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Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos
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Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos Qingzhong Yu ⇑, Yufeng Li 1, Kiril Dimitrov, Claudio L. Afonso, Stephen Spatz, Laszlo Zsak Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, United States Department of Agriculture, 934 College Station Road, Athens, GA 30605, USA
a r t i c l e
i n f o
Article history: Received 22 May 2019 Received in revised form 17 October 2019 Accepted 25 October 2019 Available online xxxx Keywords: NDV vector ILTV-gD Recombinant vaccine Genetic stability Next-generation sequencing
a b s t r a c t Previously, we have demonstrated that the recombinant Newcastle disease virus (NDV) expressing the infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) conferred protection against both virulent NDV and ILTV challenges in chickens. In this study, we evaluated the genetic stability of the recombinant vaccine after eight serial passages in embryonated chicken eggs (ECE). The vaccine master seed virus at the original egg-passage level 3 (EP3) was diluted and passaged in three separate repetitions (A, B and C) in ECE eight times (EP4 to EP11). RT-PCR analysis of the vaccine seed and egg-passaged virus stocks showed that there was no detectable insertion/deletion in the ILTV gD insert region. Next-generation sequencing analysis of the EP3 and EP11 virus stocks confirmed their genome integrity and revealed a total of thirteen single-nucleotide polymorphisms (SNPs). However, none of these SNPs were located in the ILTV gD insert or any of the known critical biological determinant positions. Virological and immunofluorescent assays provided additional evidence that the EP11 virus stocks retained their growth kinetics, low pathogenicity, and robust level of gD expression comparable to that of the vaccine master seed virus. This indicated that the SNPs were non-detrimental sporadic mutations. These results demonstrated that the insertion of ILTV gD gene into the NDV LaSota backbone did not significantly affect the genetic stability of the recombinant virus and that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least eight serial passages in ECE. Ó 2019 Published by Elsevier Ltd.
1. Introduction Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by Gallid alphaherpesvirus 1, a member of the genus Iltovirus within the family Herpseviridae, and commonly known as infectious laryngotracheitis virus (ILTV, used hereafter) [1]. The disease is mainly controlled through biosecurity and by vaccination with live-attenuated vaccines [2]. Newcastle disease (ND) is another serious infectious respiratory disease of poultry caused by virulent strains of Avian orthoavulavirus 1, a member of the family Paramyxoviridae (https://talk. ictvonline.org/taxonomy/), and commonly known as Newcastle disease virus (NDV, used hereafter) [3]. Vaccination, combined with strict biosecurity practices, has been the recommended strategy for controlling ND outbreaks [4].
⇑ Corresponding author. E-mail address: [email protected] (Q. Yu). Current address: Shandong Poultry Research Institute, Jinan, Shandong 250023, China 1
The commonly used live-attenuated vaccines for ILT were developed by either multiple passages of ILTV strains in embryonated eggs (chicken embryo origin [CEO]) or in tissue culture (tissue culture origin [TCO]) [5,6]. Although these vaccines protect against clinical disease, they have residual virulence which is exacerbated by continued infections of naïve birds from productively infected animals and latent carriers [7–9]. Moreover, the CEO vaccine strain has been demonstrated to mutate and become more virulent simply by bird-to-bird passage [10]. In high-density poultry rearing facilities, there is a continuous reservoir of viruses, both virulent and vaccinal, evolving to higher levels of virulence. These ‘‘revertants” have become the dominant field strains in poultry populations and are the cause of field ILT outbreaks [11,12]. To overcome these problems associated with live attenuated ILTV vaccine strains, we have developed an NDV LaSota vaccine strainbased recombinant virus expressing the ILTV gD protein as a bivalent vaccine and demonstrated that vaccination of chickens with the recombinant vaccine, rLS/ILTV-gD, confers complete protection against virulent NDV challenge and significant protection against pathogenic ILTV challenge [13,14].
https://doi.org/10.1016/j.vaccine.2019.10.074 0264-410X/Ó 2019 Published by Elsevier Ltd.
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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NDV contains a non-segmented, single-stranded negative-sense RNA genome of approximately 15 kb in length, which consists of six genes flanked by a 30 Leader and 50 Trailer in the order 30 - nucleocapsid protein (NP)-phosphoprotein (P)-matrix protein (M)fusion protein (F)-hemagglutinin-neuraminidase (HN)-large polymerase (L)-50 [15–17]. Unlike positive-strand RNA viruses, the naked genomic RNA of negative-strand RNA viruses (NSVs) is not infectious by itself, and it must be encapsidated with the nucleocapsid protein and associated with the phosphoprotein and the L polymerase to form an active template for viral RNA transcription and replication [18–20]. The LaSota strain is a naturally-occurring low virulent NDV that has been routinely used as a live vaccine throughout the world to prevent Newcastle disease in poultry [4,21,22]. This vaccine strain induces strong immunity, both mucosal and systemic, and can be readily administered through drinking water supplies or by direct spray [23]. The LaSota vaccine has been proven to be safe and stable with no reports of virulence reversion or recombination with field strains. However, there is a need to determine the genetic stability of the LaSota strain-based recombinant vaccine that contains foreign genes due to genome length constraints and reports of instability after excessive passages [24,25]. This study aimed to evaluate the genetic stability and biological properties of the rLS/ILTV-gD vaccine master seed after three separate repetitions of eight passages in embryonated chicken eggs (ECE). To this end, the allantoic fluids from the passages were subjected to reverse transcription-polymerase chain reaction and next-generation sequencing analyses. In addition, the biological properties of the egg-passaged (EP) virus stocks were also examined by performing standard virus titrations and pathogenicity assays [26]. The expression of ILTV gD in virus-infected DF-1 cells was assessed by immunofluorescence assay. The obtained data demonstrate that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least 8 passages from the vaccine master seed virus in ECE.
2. Materials and methods 2.1. Cells and viruses DF-1 (CRL-12203; ATCC, Manassas, VA, USA) cell line was grown in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher Scientific, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific) and antibiotics (100U/ml Penicillin, 100 mg/ml Streptomycin, 0.25 mg/ml Amphotericin B, Thermo Scientific, Suwanee, GA, USA) at 37 °C in 5% CO2 atmosphere. DF-1 cells were maintained in DMEM supplemented with 10% allantoic fluid (AF) from 10-day-old specific-pathogen-free (SPF) ECE for all subsequent infections unless otherwise indicated. The rLS/ILTV-gD vaccine candidate and the parental control virus rLaSota were generated previously [14,27]. The rLS/ILTV-gD vaccine virus was propagated in ECE and maintained at the eggpassage level 3 (EP3) as the vaccine master seed stock and stored at 70 °C.
2.3. Serial passages of the vaccine virus in ECE The rLS/ILTV-gD vaccine master seed stock (EP3) was serially passaged in ECE as illustrated in Fig. 1. Briefly, The EP3 stock was diluted 105 fold with phosphate buffer solution (PBS). Three independent repetition groups (A, B, and C) of 9-day-old SPF leghorn chicken embryonated eggs (3 eggs in each group) were inoculated with 100 ml of the diluted EP3 into the allantoic cavity of each embryo. After 4 days of incubation at 37 °C, the AF of the infected eggs was harvested and pooled within each repetition group. The pooled egg-passaged viruses were diluted 105 fold, then passaged on their corresponding groups of eggs for seven (7) more times. The serially egg-passaged viruses were designated as EP4 (A, B or C) to EP11 (A, B or C), respectively, and stored at 70 °C before use for evaluation. 2.4. Reverse transcription-polymerase chain reaction (RT-PCR) RNAs isolated from the rLS/ILTV-gD vaccine master seed (EP3), egg-passaged (EP4, EP5, EP8, and EP11) virus stocks, and the parental LaSota virus stock were used to amplify the ILTV gD insert by RT-PCR using a SuperscriptTM III One-Step RT-PCR System with Platinum Taq Hi-Fi kit (ThermoFisher Scientific) and a pair of specific primers (primer sequences available upon request) according to the manufacture instructions. The RT-PCR products were analyzed by electrophoresis on 1% agarose gel and photographed using a GelDoc-ItTS3 Imager (UVP, Upland, CA, USA). 2.5. Next-generation sequencing (NGS) RNAs isolated from the rLS/ILTV-gD EP3 and EP11 (A, B, and C) virus stocks were sequenced by using the MiSeq technology according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). Briefly, RNA was quantified with spectrophotometry and Qubit (ThermoFisher Scientific) fluorimetry. RNA was reverse transcribed, and DNA libraries were prepared using the KAPA Stranded RNA-Seq Library Preparation Kit for Illumina MiSeq platform (Kapa Biosystems, USA) according to the manufacturer’s instructions. The size distribution and concentration of DNA in the prepared libraries were checked on a Bioanalyzer 2100 and a Qubit instrument using a high-sensitivity (HS) DNA kit (Agilent Technologies, Germany) and a Qubit double-stranded DNA (dsDNA) HS assay kit (ThermoFisher Scientific), respectively. Paired-end sequencing (2 250 bp) of the generated libraries was performed on a MiSeq instrument with the 500-cycle MiSeq reagent kit version 2 (Illumina). Raw sequence data were analyzed and assembled with MIRA version 3.4.1 within a customized workflow on the Galaxy platform as described previously [28]. Obtained consensus sequences were aligned using Multiple Alignment with Fast Fourier Transformation (MAFFT v.7.221.3) [29] as implemented in the Galaxy platform [30]. The alignment and substitutions (the term single-nucleotide polymorphisms or SNPs) were visualized with Geneious v.9.1.8. In addition, the obtained sequences were also aligned with all complete genome NDV sequences available in GenBank (as of September 27th, 2019) using the same aligning tool.
