.001; **Indicates a highly significant difference at 0.001 < P ≤ 0.01; *Indicates a significant difference at 0.01 < P ≤ 0.05.
T145 18554-18675 Sentence denotes The growth kinetics of the rIBVs were assessed by inoculation with 107 copies of virus per egg in 6 eggs from each group.
T146 18676-18801 Sentence denotes The relative viral load was determined at 12, 18, 24, 36, 48, 72, and 96 h post-inoculation by quantitative real-time RT-PCR.
T147 18802-19056 Sentence denotes The growth curves of all three strains reached a relatively stable plateau at 36–48 h after infection; the level of viral RNA for the rH120-S1/YZ strain was significantly higher than that of the rIBYZ strain for the majority of the time points (Fig. 2C).
T148 19058-19069 Sentence denotes 3.2 Safety
T149 19071-19139 Sentence denotes 3.2.1 Pathogenicity of the recombinants in one-day-old SPF chickens
T150 19140-19334 Sentence denotes The earliest death was observed at 3 dpi in the QX-like strain rIBYZ group, which virus was highly pathogenic to one-day-old SPF chickens; the final mortality of this group was 63.3% (Fig. 3 A).
T151 19335-19647 Sentence denotes The morbidity of the infected chickens in the rIBYZ group reached higher than 80%, and the diseased chickens exhibited respiratory symptoms (e.g., sneezing, coughing, as well as tracheal and bronchiolar rales), and the severe cases presented with additional signs of listlessness, huddling, and ruffled feathers.
T152 19648-19841 Sentence denotes At necropsy, lesions were detected both in the respiratory and urinary system, including mucus, hyperemia, and hemorrhage in the trachea, as well as swelling and urate deposition in the kidney.
T153 19842-19916 Sentence denotes Edema and congestion were observed in the lungs in 20% of cases (Fig. 3B).
T154 19917-20110 Sentence denotes However, following inoculation with the rH120 or rH120-S1/YZ strains, the morbidity rates of the chickens was less than 20%, with moderate respiratory signs of coughing in some of the chickens.
T155 20111-20286 Sentence denotes All of the infected chickens presented no visible lesions in the kidney and the survival rate was 100% through 14 dpi in both the rH120 and rH120-S1/YZ groups (Fig. 3A and B).
T156 20287-20392 Sentence denotes No obvious clinical signs, gross lesions, or death attributable to IBV was observed in the control group.
T157 20393-20564 Sentence denotes Fig. 3 Pathogenicity of the rIBV rH120, rIBYZ, and rH120-S1/YZ strains. (A) Percent survival of SPF chickens infected at 1-day-of age during the 14-day observation period.
T158 20565-20719 Sentence denotes No mortality was observed in rH120, rH120-S1/YZ, or the control groups. (B) Gross lesions on the lungs and kidneys of the infected chickens at 5 or 7 dpi.
T159 20721-20744 Sentence denotes 3.2.2 Tissue pathology
T160 20745-20960 Sentence denotes For the chickens inoculated with the QX-like strain rIBYZ, lesions in the trachea at 5 dpi were characterized as cilia necrosis and loss, epithelial cell congestion, hemorrhage, and lymphoid infiltration (Fig. 4 C).
T161 20961-21166 Sentence denotes In the bronchial and capillary lumen of rIBYZ-infected lungs, congestion, hemorrhage, as well as erythrocyte and inflammatory cell infiltration was observed in some severe cases from 7 to 10 dpi (Fig. 4G).
T162 21167-21312 Sentence denotes Moreover, erythrocyte and lymphocyte infiltration, as well as tubular dilation was observed in the kidney of rIBYZ-inoculated chickens (Fig. 4K).
T163 21313-21470 Sentence denotes Similar to the rH120 group, chickens inoculated with rH120-S1/YZ presented with mild pathology in the trachea at the early infection time points of 3, 5 dpi.
T164 21471-21629 Sentence denotes Lesions in the trachea consisted of the loss of epithelial cells and cilia, decreased number of secretory cells, and thickening of the mucosa (Fig. 4B and D).
T165 21630-21792 Sentence denotes In contrast, there were no lesions observed in the lungs and kidneys of rH120 and rH120-S1/YZ-infected chickens during the infection period (Fig. F, H, J, and L).
T166 21793-21985 Sentence denotes No histopathological evidence of damage was detected in the bursa of all the groups and no IBV-associated lesions were found in the tissue samples of the control chickens (Fig. 4A, E, I, M–P).