2.2. RNA isolation
2.6. Virus titration and pathogenicity assessment
Viral RNA was isolated from the AF of ECE infected with EP3, each of the three repetitions of EP4, EP5, EP8, and EP11, and the parental LaSota strain, respectively. Viral RNA was isolated by using the Mag MAXTM-96 AI/ND Viral RNA Isolation Kit (ThermoFisher Scientific, Austin, TX, USA) according to the manufacturer’s instructions. Isolated RNA was submitted to further testing or stored at 70 °C until tested.
The biological properties of the vaccine master seed virus (EP3) and the egg-passaged vaccine virus stocks (EP11A, B, and C) were characterized by performing the standard hemagglutination (HA) assay and the 50% egg infective dose (EID50) titration in 9-dayold SPF chicken embryos [26]. Pathogenicity of the EP3 and EP11 (A, B, and C) viruses was assessed by performing the mean death time (MDT) test in ECE and the intracerebral pathogenicity index
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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respectively, to evaluate the co-expression of the ILTV-gD and NDV HN proteins. 3. Results 3.1. Genetic stability of the rLS/ILTV-gD vaccine
Fig. 1. Experimental design for evaluating the genetic stability of the rLS/ILTV-gD vaccine through serial passages in embryonated chicken eggs (ECE). The rLS/ILTVgD vaccine master seed stock at the original egg-passage level 3 (EP3) was diluted 105 fold with PBS. Three separate repetition groups (A, B, and C) of 9-day-old SPF ECE (3 eggs in each group) were inoculated with 100 ml of the diluted master seed virus EP3 into the allantoic cavity of each embryo. After 4 days of incubation at 37 °C, the allantoic fluid (AF) of the infected eggs was harvested, pooled (within each group), and designated as EP4A, B, and C, respectively. The EP4A, B, and C viruses were then passaged on their corresponding groups of eggs using the same passaging approach for seven (7) more times. The obtained vaccine viruses at each egg passage level were designated as EP5A, B, and C to EP11A, B, and C, respectively. The genetic stability of the egg-passaged viruses was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MiSeq sequencing at the indicated passage levels. The expression of ILTV gD and NDV HN proteins in DF-1 cells infected with rLS/ILTV-gD virus from egg-passaged (EP3, EP8 and EP11) was examined by immunofluorescence assay (IFA).
To determine the genetic stability of the rLS/ILTV-gD vaccine candidate, the vaccine master seed virus, EP3, was serially passaged in ECE in three independent repetitions as illustrated in Fig. 1. The integrity of the ILTV-gD gene insert in the NDV vector and the sequence fidelity of the egg-passaged viruses were examined by RT-PCR and MiSeq technology, respectively. As shown in Fig. 2., the RT-PCR products amplified from the rLS/ILTV-gD vaccine seed (EP3) and the EP4, EP5, EP8, and EP11 virus stocks (A, B, and C) migrated to approximately 2 Kb molecular weight marker on the agarose gel, which corresponds to the predicted molecular weight (2189 bp). These RT-PCR products include the ILTV gD insert (1,507 bp) and a part of the NDV vector (682 bp). The RT-PCR product amplified from the rLaSota virus migrated to the proximity of the predicted molecular weight position (682 bp). The migration of RT-PCR products to the expected sizes on the agarose gel indicated that there was no gross noticeable deletion/insertion (INDELS) in the ILTV-gD insertion region of the egg-passaged virus stocks. A summary of the NGS statistics is provided in Table 1. A single contiguous contig was assembled for each of the sequenced viral genomes with 100% coverage compared to the designed recombinant vaccine viral genome. The median read depth varied between 16 k and 35 k, and the highest depth reaching 68 k (Table 1). Due to this high depth of coverage, highly accurate sequencing results were achieved. Between 1.8 million and 3.6 million reads were used for final consensus calling for each of the sequenced viruses. The alignment of the final consensuses confirmed that there was no single nucleotide (nt) deletion/insertion in the viral genome of the egg-passed vaccine virus stocks (Fig. 3A). The distribution of reads across the genome assemblies was relatively uniform (Fig. 3B). Analysis of the alignment of the MiSeq-produced consensus sequences revealed thirteen (13) SNPs among the 8-time eggpassaged (EP11A, B, and C) virus stocks compared to the vaccine master seed (EP3) (Table 2). Of these, only three SNPs were shared
(ICPI) test in one-day-old SPF chickens following the established procedures [26]. The parental rLaSota virus was included in these assays as a control.
2.7. Immunofluorescence assay (IFA) The expression of ILTV gD and NDV HN proteins in DF-1 cells infected with the rLS/ILTV-gD EP3, EP8 (A, B, and C) and EP11 (A, B, and C) virus stocks, and the parental LaSota virus, respectively, was examined by IFA with anti-ILTV chicken serum (Charles River, Norwich, CT, USA) and an NDV-specific monoclonal antibody against the HN protein (Mab, a gift of Dr. Ron Iorio from University of Massachusetts Medical School, USA) as described previously [14]. Fluorescence and cytopathic effects (CPE) of the infected cells were monitored and photographed using an EVOS FL Cell Imaging System at 400X magnifications (ThermoFisher Scientific). Green and red fluorescent images that were photographed from the same field of virus-infected cells were also merged into single images,
Fig. 2. Detection of the ILTV gD insert in egg-passaged vaccine virus stocks by RTPCR and gel electrophoresis. Viral RNAs were extracted from the EP3, 4, 5, 8 and 11 stocks (A, B, and C), and rLaSota by using the Mag MAXTM-96 AI/ND Viral RNA Isolation Kit (ThermoFisher Scientific). The ILTV gD gene insert and surrounding sequences in the NDV vector were amplified from viral RNAs by using a SuperscriptTM III One Step RT-PCR system with Platinum Taq Hi-Fi kit (ThermoFisher Scientific) and a pair of specific primers. The RT-PCR products were analyzed by electrophoresis on 1% agarose gel and photographed using a GelDoc-ItTS3 Imager (UVP). The sizes of the 1 kb Plus DNA Ladder marker (NEB, Ipswich, MA, USA) are labelled with black arrows on the left side of the gel. The predicated sizes of the RTPCR products from the vaccine virus stocks (2,189 bp) and the rLaSota control (682 bp) are labeled with black arrows on the right side of the gel.
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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Table 1 Summary of sequencing and assembly data. Sample
Number of raw read pairs
Number of filtered read pairsa
Mean read length
Read length SDb
Forward reads qualityc
Reverse reads qualityc
Coverage depthd
Number of reads used for calling final consensuse
Percent coveragef
EP3 EP11A EP11B EP11C
4,416,157 3,702,512 2,308,698 4,923,314
3,912,255 3,111,295 1,936,880 4,448,806
215 153 162 180
86 75 64 76
2|37|38|38|38 2|37|38|38|38 2|37|38|38|38 2|37|38|38|38
2|36|38|38|38 2|36|38|38|38 2|37|38|38|38 2|36|38|38|38
5|27807|32861|39678|62802 7|18089|22275|27224|43200 5|13174|16109|19667|31874 9|29419|35426|43886|68527
2,948,856 2,671,506 1,800,941 3,668,669
100% 100% 100% 100%
a
the number of paired reads remaining after host and PhiX174 internal control filtering. SD = standard deviation. numbers represent distribution (minimum|lower quartile|median|upper quartile|maximum) of Phred quality scores (Q30 score is equivalent to an expected error rate of 0.001). d numbers represent distribution (minimum|lower quartile|median|upper quartile|maximum) of read depth. e numbers of paired reads used for final consensus for each sequence. f final consensus coverage compared to the genome of the designed vectored vaccine. b
c
Fig. 3. Visualization of alignment of consensus sequences and coverage depth of four rLS/ILTV-gD vaccine stocks sequenced by MiSeq sequencing. (A) Alignment of consensus sequences. Egg passage 3 (EP3) vaccine seed stock is used as a reference. Vertical black lines in EP11A, B and C represent single-nucleotide polymorphisms compared to EP3. Annotations represent Newcastle disease virus genes and ILTV glycoprotein D inserted gene. Genome positions are presented above annotations. (B) Depth coverage of MiSeq sequencing. Curve lined areas filled with blue represent relatively uniform distributions of reads across the genome assemblies. The median read death varied between 16 k (EP11B) and 35 k (EP11C). The highest depth of 68.5 k was estimated in the results of EP11C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
among the three egg-passaged (EP11A, B, and C) viral stocks. Two of these three SNPs resulted in amino acid (aa) changes, L475P in the NDV nucleoprotein and T60S in the NDV fusion protein. Six additional SNPs were non-synonymous but were not consistent between all three repetitions at passage 11 (EP11A, B, and C) and were present in only one or two of them. The remaining SNPs were either synonymous or in non-coding regions of the viral genomes (Table 2). Importantly, none of these SNPs was located in the ILTV gD insert (positions 3,310–4,614), the NDV F cleavage site (positions 6,383–6,400), the Leader (positions 1–55), the Trailer (positions 16,579–16,692), and the L polymerase gene (positions 9887–16501). The majority of the SNPs between EP3 and EP11 were randomly present in sequences from various NDV genotypes, from viruses that are of different virulence and pathogenicity, and were isolated around the world over the last several decades (Supplemental File S1).
3.2. The biological properties of the rLS/ILTV-gD egg-passaged virus stocks To determine whether the SNPs in the egg-passaged virus stocks affected their biological properties, the growth ability and pathogenicity of the rLS/ILTV-gD EP11 (A, B, and C) virus stocks were examined by virus titration and the MDT and ICPI tests. As listed in Table 3, the HA and EID50 titers of the eggpassaged vaccine viruses (EP11A, B, and C) were comparable with those of the vaccine master seed stock (EP3) and the parental rLaSota virus. These egg-passaged vaccine virus stocks (EP11A, B, and C) retained the low virulence as the vaccine master seed virus (EP3) with a similar MDT. The ICPI values for all eggpassaged virus stocks were zero, which confirmed that the virus stocks are not pathogenic for chickens. These collective data demonstrated that the SNPs did not have any detectable impact
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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Table 2 Summary of single-nucleotide polymorphism (SNPs) observed in the rLS/ILTV-gD vaccine after eight passages in embryonated chicken eggs. Shaded cells represent SNPs that were shared in all passage repetitions. Genome nt position
Location
61 67 598 1371 1402 1545 2842 3306 5478 6227 6491 6492 6493 9095
NP gene, non-CDS
Amino acid change
NP CDS
NO change T417I NO change L475P G319E
P CDS gD gene non-CDS M CDS F CDS F CDS
D228G T60S D148A*or D148N
HN CDS
N393S
EP3
EP11A
EP11B
EP11C
T A C C A T G C A A G A T A
A G T C G C G C A T G C T A
T G C T A C G T G T A A T G
T G C T A C A C G T A A T A
D
A*
N
N
NP = NDV nucleoprotein. P = NDV phosphoprotein. gD = ILTV glycoprotein D. M = NDV matrix protein. F = NDV fusion protein. HN = NDV hemagglutinin-neuraminidase. CDS = non coding sequence. nt = nucleotide. A* = alanine.
Table 3 Biological assessments of the rLS/ILTV-gD EP11 and parental viruses. Viruses rLaSota rLS/ILTV-gD rLS/ILTV-gD rLS/ILTV-gD rLS/ILTV-gD a b c d
EP3 EP11A EP11B EP11C
MDTa
ICPIb
HAc
EID50d
110hs 138hs 130hs 133hs 135hs
0.15 0 0 0 0
1024 512 512 512 512
2.37 108 3.16 108 1.47 108 1.47 108 2.37 108
MDT: Mean death time assay in embryonated chicken eggs. ICPI: Intracerebral pathogenicity index assay in day-old chickens. HA: Hemagglutination assay. EID50: The 50% egg infective dose assay in embryonated eggs.
on the phenotype and biological properties of the rLS/ILTV-gD egg-passaged virus stocks. 3.3. The expression of ILTV gD protein from the passaged virus To evaluate whether the SNPs in the egg-passaged virus stocks affected the foreign gene expression, we examined the expression of ILTV gD and NDV HN proteins in DF-1 cells infected with the vaccine master seed (EP3) and egg-passaged (EP 8 and EP11) virus stocks, respectively, by IFA with ILTV- and NDV-specific antibodies. The result showed that the ILTV gD protein (green fluorescence) was expressed from the EP8A, B, and C and EP11A, B and C vaccine virus-infected cells at a similar level of green fluorescence intensity as that of EP3-infected cells after 24 h of infection (Fig. 4). No green fluorescence was detected from the parental rLaSota virus-infected cells, confirming the specificity of the ILTV antibody. The NDV HN protein (red fluorescence) was detected from rLaSota and all eggpassaged rLS/ILTV-gD vaccine virus-infected cells by the NDV HN-specific monoclonal antibody (Fig. 4). After merging the fluorescence images, taken in the same field of infected cells, the green and red fluorescence co-localized to all visibly infected cells, indicating that the antigenic ILTV gD protein was expressed from the NDV vector in the infected cells. This result demonstrated that the rLS/ILTV-gD vaccine virus was highly stable and retained the capability to express ILTV glycoprotein D after at least 8 passages in ECE.
4. Discussion The genetic stability of a live virus vaccine is paramount for vaccine safety and efficacy. There is a need for better vaccines against infectious laryngotracheitis since the widely used modified live ILTV vaccines [chicken embryo origin (CEO)] are genetically unstable and can revert to virulence through mutations or recombination during the vaccine virus replication in chicken embryos or birds and cause disease outbreaks [11,12,31]. In contrast, the LaSota strain, a widely used low virulent NDV vaccine, has been proven to be safe and stable with no reports of virulence reversion or recombination with field strains [4,32]. It has been routinely used as a live vaccine to control Newcastle disease throughout the world for more than 60 years [4]. According to the US Code of Federal Regulations (CFR) for Newcastle disease vaccine production (9 CFR § 113.329), the vaccine production seed virus shall be prepared from the first through the fifth passage from the master seed virus (https://www.ecfr. gov/cgi-bin/text-idx?SID = c545c93903e2c15d8a4a328b877cccd5 &mc = true&node = se9.1.113_1329&rgn = div8). The final vaccine product will be prepared from the vaccine production seed virus through propagation in ECE or cell cultures. Therefore, the NDV vectored vaccine master seed virus should maintain its safety, genome integrity, and immunogenicity after 1–5 rounds of propagation in ECE. In this study, we investigated the genetic stability of the rLS/ILTV-gD vaccine candidate after 8 serial passages in ECE
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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Fig. 4. Detection of ILTV gD and NDV HN protein expression by IFA. DF-1 cells were infected with the rLaSota, rLS/ILTV-gD EP3, EP8 (A, B, C), and EP11 (A, B, C) viruses, respectively, at a multiplicity of infection (M.O.I.) of 0.1. At 24 h post-infection, the cells were fixed and stained with a mixture of chicken anti-ILTV serum and mouse antiNDV HN Mab, followed by a mixture of the FITC-labeled goat anti-chicken IgG and Alexa FluorÒ 568 labeled goat anti- mouse IgG. Fluorescence and CPE were monitored and photographed using an EVOS FL Cell Imaging System at 400X magnifications (ThermoFisher Scientific). Green and red fluorescent images that were photographed from the same field of virus-infected cells were merged into single images, respectively. Bars represent 100 mm in size. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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from the master seed virus (EP3), which exceeds the requirement of 5 passages for extra assurance. The data obtained from the RTPCR analysis suggested that the rLS/ILTV-gD vaccine candidate did not have visible size differences in the ILTV gD insertion region during passages in ECE. MiSeq sequence analysis confirmed that the genome integrity of the passaged viruses was maintained without a single nucleotide insertion/deletion. This analysis also revealed 13 SNPs between the vaccine master seed and the eggpassaged virus stocks (EP11 A, B, and C). However, none of these SNPs were located in the ILTV gD insert or any of the known critical biological determinant positions, such as the NDV F cleavage site, cis- and trans-acting elements, and the large polymerase gene regions [15–17]. Furthermore, theses SNPs had no impact on the passaged viruses’ growth kinetics and virulence, nor did they impede the levels of ILTV glycoprotein D expression, suggesting that these SNPs are non-functional sporadic mutations. Negative strand non-segmented RNA viruses (NSVs) have evolved a variety of mechanisms to protect their genome integrity. These include mechanisms to promote replicase fidelity, RNA editing and ‘‘rule-of-six”; and mechanisms to repair the genome termini [33]. RNA dependent RNA polymerase (RdRp) of NSVs lacks proofreading activity and the error rate of the viral RdRp is estimated to be between 1.5 103 bp and 7.2 105 bp [34]. This relaxed polymerase fidelity is an important facet of RNA virus biology, providing a source of sequence diversity that can allow virus quasi-species to form, enabling the virus to adapt successfully to changing environments. However, on the other hand, polymerase error can also lead to the generation of nonviable templates that reduce overall viral fitness. If too many nonviable templates are generated by an RdRp error rate, a virus population becomes unsustainable, and it is believed that many viral RdRps operate close to this threshold [35]. Most viruses in the family paramyxoviridae have a strict requirement for their genome nucleotide length to be divisible by six, a phenomenon known as the ‘‘rule of six” [36,37]. The ‘‘rule of six” regulates viral RNA replication and enables this group of viruses to maintain genome integrity. The NDV vector that we