T167 21986-22161 Sentence denotes Fig. 4 Histopathological changes in the trachea, lungs, kidneys, and bursa of SPF chickens infected with the recombinant rH120, rIBYZ, and rH120-S1/YZ strains at 1-day-of-age.
T168 22162-22223 Sentence denotes Black arrows in the trachea indicate cilia and necrosis loss.
T169 22224-22285 Sentence denotes Black arrows in the lungs indicate congestion and hemorrhage.
T170 22286-22451 Sentence denotes Trachea samples were collected at 3, 5 dpi (A–D); lung and bursa samples were collected at 7, 10 dpi (E–H; M–P); and kidney samples were collected at 5, 7 dpi (I–L).
T171 22452-22532 Sentence denotes Black arrows in kidneys indicate tubular dilation, congestion, and inflammation.
T172 22533-22562 Sentence denotes Scale bar = 100 μm or 200 μm.
T173 22564-22615 Sentence denotes 3.2.3 Growth in tracheal, lung, and kidney tissues
T174 22616-22793 Sentence denotes The time-dependent levels of viral replication in the different tissues determined by real-time RT-PCR presented in Fig. 5 are consistent with the tissue histopathology results.
T175 22794-22977 Sentence denotes Compared to rH120 and rH120-S1/YZ groups, one day after inoculation with the QX-like strain, rIBYZ, the viral load in all organs began to rise rapidly, except at 5 dpi in the trachea.
T176 22978-23068 Sentence denotes The viral load in tracheal samples in the rH120-S1/YZ group increased and peaked at 5 dpi.
T177 23069-23171 Sentence denotes The level of viral RNA was significantly lower than the rH120 group both at 1 dpi and 7 dpi (Fig. 5A).
T178 23172-23394 Sentence denotes The viral load in lung samples from all infection groups increased starting at 3 dpi, and the viral RNA in the lungs of the rH120-S1/YZ group was significantly lower compared with both the rH120 and rIBYZ groups (Fig. 5B).
T179 23395-23475 Sentence denotes The viral load in the kidney of all IBV-inoculated groups was detected at 1 dpi.
T180 23476-23652 Sentence denotes In contrast to the rH120 and rH120-S1/YZ groups, the viral growth in kidney of rIBYZ-infected chickens exhibited an extremely significant increase at 3 dpi and peaked at 5 dpi.
T181 23653-23763 Sentence denotes The viral RNA copies of the rH120-S1/YZ group peaked at 1 dpi, after which they gradually decreased (Fig. 5C).
T182 23764-23840 Sentence denotes No viral load was detected in the control samples at any of the time points.
T183 23841-24005 Sentence denotes Fig. 5 Viral load in the different organs at 1, 3, 5, and 7 dpi of SPF chickens infected with the recombinant strains rH120, rIBYZ, and rH120-S1/YZ at 1-day-of age.
T184 24006-24236 Sentence denotes The bars indicate means ± standard deviations. ***Indicates an extremely significant difference at P ≤ 0.001; **Indicates a highly significant difference at 0.001 < P ≤ 0.01; *Indicates a significant difference at 0.01 < P ≤ 0.05.
T185 24238-24285 Sentence denotes 3.2.4 Viral load in the oral and cloacal swabs
T186 24286-24421 Sentence denotes In all IBV infection groups, the viral load in both the oral and cloacal swabs generally showed a gradual decline from 7 dpi to 26 dpi.
T187 24422-24762 Sentence denotes Compared with the rH120 group, The reduction in viral load between 14 or 18 dpi was significantly greater for chickens infected with rH120-S1/YZ than for chickens infected with rH120, and the viral RNA of the QX-like strain rIBYZ was lower than that of the rH120 group at 7, 14, and 18 dpi but with no statistically significance (Fig. 6 A).
T188 24763-24910 Sentence denotes In contrast, the viral load in the cloacal swab of the rIBYZ group was significantly higher than that of the rH120 and rH120-S1/YZ groups at 7 dpi.
T189 24911-25053 Sentence denotes Compared with other groups, the viral RNA in the cloacal swab of the rH120-S1/YZ group was significantly lower at 7, 14, and 22 dpi (Fig. 6B).
T190 25054-25115 Sentence denotes No virus was detected in the control group at any time point.
T191 25116-25274 Sentence denotes Fig. 6 Viral loads at 7, 14, 18, 22, and 26 dpi in the oral and cloacal swabs of chickens infected with the recombinant rH120, rIBYZ, and rH120-S1/YZ strains.