used to express the ILTV gD antigen belongs to this group of viruses. The genome length of the rLS/ ILTV-gD recombinant vaccine is also divisible by six [14], which would regulate the viral RNA replication to maintain its genome integrity. In the meantime, the NDV RdRp error may also generate some sporadic mutations in the recombinant viral genome during virus propagation. Besides the virus vector, a foreign gene insert may also influence the genetic stability of the recombinant virus. It has been reported that the human respiratory virus (RSV) F and G gene inserts in the Parainfluenza virus 5 (PIV5) vector had different impacts on the recombinant virus sequence fidelity [38]. Both PIV5 and RSV are non-segmented NSVs, and PIV5 obeys the ‘‘rule of six” but RSV doesn’t [18,39]. The RSV G and F proteins are responsible for virus attachment and membrane fusion with host cells but are not required for viral RNA replication [20,39]. Therefore, one would assume that the RSV G and F genes might not have had different impacts on the genetic stability of the PIV5 based recombinant viruses. However, Phan et al. [38] reported that the PIV5-RSV-G recombinant vaccine candidate generated more sequence mutations than that of PIV5-RSV-F although both viruses maintained their genome integrity after 11 passages in Vero cells. It was not clear why the RSV G gene had more impacts on the stability of the PIV5-RSV-G genome than the F gene, but it warrants that the recombinant viruses with different foreign gene inserts need to be evaluated individually for their impacts on genomic sequence fidelity. The ILTV glycoprotein D, a membraneassociated glycoprotein, is essential for receptor binding and virus entry to host cells [40,41]. The data presented in this study showed that the insertion of the gD gene into the LaSota vaccine backbone
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did not affect the recombinant virus genome integrity nor affect gD expression after 8 passages in ECE. At present, it is not clear how many of the sporadic mutations found in the rLS/ILTV-gD EP11 virus stocks were introduced by the NDV RdRp error or the ILTV gD gene insert. However, the SNPs observed in the studied viral passages are also present in multiple NDV from various genotypes, pathogenicity, and virulence, providing further evidence that these mutations are random. Based on data obtained from this study and others [38], we postulated that the viral vector played a dominant role, whereas the inserted gene played a minor role (at most), in controlling the sequence fidelity of the rLS/ILTV-gD vaccine candidate. The genetic stability of the rLS/ILTV-gD vaccine in birds has not been examined in the present study. Based on the genetic stability of the vaccine candidate in chicken embryos and the fact that no virulence reversion or recombination of LaSota vaccine with field strains was reported during last 60 years of the LaSota vaccine application [4], we anticipated that the rLS/ILTV-gD vaccine would be very unlike to mutate to a virulent form or undergo recombination with field strains in birds. Additional in vivo study with a bird to bird passage experiment is needed to confirm this. In summary, in the present study, the genetic stability and biological properties of the NDV LaSota vaccine vectored ILTV vaccine candidate were evaluated after serial passages in embryonated chicken eggs to mimic NDV vaccine production. NGS analysis confirmed that the rLS/ILTV-gD vaccine virus maintained its genome integrity and revealed thirteen SNPs in the egg-passaged virus stocks. However, these SNPs did not have any significant impacts on the virus growth ability, virulence, and expression of ILTV gD protein. These results demonstrated that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least eight serial passages in ECE. Declaration of Competing Interest 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. Acknowledgments The authors wish to thank Xiuqin Xia, Fenglan Li and Dawn Williams-Coplin for excellent technical assistance, Jesse Gallagher for MiSeq next-generation sequencing, and Ron Iorio for a gift of anti-NDV HN monoclonal antibody. This research was supported by USDA, ARS projects 6040-32000-073-00D and 6040-32000074-00D. Mention of trade names or commercial products in this publication is solely to provide specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2019.10.074. References [1] Garcia M, Spatz S, Guy JS. Infectious laryngotracheitis. In: Swayne DE, Glisson JR, McDougald LR, Nolan LK, Suarez DL, Nair V, editors. Diseases of Poultry. Ames, Iowa, USA: Wiley-Blackwell Publishing; 2013. p. 161–79. [2] Coppo MJ, Noormohammadi AH, Browning GF, Devlin JM. Challenges and recent advancements in infectious laryngotracheitis virus vaccines. Avian Pathol 2013;42(3):195–205. [3] Miller PJ, Koch G. Newcastle Disease. In: Swayne DE, Glisson JR, McDougald LR, Nolan LK, Suarez DL, Nair V, editors. Diseases of Poultry. Ames, Iowa, USA: Wiley-Blackwell Publishing; 2013. p. 98–107.
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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[4] Dimitrov KM, Afonso CL, Yu Q, Miller PJ. Newcastle disease vaccines-A solved problem or a continuous challenge? Vet Microbiol 2017;206:126–36. [5] Gelenczei E, Marty E. Strain stability and immunologic characterizatics of tissue-culture modified infectious laryngotracheitis virus. Avian Dis 1962;9:44–56. [6] Samberg Y, Aronovici I. The devlopment of a vaccine against avian infectious laryngotracheitis. I. Modification of a laryngotracheitis virus. Refu Vet 1969;26:54–9. [7] Menendez KR, Garcia M, Spatz S, Tablante NL. Molecular epidemiology of infectious laryngotracheitis: a review. Avian Pathol 2014. [8] Hughes CS, Gaskell RM, Jones RC, Bradbury JM, Jordan FT. Effects of certain stress factors on the re-excretion of infectious laryngotracheitis virus from latently infected carrier birds. Res Vet Sci 1989;46(2):274–6. [9] Hughes CS, Williams RA, Gaskell RM, Jordan FT, Bradbury JM, Bennett M, et al. Latency and reactivation of infectious laryngotracheitis vaccine virus. Arch Virol 1991;121(1–4):213–8. [10] Guy JS, Barnes HJ, Smith L. Increased virulence of modified-live infectious laryngotracheitis vaccine virus following bird-to-bird passage. Avian Dis 1991;35(2):348–55. [11] Guy JS, Barnes HJ, Morgan LM. Virulence of infectious laryngotracheitis viruses: comparison of modified-live vaccine viruses and North Carolina field isolates. Avian Dis 1990;34(1):106–13. [12] Kotiw M, Wilks CR, May JT. The effect of serial in vivo passage on the expression of virulence and DNA stability of an infectious laryngotracheitis virus strain of low virulence. Vet Microbiol 1995;45(1):71–80. [13] Yu Q, Spatz S, Li Y, Yang J, Zhao W, Zhang Z, et al. Newcastle disease virus vectored infectious laryngotracheitis vaccines protect commercial broiler chickens in the presence of maternally derived antibodies. Vaccine 2017;35 (5):789–95. [14] Zhao W, Spatz S, Zhang Z, Wen G, Garcia M, Zsak L, et al. Newcastle disease virus (NDV) recombinants expressing infectious laryngotracheitis virus (ILTV) glycoproteins gB and gD protect chickens against ILTV and NDV challenges. J Virol 2014;88(15):8397–406. [15] de Leeuw O, Peeters B. Complete nucleotide sequence of Newcastle disease virus: evidence for the existence of a new genus within the subfamily Paramyxovirinae. J Gen Virol 1999;80(Pt 1):131–6. [16] Peeters BP, de Leeuw OS, Koch G, Gielkens AL. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. J Virol 1999;73(6):5001–9. [17] Samal SK. Newcastle disease and ralated avian paramyxoviruses. In: Samal SK, editor. The Biology of Paramyxoviruses. Norfolk, UK: Caister Academic press; 2011. p. 69–114. [18] Lamb RA, Parks GD. Paramyxoviridae. In: Knipe DM, Howley PM, Cohen JI, Griffin DE, Lamb RA, Marti MA, editors. Fields Virology. Philadelphia, PA, USA: Lippincott Williams & Wilkins; 2013. p. 957–95. [19] Ortin J, Martin-Benito J. The RNA synthesis machinery of negative-stranded RNA viruses. Virology 2015;479–480:532–44. [20] Yu Q, Hardy RW, Wertz GW. Functional cDNA clones of the human respiratory syncytial (RS) virus N, P, and L proteins support replication of RS virus genomic RNA analogs and define minimal trans-acting requirements for RNA replication. J Virol 1995;69(4):2412–9. [21] Ayala AJ, Dimitrov KM, Becker CR, Goraichuk IV, Arns CW, Bolotin VI, et al. Presence of vaccine-derived newcastle disease viruses in wild birds. PLoS ONE 2016;11(9):e0162484. [22] Goldhaft TM. Historical note on the origin of the LaSota strain of Newcastle disease virus. Avian Dis 1980;24(2):297–301.
[23] Meulemans G. Control by vaccination. In: Alexander DJ, editor. Newcastle Disease. Boston, MA: Kluwer Academic Publishers; 1988. p. 318–32. [24] Bello MB, Yusoff K, Ideris A, Hair-Bejo M, Peeters BPH, Omar AR. Diagnostic and Vaccination Approaches for Newcastle Disease Virus in Poultry: The Current and Emerging Perspectives. Biomed Res Int 2018;2018:7278459. [25] Meng C, Qiu X, Yu S, Li C, Sun Y, Chen Z, et al. Evolution of newcastle disease virus quasispecies diversity and enhanced virulence after passage through chicken air sacs. J Virol 2016;90(4):2052–63. [26] Alexander DJ. Newcastle Disease Virus and other Avian Paramyxoviruses. In: Swayne D, Glisson JR, Jackwood MW, Pearson JE, Reed WM, editors. A laboratory manual for the isolation and identification of avian pathogens. 4th Edition ed. Kennett Square, PA: American Association of Avian Pathologists; 1998, p. 156-63. [27] Hu H, Roth JP, Estevez CN, Zsak L, Liu B, Yu Q. Generation and evaluation of a recombinant Newcastle disease virus expressing the glycoprotein (G) of avian metapneumovirus subgroup C as a bivalent vaccine in turkeys. Vaccine 2011;29(47):8624–33. [28] Dimitrov KM, Sharma P, Volkening JD, Goraichuk IV, Wajid A, Rehmani SF, et al. A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses. Virol J 2017;14(1):72. [29] Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 2013;30 (4):772–80. [30] Goecks J, Nekrutenko A, Taylor J, Galaxy T. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 2010;11(8):R86. [31] Lee SW, Markham PF, Coppo MJ, Legione AR, Markham JF, Noormohammadi AH, et al. Attenuated vaccines can recombine to form virulent field viruses. Science 2012;337(6091):188. [32] Afonso CL. Not so fast on recombination analysis of Newcastle disease virus. J Virol 2008;82(18):9303. [33] Barr JN, Fearns R. How RNA viruses maintain their genome integrity. J Gen Virol 2010;91(Pt 6):1373–87. [34] Drake JW. Rates of spontaneous mutation among RNA viruses. Proc Natl Acad Sci USA 1993;90(9):4171–5. [35] Crotty S, Andino R. Implications of high RNA virus mutation rates: lethal mutagenesis and the antiviral drug ribavirin. Microb Infect 2002;4 (13):1301–7. [36] Calain P, Roux L. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol 1993;67(8):4822–30. [37] Kolakofsky D, Roux L, Garcin D, Ruigrok RW. Paramyxovirus mRNA editing, the ‘‘rule of six” and error catastrophe: a hypothesis. J Gen Virol 2005;86(Pt 7):1869–77. [38] Phan SI, Adam CM, Chen Z, Citron M, Liang X, Espeseth AS. Genetic stability of parainfluenza virus 5-vectored human respiratory syncytial virus vaccine candidates after in vitro and in vivo passage. J. Virol. 2017;91(19). https://doi. org/10.1128/JVI.00559-17. [39] Collins PL, Fearns R, Graham BS. Respiratory syncytial virus: virology, reverse genetics, and pathogenesis of disease. Curr Top Microbiol Immunol 2013;372:3–38. [40] Fuchs W, Veits J, Helferich D, Granzow H, Teifke JP, Mettenleiter TC. Molecular biology of avian infectious laryngotracheitis virus. Vet Res 2007;38(2):261–79. [41] Pavlova S, Veits J, Mettenleiter TC, Fuchs W. Identification and functional analysis of membrane proteins gD, gE, gI, and pUS9 of Infectious laryngotracheitis virus. Avian Dis 2013;57(2 Suppl):416–26.
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos Qingzhong Yu ⇑, Yufeng Li 1, Kiril Dimitrov, Claudio L. Afonso, Stephen Spatz, Laszlo Zsak Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, United States Department of Agriculture, 934 College Station Road, Athens, GA 30605, USA
a r t i c l e
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Article history: Received 22 May 2019 Received in revised form 17 October 2019 Accepted 25 October 2019 Available online xxxx Keywords: NDV vector ILTV-gD Recombinant vaccine Genetic stability Next-generation sequencing
a b s t r a c t Previously, we have demonstrated that the recombinant Newcastle disease virus (NDV) expressing the infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) conferred protection against both virulent NDV and ILTV challenges in chickens. In this study, we evaluated the genetic stability of the recombinant vaccine after eight serial passages in embryonated chicken eggs (ECE). The vaccine master seed virus at the original egg-passage level 3 (EP3) was diluted and passaged in three separate repetitions (A, B and C) in ECE eight times (EP4 to EP11). RT-PCR analysis of the vaccine seed and egg-passaged virus stocks showed that there was no detectable insertion/deletion in the ILTV gD insert region. Next-generation sequencing analysis of the EP3 and EP11 virus stocks confirmed their genome integrity and revealed a total of thirteen single-nucleotide polymorphisms (SNPs). However, none of these SNPs were located in the ILTV gD insert or any of the known critical biological determinant positions. Virological and immunofluorescent assays provided additional evidence that the EP11 virus stocks retained their growth kinetics, low pathogenicity, and robust level of gD expression comparable to that of the vaccine master seed virus. This indicated that the SNPs were non-detrimental sporadic mutations. These results demonstrated that the insertion of ILTV gD gene into the NDV LaSota backbone did not significantly affect the genetic stability of the recombinant virus and that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least eight serial passages in ECE. Ó 2019 Published by Elsevier Ltd.
1. Introduction Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease of chickens caused by Gallid alphaherpesvirus 1, a member of the genus Iltovirus within the family Herpseviridae, and commonly known as infectious laryngotracheitis virus (ILTV, used hereafter) [1]. The disease is mainly controlled through biosecurity and by vaccination with live-attenuated vaccines [2]. Newcastle disease (ND) is another serious infectious respiratory disease of poultry caused by virulent strains of Avian orthoavulavirus 1, a member of the family Paramyxoviridae (https://talk. ictvonline.org/taxonomy/), and commonly known as Newcastle disease virus (NDV, used hereafter) [3]. Vaccination, combined with strict biosecurity practices, has been the recommended strategy for controlling ND outbreaks [4].
⇑ Corresponding author. E-mail address: [email protected] (Q. Yu). Current address: Shandong Poultry Research Institute, Jinan, Shandong 250023, China 1
The commonly used live-attenuated vaccines for ILT were developed by either multiple passages of ILTV strains in embryonated eggs (chicken embryo origin [CEO]) or in tissue culture (tissue culture origin [TCO]) [5,6]. Although these vaccines protect against clinical disease, they have residual virulence which is exacerbated by continued infections of naïve birds from productively infected animals and latent carriers [7–9]. Moreover, the CEO vaccine strain has been demonstrated to mutate and become more virulent simply by bird-to-bird passage [10]. In high-density poultry rearing facilities, there is a continuous reservoir of viruses, both virulent and vaccinal, evolving to higher levels of virulence. These ‘‘revertants” have become the dominant field strains in poultry populations and are the cause of field ILT outbreaks [11,12]. To overcome these problems associated with live attenuated ILTV vaccine strains, we have developed an NDV LaSota vaccine strainbased recombinant virus expressing the ILTV gD protein as a bivalent vaccine and demonstrated that vaccination of chickens with the recombinant vaccine, rLS/ILTV-gD, confers complete protection against virulent NDV challenge and significant protection against pathogenic ILTV challenge [13,14].
https://doi.org/10.1016/j.vaccine.2019.10.074 0264-410X/Ó 2019 Published by Elsevier Ltd.
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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NDV contains a non-segmented, single-stranded negative-sense RNA genome of approximately 15 kb in length, which consists of six genes flanked by a 30 Leader and 50 Trailer in the order 30 - nucleocapsid protein (NP)-phosphoprotein (P)-matrix protein (M)fusion protein (F)-hemagglutinin-neuraminidase (HN)-large polymerase (L)-50 [15–17]. Unlike positive-strand RNA viruses, the naked genomic RNA of negative-strand RNA viruses (NSVs) is not infectious by itself, and it must be encapsidated with the nucleocapsid protein and associated with the phosphoprotein and the L polymerase to form an active template for viral RNA transcription and replication [18–20]. The LaSota strain is a naturally-occurring low virulent NDV that has been routinely used as a live vaccine throughout the world to prevent Newcastle disease in poultry [4,21,22]. This vaccine strain induces strong immunity, both mucosal and systemic, and can be readily administered through drinking water supplies or by direct spray [23]. The LaSota vaccine has been proven to be safe and stable with no reports of virulence reversion or recombination with field strains. However, there is a need to determine the genetic stability of the LaSota strain-based recombinant vaccine that contains foreign genes due to genome length constraints and reports of instability after excessive passages [24,25]. This study aimed to evaluate the genetic stability and biological properties of the rLS/ILTV-gD vaccine master seed after three separate repetitions of eight passages in embryonated chicken eggs (ECE). To this end, the allantoic fluids from the passages were subjected to reverse transcription-polymerase chain reaction and next-generation sequencing analyses. In addition, the biological properties of the egg-passaged (EP) virus stocks were also examined by performing standard virus titrations and pathogenicity assays [26]. The expression of ILTV gD in virus-infected DF-1 cells was assessed by immunofluorescence assay. The obtained data demonstrate that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least 8 passages from the vaccine master seed virus in ECE.
2. Materials and methods 2.1. Cells and viruses DF-1 (CRL-12203; ATCC, Manassas, VA, USA) cell line was grown in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher Scientific, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific) and antibiotics (100U/ml Penicillin, 100 mg/ml Streptomycin, 0.25 mg/ml Amphotericin B, Thermo Scientific, Suwanee, GA, USA) at 37 °C in 5% CO2 atmosphere. DF-1 cells were maintained in DMEM supplemented with 10% allantoic fluid (AF) from 10-day-old specific-pathogen-free (SPF) ECE for all subsequent infections unless otherwise indicated. The rLS/ILTV-gD vaccine candidate and the parental control virus rLaSota were generated previously [14,27]. The rLS/ILTV-gD vaccine virus was propagated in ECE and maintained at the eggpassage level 3 (EP3) as the vaccine master seed stock and stored at 70 °C.