T192 25275-25527 Sentence denotes All data are presented as the mean ± standard error of the mean (SEM); ***Indicates an extremely significant difference at P ≤ 0.001; **Indicates a highly significant difference at 0.001 < P ≤ 0.01; *Indicates significant difference at 0.01 < P ≤ 0.05.
T193 25529-25550 Sentence denotes 3.3 Vaccine efficacy
T194 25552-25639 Sentence denotes 3.3.1 Efficacy of the different vaccines against QX-like IBV challenge in SPF chickens
T195 25640-25741 Sentence denotes The rH120-S1/YZ group exhibited effective protection against challenge with the QX-like strain rIBYZ.
T196 25742-25886 Sentence denotes The mortality rate in the rH120-S1/YZ group was 0%, and no clinical signs or lesions were observed during the rIBYZ challenge period (Fig. 7 A).
T197 25887-26197 Sentence denotes In contrast, chickens vaccinated with the rH120 strain began to exhibit clinical signs (e.g., coughing and nasal discharge) at 3 days post-challenge (dpc), and some chickens showed severe signs of listlessness, huddling, and ruffled feathers, with the mortality rate reaching 50% during the observation period.
T198 26198-26335 Sentence denotes At necropsy, the dead animals displayed typical kidney lesions characterized by swelling and urate deposition in the tubules and ureters.
T199 26336-26597 Sentence denotes In contrast to chickens vaccinated with rH120-S1/YZ, the chickens in the unvaccinated control group exhibited clinical signs at 3 dpc, and a 60% mortality rate; the gross lesions of the dead birds in this group were similar to that of the rH120 group (Fig. 7B).
T200 26598-26783 Sentence denotes Fig. 7 Efficacy of the rH120 and rH120-S1/YZ vaccines. (A) Percent survival of the vaccinated chickens challenged with the QX-like IBV rIBYZ strain during the 14-day observation period.
T201 26784-27039 Sentence denotes No mortality was observed in the chickens vaccinated with the rH120-S1/YZ strain. (B) Gross lesions observed on the kidneys of the vaccinated chickens challenged with the rIBYZ strain at 5 or 7 dpc. (C) The mean antibody OD value at 7, 14, 21, and 28 dpv.
T202 27040-27110 Sentence denotes All data are presented as the mean ± standard error of the mean (SEM).
T203 27112-27136 Sentence denotes 3.3.2 Antibody response
T204 27137-27204 Sentence denotes Antibodies against the IBVs were measured using an ELISA (Fig. 7C).
T205 27205-27452 Sentence denotes None of the birds in the rH120 or rH120-S1/YZ groups showed a positive antibody response at 7 dpv, whereas 100% of the birds had an antibody response detectable at 14 dpv, with the antibody level gradually increasing during the observation period.
T206 27453-27601 Sentence denotes However, there was no significant difference in the level of serum antibodies between rH120-vaccinated chickens and rH120-S1/YZ-vaccinated chickens.
T207 27602-27682 Sentence denotes No positive serum antibody response was detected in the non-vaccinated controls.
T208 27684-27697 Sentence denotes 4 Discussion
T209 27698-27943 Sentence denotes Vaccines, particularly live-attenuated vaccines, remain the most effective means of protection against IBV challenge (e.g., the Mass serotype strains H120, H52, Ma5, and W93, which are still widely used in the poultry industry) [38], [39], [40].
T210 27944-28162 Sentence denotes However, due to the poor cross-protection provided between the different serotypes, these attenuated serotype-specific vaccines cannot provide complete protection, and the IB endemic in China cannot be controlled [22].
T211 28163-28323 Sentence denotes Genetic mutations and immune pressure during the replication process of the single stranded RNA virus often results in the emergence of IBV variants [41], [42].
T212 28324-28501 Sentence denotes To date, at least six genotypes of IBV strains have been identified in China, of which the QX-type (first isolated in 1999) remains the most prevalent genotype [10], [42], [43].
T213 28502-28770 Sentence denotes The poor relationship between the QX-type strains, which are abundant and display variable virulence in various parts of China, and the Mass-type vaccine strains could explain the failure of the Mass-type vaccination programs to control IBV in these flocks [19], [39].
T214 28771-28913 Sentence denotes Consequently, the development of novel vaccines against circulating IBV strains in China using local IBV strains is required [20], [43], [44].
T215 28914-29074 Sentence denotes Optimal vaccination against circulating IBV strains in China requires the development of attenuated vaccines designed from local strains [20], [21], [22], [45].