2.3. Serial passages of the vaccine virus in ECE The rLS/ILTV-gD vaccine master seed stock (EP3) was serially passaged in ECE as illustrated in Fig. 1. Briefly, The EP3 stock was diluted 105 fold with phosphate buffer solution (PBS). Three independent repetition groups (A, B, and C) of 9-day-old SPF leghorn chicken embryonated eggs (3 eggs in each group) were inoculated with 100 ml of the diluted EP3 into the allantoic cavity of each embryo. After 4 days of incubation at 37 °C, the AF of the infected eggs was harvested and pooled within each repetition group. The pooled egg-passaged viruses were diluted 105 fold, then passaged on their corresponding groups of eggs for seven (7) more times. The serially egg-passaged viruses were designated as EP4 (A, B or C) to EP11 (A, B or C), respectively, and stored at 70 °C before use for evaluation. 2.4. Reverse transcription-polymerase chain reaction (RT-PCR) RNAs isolated from the rLS/ILTV-gD vaccine master seed (EP3), egg-passaged (EP4, EP5, EP8, and EP11) virus stocks, and the parental LaSota virus stock were used to amplify the ILTV gD insert by RT-PCR using a SuperscriptTM III One-Step RT-PCR System with Platinum Taq Hi-Fi kit (ThermoFisher Scientific) and a pair of specific primers (primer sequences available upon request) according to the manufacture instructions. The RT-PCR products were analyzed by electrophoresis on 1% agarose gel and photographed using a GelDoc-ItTS3 Imager (UVP, Upland, CA, USA). 2.5. Next-generation sequencing (NGS) RNAs isolated from the rLS/ILTV-gD EP3 and EP11 (A, B, and C) virus stocks were sequenced by using the MiSeq technology according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). Briefly, RNA was quantified with spectrophotometry and Qubit (ThermoFisher Scientific) fluorimetry. RNA was reverse transcribed, and DNA libraries were prepared using the KAPA Stranded RNA-Seq Library Preparation Kit for Illumina MiSeq platform (Kapa Biosystems, USA) according to the manufacturer’s instructions. The size distribution and concentration of DNA in the prepared libraries were checked on a Bioanalyzer 2100 and a Qubit instrument using a high-sensitivity (HS) DNA kit (Agilent Technologies, Germany) and a Qubit double-stranded DNA (dsDNA) HS assay kit (ThermoFisher Scientific), respectively. Paired-end sequencing (2 250 bp) of the generated libraries was performed on a MiSeq instrument with the 500-cycle MiSeq reagent kit version 2 (Illumina). Raw sequence data were analyzed and assembled with MIRA version 3.4.1 within a customized workflow on the Galaxy platform as described previously [28]. Obtained consensus sequences were aligned using Multiple Alignment with Fast Fourier Transformation (MAFFT v.7.221.3) [29] as implemented in the Galaxy platform [30]. The alignment and substitutions (the term single-nucleotide polymorphisms or SNPs) were visualized with Geneious v.9.1.8. In addition, the obtained sequences were also aligned with all complete genome NDV sequences available in GenBank (as of September 27th, 2019) using the same aligning tool.
2.2. RNA isolation
2.6. Virus titration and pathogenicity assessment
Viral RNA was isolated from the AF of ECE infected with EP3, each of the three repetitions of EP4, EP5, EP8, and EP11, and the parental LaSota strain, respectively. Viral RNA was isolated by using the Mag MAXTM-96 AI/ND Viral RNA Isolation Kit (ThermoFisher Scientific, Austin, TX, USA) according to the manufacturer’s instructions. Isolated RNA was submitted to further testing or stored at 70 °C until tested.
The biological properties of the vaccine master seed virus (EP3) and the egg-passaged vaccine virus stocks (EP11A, B, and C) were characterized by performing the standard hemagglutination (HA) assay and the 50% egg infective dose (EID50) titration in 9-dayold SPF chicken embryos [26]. Pathogenicity of the EP3 and EP11 (A, B, and C) viruses was assessed by performing the mean death time (MDT) test in ECE and the intracerebral pathogenicity index
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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respectively, to evaluate the co-expression of the ILTV-gD and NDV HN proteins. 3. Results 3.1. Genetic stability of the rLS/ILTV-gD vaccine
Fig. 1. Experimental design for evaluating the genetic stability of the rLS/ILTV-gD vaccine through serial passages in embryonated chicken eggs (ECE). The rLS/ILTVgD vaccine master seed stock at the original egg-passage level 3 (EP3) was diluted 105 fold with PBS. Three separate repetition groups (A, B, and C) of 9-day-old SPF ECE (3 eggs in each group) were inoculated with 100 ml of the diluted master seed virus EP3 into the allantoic cavity of each embryo. After 4 days of incubation at 37 °C, the allantoic fluid (AF) of the infected eggs was harvested, pooled (within each group), and designated as EP4A, B, and C, respectively. The EP4A, B, and C viruses were then passaged on their corresponding groups of eggs using the same passaging approach for seven (7) more times. The obtained vaccine viruses at each egg passage level were designated as EP5A, B, and C to EP11A, B, and C, respectively. The genetic stability of the egg-passaged viruses was determined by reverse transcription-polymerase chain reaction (RT-PCR) and MiSeq sequencing at the indicated passage levels. The expression of ILTV gD and NDV HN proteins in DF-1 cells infected with rLS/ILTV-gD virus from egg-passaged (EP3, EP8 and EP11) was examined by immunofluorescence assay (IFA).
To determine the genetic stability of the rLS/ILTV-gD vaccine candidate, the vaccine master seed virus, EP3, was serially passaged in ECE in three independent repetitions as illustrated in Fig. 1. The integrity of the ILTV-gD gene insert in the NDV vector and the sequence fidelity of the egg-passaged viruses were examined by RT-PCR and MiSeq technology, respectively. As shown in Fig. 2., the RT-PCR products amplified from the rLS/ILTV-gD vaccine seed (EP3) and the EP4, EP5, EP8, and EP11 virus stocks (A, B, and C) migrated to approximately 2 Kb molecular weight marker on the agarose gel, which corresponds to the predicted molecular weight (2189 bp). These RT-PCR products include the ILTV gD insert (1,507 bp) and a part of the NDV vector (682 bp). The RT-PCR product amplified from the rLaSota virus migrated to the proximity of the predicted molecular weight position (682 bp). The migration of RT-PCR products to the expected sizes on the agarose gel indicated that there was no gross noticeable deletion/insertion (INDELS) in the ILTV-gD insertion region of the egg-passaged virus stocks. A summary of the NGS statistics is provided in Table 1. A single contiguous contig was assembled for each of the sequenced viral genomes with 100% coverage compared to the designed recombinant vaccine viral genome. The median read depth varied between 16 k and 35 k, and the highest depth reaching 68 k (Table 1). Due to this high depth of coverage, highly accurate sequencing results were achieved. Between 1.8 million and 3.6 million reads were used for final consensus calling for each of the sequenced viruses. The alignment of the final consensuses confirmed that there was no single nucleotide (nt) deletion/insertion in the viral genome of the egg-passed vaccine virus stocks (Fig. 3A). The distribution of reads across the genome assemblies was relatively uniform (Fig. 3B). Analysis of the alignment of the MiSeq-produced consensus sequences revealed thirteen (13) SNPs among the 8-time eggpassaged (EP11A, B, and C) virus stocks compared to the vaccine master seed (EP3) (Table 2). Of these, only three SNPs were shared
(ICPI) test in one-day-old SPF chickens following the established procedures [26]. The parental rLaSota virus was included in these assays as a control.
2.7. Immunofluorescence assay (IFA) The expression of ILTV gD and NDV HN proteins in DF-1 cells infected with the rLS/ILTV-gD EP3, EP8 (A, B, and C) and EP11 (A, B, and C) virus stocks, and the parental LaSota virus, respectively, was examined by IFA with anti-ILTV chicken serum (Charles River, Norwich, CT, USA) and an NDV-specific monoclonal antibody against the HN protein (Mab, a gift of Dr. Ron Iorio from University of Massachusetts Medical School, USA) as described previously [14]. Fluorescence and cytopathic effects (CPE) of the infected cells were monitored and photographed using an EVOS FL Cell Imaging System at 400X magnifications (ThermoFisher Scientific). Green and red fluorescent images that were photographed from the same field of virus-infected cells were also merged into single images,
Fig. 2. Detection of the ILTV gD insert in egg-passaged vaccine virus stocks by RTPCR and gel electrophoresis. Viral RNAs were extracted from the EP3, 4, 5, 8 and 11 stocks (A, B, and C), and rLaSota by using the Mag MAXTM-96 AI/ND Viral RNA Isolation Kit (ThermoFisher Scientific). The ILTV gD gene insert and surrounding sequences in the NDV vector were amplified from viral RNAs by using a SuperscriptTM III One Step RT-PCR system with Platinum Taq Hi-Fi kit (ThermoFisher Scientific) and a pair of specific primers. The RT-PCR products were analyzed by electrophoresis on 1% agarose gel and photographed using a GelDoc-ItTS3 Imager (UVP). The sizes of the 1 kb Plus DNA Ladder marker (NEB, Ipswich, MA, USA) are labelled with black arrows on the left side of the gel. The predicated sizes of the RTPCR products from the vaccine virus stocks (2,189 bp) and the rLaSota control (682 bp) are labeled with black arrows on the right side of the gel.
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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Table 1 Summary of sequencing and assembly data. Sample
Number of raw read pairs
Number of filtered read pairsa
Mean read length
Read length SDb
Forward reads qualityc
Reverse reads qualityc
Coverage depthd
Number of reads used for calling final consensuse
Percent coveragef
EP3 EP11A EP11B EP11C
4,416,157 3,702,512 2,308,698 4,923,314
3,912,255 3,111,295 1,936,880 4,448,806
215 153 162 180
86 75 64 76
2|37|38|38|38 2|37|38|38|38 2|37|38|38|38 2|37|38|38|38
2|36|38|38|38 2|36|38|38|38 2|37|38|38|38 2|36|38|38|38
5|27807|32861|39678|62802 7|18089|22275|27224|43200 5|13174|16109|19667|31874 9|29419|35426|43886|68527
2,948,856 2,671,506 1,800,941 3,668,669
100% 100% 100% 100%
a
the number of paired reads remaining after host and PhiX174 internal control filtering. SD = standard deviation. numbers represent distribution (minimum|lower quartile|median|upper quartile|maximum) of Phred quality scores (Q30 score is equivalent to an expected error rate of 0.001). d numbers represent distribution (minimum|lower quartile|median|upper quartile|maximum) of read depth. e numbers of paired reads used for final consensus for each sequence. f final consensus coverage compared to the genome of the designed vectored vaccine. b
c
Fig. 3. Visualization of alignment of consensus sequences and coverage depth of four rLS/ILTV-gD vaccine stocks sequenced by MiSeq sequencing. (A) Alignment of consensus sequences. Egg passage 3 (EP3) vaccine seed stock is used as a reference. Vertical black lines in EP11A, B and C represent single-nucleotide polymorphisms compared to EP3. Annotations represent Newcastle disease virus genes and ILTV glycoprotein D inserted gene. Genome positions are presented above annotations. (B) Depth coverage of MiSeq sequencing. Curve lined areas filled with blue represent relatively uniform distributions of reads across the genome assemblies. The median read death varied between 16 k (EP11B) and 35 k (EP11C). The highest depth of 68.5 k was estimated in the results of EP11C. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
among the three egg-passaged (EP11A, B, and C) viral stocks. Two of these three SNPs resulted in amino acid (aa) changes, L475P in the NDV nucleoprotein and T60S in the NDV fusion protein. Six additional SNPs were non-synonymous but were not consistent between all three repetitions at passage 11 (EP11A, B, and C) and were present in only one or two of them. The remaining SNPs were either synonymous or in non-coding regions of the viral genomes (Table 2). Importantly, none of these SNPs was located in the ILTV gD insert (positions 3,310–4,614), the NDV F cleavage site (positions 6,383–6,400), the Leader (positions 1–55), the Trailer (positions 16,579–16,692), and the L polymerase gene (positions 9887–16501). The majority of the SNPs between EP3 and EP11 were randomly present in sequences from various NDV genotypes, from viruses that are of different virulence and pathogenicity, and were isolated around the world over the last several decades (Supplemental File S1).
3.2. The biological properties of the rLS/ILTV-gD egg-passaged virus stocks To determine whether the SNPs in the egg-passaged virus stocks affected their biological properties, the growth ability and pathogenicity of the rLS/ILTV-gD EP11 (A, B, and C) virus stocks were examined by virus titration and the MDT and ICPI tests. As listed in Table 3, the HA and EID50 titers of the eggpassaged vaccine viruses (EP11A, B, and C) were comparable with those of the vaccine master seed stock (EP3) and the parental rLaSota virus. These egg-passaged vaccine virus stocks (EP11A, B, and C) retained the low virulence as the vaccine master seed virus (EP3) with a similar MDT. The ICPI values for all eggpassaged virus stocks were zero, which confirmed that the virus stocks are not pathogenic for chickens. These collective data demonstrated that the SNPs did not have any detectable impact
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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Table 2 Summary of single-nucleotide polymorphism (SNPs) observed in the rLS/ILTV-gD vaccine after eight passages in embryonated chicken eggs. Shaded cells represent SNPs that were shared in all passage repetitions. Genome nt position
Location
61 67 598 1371 1402 1545 2842 3306 5478 6227 6491 6492 6493 9095
NP gene, non-CDS
Amino acid change
NP CDS
NO change T417I NO change L475P G319E
P CDS gD gene non-CDS M CDS F CDS F CDS
D228G T60S D148A*or D148N
HN CDS
N393S
EP3
EP11A
EP11B
EP11C
T A C C A T G C A A G A T A
A G T C G C G C A T G C T A
T G C T A C G T G T A A T G
T G C T A C A C G T A A T A
D
A*
N
N
NP = NDV nucleoprotein. P = NDV phosphoprotein. gD = ILTV glycoprotein D. M = NDV matrix protein. F = NDV fusion protein. HN = NDV hemagglutinin-neuraminidase. CDS = non coding sequence. nt = nucleotide. A* = alanine.
Table 3 Biological assessments of the rLS/ILTV-gD EP11 and parental viruses. Viruses rLaSota rLS/ILTV-gD rLS/ILTV-gD rLS/ILTV-gD rLS/ILTV-gD a b c d
EP3 EP11A EP11B EP11C
MDTa
ICPIb
HAc
EID50d
110hs 138hs 130hs 133hs 135hs
0.15 0 0 0 0
1024 512 512 512 512
2.37 108 3.16 108 1.47 108 1.47 108 2.37 108
MDT: Mean death time assay in embryonated chicken eggs. ICPI: Intracerebral pathogenicity index assay in day-old chickens. HA: Hemagglutination assay. EID50: The 50% egg infective dose assay in embryonated eggs.
on the phenotype and biological properties of the rLS/ILTV-gD egg-passaged virus stocks. 3.3. The expression of ILTV gD protein from the passaged virus To evaluate whether the SNPs in the egg-passaged virus stocks affected the foreign gene expression, we examined the expression of ILTV gD and NDV HN proteins in DF-1 cells infected with the vaccine master seed (EP3) and egg-passaged (EP 8 and EP11) virus stocks, respectively, by IFA with ILTV- and NDV-specific antibodies. The result showed that the ILTV gD protein (green fluorescence) was expressed from the EP8A, B, and C and EP11A, B and C vaccine virus-infected cells at a similar level of green fluorescence intensity as that of EP3-infected cells after 24 h of infection (Fig. 4). No green fluorescence was detected from the parental rLaSota virus-infected cells, confirming the specificity of the ILTV antibody. The NDV HN protein (red fluorescence) was detected from rLaSota and all eggpassaged rLS/ILTV-gD vaccine virus-infected cells by the NDV HN-specific monoclonal antibody (Fig. 4). After merging the fluorescence images, taken in the same field of infected cells, the green and red fluorescence co-localized to all visibly infected cells, indicating that the antigenic ILTV gD protein was expressed from the NDV vector in the infected cells. This result demonstrated that the rLS/ILTV-gD vaccine virus was highly stable and retained the capability to express ILTV glycoprotein D after at least 8 passages in ECE.
4. Discussion The genetic stability of a live virus vaccine is paramount for vaccine safety and efficacy. There is a need for better vaccines against infectious laryngotracheitis since the widely used modified live ILTV vaccines [chicken embryo origin (CEO)] are genetically unstable and can revert to virulence through mutations or recombination during the vaccine virus replication in chicken embryos or birds and cause disease outbreaks [11,12,31]. In contrast, the LaSota strain, a widely used low virulent NDV vaccine, has been proven to be safe and stable with no reports of virulence reversion or recombination with field strains [4,32]. It has been routinely used as a live vaccine to control Newcastle disease throughout the world for more than 60 years [4]. According to the US Code of Federal Regulations (CFR) for Newcastle disease vaccine production (9 CFR § 113.329), the vaccine production seed virus shall be prepared from the first through the fifth passage from the master seed virus (https://www.ecfr. gov/cgi-bin/text-idx?SID = c545c93903e2c15d8a4a328b877cccd5 &mc = true&node = se9.1.113_1329&rgn = div8). The final vaccine product will be prepared from the vaccine production seed virus through propagation in ECE or cell cultures. Therefore, the NDV vectored vaccine master seed virus should maintain its safety, genome integrity, and immunogenicity after 1–5 rounds of propagation in ECE. In this study, we investigated the genetic stability of the rLS/ILTV-gD vaccine candidate after 8 serial passages in ECE
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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Fig. 4. Detection of ILTV gD and NDV HN protein expression by IFA. DF-1 cells were infected with the rLaSota, rLS/ILTV-gD EP3, EP8 (A, B, C), and EP11 (A, B, C) viruses, respectively, at a multiplicity of infection (M.O.I.) of 0.1. At 24 h post-infection, the cells were fixed and stained with a mixture of chicken anti-ILTV serum and mouse antiNDV HN Mab, followed by a mixture of the FITC-labeled goat anti-chicken IgG and Alexa FluorÒ 568 labeled goat anti- mouse IgG. Fluorescence and CPE were monitored and photographed using an EVOS FL Cell Imaging System at 400X magnifications (ThermoFisher Scientific). Green and red fluorescent images that were photographed from the same field of virus-infected cells were merged into single images, respectively. Bars represent 100 mm in size. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
Q. Yu et al. / Vaccine xxx (xxxx) xxx