T216 29075-29418 Sentence denotes However, due to the particularity of RNA virus replication, attenuated vaccine strains generated by continuous passage remain associated with the risk of reversion to virulence or potential recombination between vaccine strains and virulent field strains [27], [46], [47], which represents a substantial hidden concern to the poultry industry.
T217 29419-29700 Sentence denotes To reduce the problems associated with vaccine reversion, researchers have explored the option of creating vaccine viruses using reverse genetic technology (e.g., Beaudette strains carrying the S1 gene of the H120 vaccine strain, virulent M41 strain, or QX-like strain) [30], [48].
T218 29701-29885 Sentence denotes However, the titer of the Beaudette strains carrying the S1 gene of the vaccine H120 strain only reaches 106.13 ± 0.23 EID50 [49], which is lower than that of the H120 backbone strain.
T219 29886-30127 Sentence denotes Therefore, we aimed to develop a recombinant rH120-S1/YZ strain based on the H120 vaccine strain that carries the S1 gene of the ck/CH/IBYZ/2011 strain (a Chinese QX-like nephoropathogenic strain) using reverse genetic technology [35], [36].
T220 30128-30204 Sentence denotes In the present study, we isolated a nephoropathogenic strain (IBYZ) in 2011.
T221 30205-30342 Sentence denotes This strain was isolated, identified, and preserved from a large-scale chicken farm where an outbreak of IBV emerged in Jiangsu province.
T222 30343-30446 Sentence denotes Chickens infected with the IBYZ strain showed severe kidney damage and a high mortality rate (Fig. 3B).
T223 30447-30551 Sentence denotes The complete sequence has been deposited in the GenBank database under the accession number of KF663561.
T224 30552-30770 Sentence denotes The S1 gene sequencing data indicated that the IBYZ strain belonged to the QX-like genotype (Fig. 8 ), with a nucleotide homology with the attenuated vaccine H120 strain of only 77.4%, and amino acid homology of 77.1%.
T225 30771-30880 Sentence denotes Moreover, the S1/S2 cleavage site of IBYZ is HRRRR/S, which differed from RRFRR/S of the H120 vaccine strain.
T226 30881-30999 Sentence denotes After whole-genome sequencing, we constructed a molecular clone strain using reverse genetic technology, termed rIBYZ.
T227 31000-31172 Sentence denotes In addition, another strain used in this study is the live attenuated IBV vaccine, H120, and the molecular clone strain, rH120, which was constructed using the same method.
T228 31173-31344 Sentence denotes Fig. 8 Phylogenetic tree constructed based on the S1 gene of isolate ck/CH/IBYZ/2011 and 28 published IBV reference strains using the neighbor-joining method (Mega10.1.6).
T229 31345-31477 Sentence denotes The spike protein is the coronavirus structural proteins, which plays an important role on pathogenicity in coronaviruses [6], [50].
T230 31478-31589 Sentence denotes However, replacing the S gene of Beaudette strain by the virulent strains does not make it virulent [51], [52].
T231 31590-31750 Sentence denotes The S protein can be cleaved into S1 and S2 subunits at the S1/S2 cleavage site [53], the S1 subunit is responsible for receptor binding to the host cells [54].
T232 31751-31826 Sentence denotes Whether the S1 gene has an effect on the virulence of the virus is unknown.
T233 31827-32046 Sentence denotes In this study, the recombinant strain, rH120-S1/YZ, expressed the S1 gene of the QX-like strain, which retains the S1/S2 cleavage site of H120, maintained the ability to replicate and exhibited pathogenicity in embryos.
T234 32047-32140 Sentence denotes However, providing IBYZ S1 sequences to H120 did not increase its virulence to that of rIBYZ.
T235 32141-32270 Sentence denotes IBVs replicate in many epithelial cells, including respiratory tissues, the alimentary canal, kidney, gonads, and bursa [2], [6].
T236 32271-32400 Sentence denotes The QX-like rIBYZ strain is capable of considerable growth in the trachea, lung, and kidney, which leads to severe tissue damage.
T237 32401-32621 Sentence denotes In addition, this strain caused cilia necrosis and loss, lung congestion and hemorrhage, nephritis, as well as inflammatory cell infiltration in these tissues in infected chickens at different time points post-infection.
T238 32622-32895 Sentence denotes The ability of IBV to replicate within many respiratory, enteric, and other epithelial surfaces may be partially related to the fact that the attachment of IBV to host cells is dependent on sialic acid on the cell surface being recognized by the S1 subunit [6], [54], [55].
T239 32896-33031 Sentence denotes However, the binding of virus to the host cell is the first step in determining tropism and S2 is responsible for membrane fusion [56].
T240 33032-33213 Sentence denotes Thus, to further explore the different tissue tropism between rH120-S1/YZ and rIBYZ, we examined the viral loads and histopathology in the tissues of the rH120-S1/YZ-infected group.
T241 33214-33487 Sentence denotes It was found that rH120-S1/YZ remains moderately pathogenic in the trachea (e.g., some cilia necrosis and loss and decreased secretory cells) but no lesions were observed in the lungs and kidneys of infected chickens, in contrast to chickens infected with the rIBYZ strain.
T242 33488-33626 Sentence denotes This finding suggests that both the S2 and S1 genes might play an important role in viral tissue tropism as also suggested by others [57].
T243 33627-33822 Sentence denotes Another possibility is that the lack of viral replication and damage in kidney following rH120-S1/YZ is due to lower replication ability of backbone except S2 gene from its parental rH120 strain.
T244 33823-34171 Sentence denotes The viral load of the respiratory-type strain, rH120 in the different tissues indicated that the viral levels in the trachea, early following infection were significantly higher than those of rIBYZ, which indicates that the ability of the virus to attached to trachea epithelial cells of the rH120 strain was stronger than that of the rIBYZ strain.
T245 34172-34543 Sentence denotes However, during the later time points post-infection, the level of the rH120 strain was significantly lower than rIBYZ, which suggests that the virulence of IBV depends both on tissue invasion and viral replication ability, as well as other functions mediated by other viral proteins (e.g., non-structural, structural, and accessory proteins) [6], [29], [58], [59], [60].
T246 34544-34704 Sentence denotes The reduction of the viral load at 5 dpi in the group infected with the rIBYZ strain may be associated with the necrosis and shedding of the trachea epithelium.
T247 34705-35174 Sentence denotes Moreover, the ability to replication in the different organs of chickens infected with rH120-S1/YZ as reflected by viral loads at multiple time points was lower than that exhibited in the chickens infected with the rIBYZ strain, which demonstrated that the S1 subunit was not the only determinant of viral tropism that was consistent with results by others [57], [61], [62], and the significant difference in viral load was related to differences in the viral backbone.
T248 35175-35612 Sentence denotes However, when comparing the rH120-S1/YZ and rH120 strains which carry the same backbone, it was found that the viral RNA of rH120-S1/YZ in the lungs and kidneys was significantly lower than that of rH120, which may due to the replacement of the IBYZ S1 gene in the recombinant virus, whose structure on the surface contains some degree of changes, which we speculated that may affects the functionality of the S protein of coronaviruses.
T249 35613-35769 Sentence denotes Although cryo-EM structure of IBV spike protein has been determined, the relation between structure and function of different IBV S1 genes is unknown. [63].
T250 35770-36026 Sentence denotes In addition to the growth in epithelial tissues observed during the early infection period, IBV can establish long-term persistent infections in chicken flocks via oral and cloacal shedding, which represents a substantial challenge to IBV control [1], [6].
T251 36027-36172 Sentence denotes In this study, we detected viral RNA in the oral and cloacal swabs isolated from infected chickens at 7, 14, 18, 22, and 26 dpi by real-time PCR.
T252 36173-36348 Sentence denotes Compared with rH120 and rIBYZ, the viral load in both the oral and cloacal swabs of the rH120-S1/YZ group at different time points after infection was significantly decreased.
T253 36349-36550 Sentence denotes However, while it has been reported that the persistent virus will be re-excreted at the point of lay [1], the persistence of rH120-S1/YZ strain in the egg-laying chickens needs to be further verified.
T254 36551-36776 Sentence denotes In a word, due to its low virulence i presented in virus loading and lesion in different tissue, oral and cloacal shedding of host animals, rH120-S1/YZ can be considered a safe attenuated vaccine candidate for young chickens.
T255 36777-37029 Sentence denotes In addition to its role in tissue tropism, the S1 glycoprotein also has an important function in inducing a neutralizing antibody response [64], [65], and small differences in the S1 contribute to poor cross protectionby neutralization test [64], [66].
T256 37030-37240 Sentence denotes To evaluate the protective efficacy of this candidate vaccine, we selected the QX-like virulent rIBYZ strain to infect chickens at 14 dpv that were vaccinated with either the rH120-S1/YZ strain or rH120 strain.
T257 37241-37429 Sentence denotes We found that little protection against rIBYZ virulent strain challenge in the rH120 vaccinated group was induced, with a 50% survival rate and severe clinical symptoms associated with IB.
T258 37430-37521 Sentence denotes This finding could be explained by differences in the antigenicity between rH120 and rIBYZ.
T259 37522-37715 Sentence denotes No death, clinical signs, or lesions were observed in the rH120-S1/YZ-vaccinated group, indicating that this strain could provide effective protection against rIBYZ challenge in young chickens.
T260 37716-37966 Sentence denotes Since high humoral antibody titers can contribute to reduced viral replication in various organs and disease recovery [67], [68], the level of specific IgG antibodies remains an important standard for evaluating the immune response to an IBV vaccine.
T261 37967-38139 Sentence denotes In the present study, the humoral antibody level of chickens vaccinated with the rH120-S1/YZ strain was observed to gradually increase compared to the non-vaccinated group.
T262 38140-38480 Sentence denotes However, although the rH120 strain was able to induce similar levels of humoral antibodies, it provided poor protection against rIBYZ, suggesting that protection against IBV infection is not only with humoral immunity, but also with both mucosal immunity combined with the local tracheal and cellular immune response [69], [70], [71], [72].
T263 38481-38597 Sentence denotes Thus, the detection of mucosal and cellular immunity in rH120-S1/YZ-vaccinated animals requires further exploration.
T264 38598-38851 Sentence denotes Nevertheless, some studies have shown that a peptide located near the amino terminal end of S2 can be recognized by neutralizing monoclonal antibodies [73], and the S2 domain also plays an important role in inducing protective immunity [28], [61], [74].
T265 38852-39148 Sentence denotes Because the S2 domain plays an important role in cell tropism [57], we also considered that expressing the S1 subunit would provide greater safety than the entire S gene, which could lead to further destruction in some organs of the vaccinated chickens; however, further experiments are required.
T266 39150-39163 Sentence denotes 5 Conclusion
T267 39164-39436 Sentence denotes In conclusion, we constructed the recombinant strain, rH120-S1/YZ, which was based on the backbone of the H120 vaccine strain, and replaced the S1 gene with that of the QX-like nephropathogenic strain, ck/CH/IBYZ/2011, isolated from China using the reverse genetic system.
T268 39437-39642 Sentence denotes The safety results showed that this recombinant strain was not lethal to one-day-old chicks and it had not gained the ability to tissue invasion or replication in the kidneys and lungs of infected animals.
T269 39643-39754 Sentence denotes Moreover, the viral load of the chickens was significantly decreased compared with the rH120 and rIBYZ strains.
T270 39755-39899 Sentence denotes Furthermore, rH120-S1/YZ was found to provide effective protection against challenge with the QX-like nephropathogenic strain in young chickens.
T271 39900-40025 Sentence denotes The level of antibody production in the serum of the vaccinated chickens detected by ELISA continued to increase after 7 dpi.
T272 40026-40167 Sentence denotes Thus, rH120-S1/YZ may be considered a potential live vaccine candidate for protection against Chinese QX-like nephropathogenic IBV infection.
T273 40169-40188 Sentence denotes 6 Ethics statement
T274 40189-40476 Sentence denotes All of the animals were cared for in accordance with the animal ethics guidelines and approved protocols, and the experimental protocols were performed with the approval of the Animal Welfare and Ethical Censor Committee of the Poultry Institute, Chinese Academy of Agriculture Sciences.
T275 40478-40518 Sentence denotes CRediT authorship contribution statement
T276 40519-40528 Sentence denotes Yi Jiang:
T277 40529-40713 Sentence denotes Conceptualization, Methodology, Software, Investigation, Validation, Visualization, Resources, Writing - original draft, Writing - review & editing, Visualization, Funding acquisition.
T278 40714-40723 Sentence denotes Xu Cheng:
T279 40724-40753 Sentence denotes Investigation, Data curation.
T280 40754-40766 Sentence denotes Xiumei Zhao:
T281 40767-40796 Sentence denotes Visualization, Investigation.
T282 40797-40804 Sentence denotes Yan Yu:
T283 40805-40829 Sentence denotes Investigation, Software.
T284 40830-40842 Sentence denotes Mingyan Gao:
T285 40843-40857 Sentence denotes Investigation.
T286 40858-40869 Sentence denotes Sheng Zhou:
T287 40870-41023 Sentence denotes Conceptualization, Validation, Resources, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition.
T288 41025-41058 Sentence denotes Declaration of Competing Interest
T289 41059-41229 Sentence denotes The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.