from the master seed virus (EP3), which exceeds the requirement of 5 passages for extra assurance. The data obtained from the RTPCR analysis suggested that the rLS/ILTV-gD vaccine candidate did not have visible size differences in the ILTV gD insertion region during passages in ECE. MiSeq sequence analysis confirmed that the genome integrity of the passaged viruses was maintained without a single nucleotide insertion/deletion. This analysis also revealed 13 SNPs between the vaccine master seed and the eggpassaged virus stocks (EP11 A, B, and C). However, none of these SNPs were located in the ILTV gD insert or any of the known critical biological determinant positions, such as the NDV F cleavage site, cis- and trans-acting elements, and the large polymerase gene regions [15–17]. Furthermore, theses SNPs had no impact on the passaged viruses’ growth kinetics and virulence, nor did they impede the levels of ILTV glycoprotein D expression, suggesting that these SNPs are non-functional sporadic mutations. Negative strand non-segmented RNA viruses (NSVs) have evolved a variety of mechanisms to protect their genome integrity. These include mechanisms to promote replicase fidelity, RNA editing and ‘‘rule-of-six”; and mechanisms to repair the genome termini [33]. RNA dependent RNA polymerase (RdRp) of NSVs lacks proofreading activity and the error rate of the viral RdRp is estimated to be between 1.5 103 bp and 7.2 105 bp [34]. This relaxed polymerase fidelity is an important facet of RNA virus biology, providing a source of sequence diversity that can allow virus quasi-species to form, enabling the virus to adapt successfully to changing environments. However, on the other hand, polymerase error can also lead to the generation of nonviable templates that reduce overall viral fitness. If too many nonviable templates are generated by an RdRp error rate, a virus population becomes unsustainable, and it is believed that many viral RdRps operate close to this threshold [35]. Most viruses in the family paramyxoviridae have a strict requirement for their genome nucleotide length to be divisible by six, a phenomenon known as the ‘‘rule of six” [36,37]. The ‘‘rule of six” regulates viral RNA replication and enables this group of viruses to maintain genome integrity. The NDV vector that we used to express the ILTV gD antigen belongs to this group of viruses. The genome length of the rLS/ ILTV-gD recombinant vaccine is also divisible by six [14], which would regulate the viral RNA replication to maintain its genome integrity. In the meantime, the NDV RdRp error may also generate some sporadic mutations in the recombinant viral genome during virus propagation. Besides the virus vector, a foreign gene insert may also influence the genetic stability of the recombinant virus. It has been reported that the human respiratory virus (RSV) F and G gene inserts in the Parainfluenza virus 5 (PIV5) vector had different impacts on the recombinant virus sequence fidelity [38]. Both PIV5 and RSV are non-segmented NSVs, and PIV5 obeys the ‘‘rule of six” but RSV doesn’t [18,39]. The RSV G and F proteins are responsible for virus attachment and membrane fusion with host cells but are not required for viral RNA replication [20,39]. Therefore, one would assume that the RSV G and F genes might not have had different impacts on the genetic stability of the PIV5 based recombinant viruses. However, Phan et al. [38] reported that the PIV5-RSV-G recombinant vaccine candidate generated more sequence mutations than that of PIV5-RSV-F although both viruses maintained their genome integrity after 11 passages in Vero cells. It was not clear why the RSV G gene had more impacts on the stability of the PIV5-RSV-G genome than the F gene, but it warrants that the recombinant viruses with different foreign gene inserts need to be evaluated individually for their impacts on genomic sequence fidelity. The ILTV glycoprotein D, a membraneassociated glycoprotein, is essential for receptor binding and virus entry to host cells [40,41]. The data presented in this study showed that the insertion of the gD gene into the LaSota vaccine backbone
7
did not affect the recombinant virus genome integrity nor affect gD expression after 8 passages in ECE. At present, it is not clear how many of the sporadic mutations found in the rLS/ILTV-gD EP11 virus stocks were introduced by the NDV RdRp error or the ILTV gD gene insert. However, the SNPs observed in the studied viral passages are also present in multiple NDV from various genotypes, pathogenicity, and virulence, providing further evidence that these mutations are random. Based on data obtained from this study and others [38], we postulated that the viral vector played a dominant role, whereas the inserted gene played a minor role (at most), in controlling the sequence fidelity of the rLS/ILTV-gD vaccine candidate. The genetic stability of the rLS/ILTV-gD vaccine in birds has not been examined in the present study. Based on the genetic stability of the vaccine candidate in chicken embryos and the fact that no virulence reversion or recombination of LaSota vaccine with field strains was reported during last 60 years of the LaSota vaccine application [4], we anticipated that the rLS/ILTV-gD vaccine would be very unlike to mutate to a virulent form or undergo recombination with field strains in birds. Additional in vivo study with a bird to bird passage experiment is needed to confirm this. In summary, in the present study, the genetic stability and biological properties of the NDV LaSota vaccine vectored ILTV vaccine candidate were evaluated after serial passages in embryonated chicken eggs to mimic NDV vaccine production. NGS analysis confirmed that the rLS/ILTV-gD vaccine virus maintained its genome integrity and revealed thirteen SNPs in the egg-passaged virus stocks. However, these SNPs did not have any significant impacts on the virus growth ability, virulence, and expression of ILTV gD protein. These results demonstrated that the rLS/ILTV-gD virus is a safe and genetically stable vaccine candidate after at least eight serial passages in ECE. Declaration of Competing Interest 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. Acknowledgments The authors wish to thank Xiuqin Xia, Fenglan Li and Dawn Williams-Coplin for excellent technical assistance, Jesse Gallagher for MiSeq next-generation sequencing, and Ron Iorio for a gift of anti-NDV HN monoclonal antibody. This research was supported by USDA, ARS projects 6040-32000-073-00D and 6040-32000074-00D. Mention of trade names or commercial products in this publication is solely to provide specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2019.10.074. References [1] Garcia M, Spatz S, Guy JS. Infectious laryngotracheitis. In: Swayne DE, Glisson JR, McDougald LR, Nolan LK, Suarez DL, Nair V, editors. Diseases of Poultry. Ames, Iowa, USA: Wiley-Blackwell Publishing; 2013. p. 161–79. [2] Coppo MJ, Noormohammadi AH, Browning GF, Devlin JM. Challenges and recent advancements in infectious laryngotracheitis virus vaccines. Avian Pathol 2013;42(3):195–205. [3] Miller PJ, Koch G. Newcastle Disease. In: Swayne DE, Glisson JR, McDougald LR, Nolan LK, Suarez DL, Nair V, editors. Diseases of Poultry. Ames, Iowa, USA: Wiley-Blackwell Publishing; 2013. p. 98–107.
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074
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Q. Yu et al. / Vaccine xxx (xxxx) xxx
[4] Dimitrov KM, Afonso CL, Yu Q, Miller PJ. Newcastle disease vaccines-A solved problem or a continuous challenge? Vet Microbiol 2017;206:126–36. [5] Gelenczei E, Marty E. Strain stability and immunologic characterizatics of tissue-culture modified infectious laryngotracheitis virus. Avian Dis 1962;9:44–56. [6] Samberg Y, Aronovici I. The devlopment of a vaccine against avian infectious laryngotracheitis. I. Modification of a laryngotracheitis virus. Refu Vet 1969;26:54–9. [7] Menendez KR, Garcia M, Spatz S, Tablante NL. Molecular epidemiology of infectious laryngotracheitis: a review. Avian Pathol 2014. [8] Hughes CS, Gaskell RM, Jones RC, Bradbury JM, Jordan FT. Effects of certain stress factors on the re-excretion of infectious laryngotracheitis virus from latently infected carrier birds. Res Vet Sci 1989;46(2):274–6. [9] Hughes CS, Williams RA, Gaskell RM, Jordan FT, Bradbury JM, Bennett M, et al. Latency and reactivation of infectious laryngotracheitis vaccine virus. Arch Virol 1991;121(1–4):213–8. [10] Guy JS, Barnes HJ, Smith L. Increased virulence of modified-live infectious laryngotracheitis vaccine virus following bird-to-bird passage. Avian Dis 1991;35(2):348–55. [11] Guy JS, Barnes HJ, Morgan LM. Virulence of infectious laryngotracheitis viruses: comparison of modified-live vaccine viruses and North Carolina field isolates. Avian Dis 1990;34(1):106–13. [12] Kotiw M, Wilks CR, May JT. The effect of serial in vivo passage on the expression of virulence and DNA stability of an infectious laryngotracheitis virus strain of low virulence. Vet Microbiol 1995;45(1):71–80. [13] Yu Q, Spatz S, Li Y, Yang J, Zhao W, Zhang Z, et al. Newcastle disease virus vectored infectious laryngotracheitis vaccines protect commercial broiler chickens in the presence of maternally derived antibodies. Vaccine 2017;35 (5):789–95. [14] Zhao W, Spatz S, Zhang Z, Wen G, Garcia M, Zsak L, et al. Newcastle disease virus (NDV) recombinants expressing infectious laryngotracheitis virus (ILTV) glycoproteins gB and gD protect chickens against ILTV and NDV challenges. J Virol 2014;88(15):8397–406. [15] de Leeuw O, Peeters B. Complete nucleotide sequence of Newcastle disease virus: evidence for the existence of a new genus within the subfamily Paramyxovirinae. J Gen Virol 1999;80(Pt 1):131–6. [16] Peeters BP, de Leeuw OS, Koch G, Gielkens AL. Rescue of Newcastle disease virus from cloned cDNA: evidence that cleavability of the fusion protein is a major determinant for virulence. J Virol 1999;73(6):5001–9. [17] Samal SK. Newcastle disease and ralated avian paramyxoviruses. In: Samal SK, editor. The Biology of Paramyxoviruses. Norfolk, UK: Caister Academic press; 2011. p. 69–114. [18] Lamb RA, Parks GD. Paramyxoviridae. In: Knipe DM, Howley PM, Cohen JI, Griffin DE, Lamb RA, Marti MA, editors. Fields Virology. Philadelphia, PA, USA: Lippincott Williams & Wilkins; 2013. p. 957–95. [19] Ortin J, Martin-Benito J. The RNA synthesis machinery of negative-stranded RNA viruses. Virology 2015;479–480:532–44. [20] Yu Q, Hardy RW, Wertz GW. Functional cDNA clones of the human respiratory syncytial (RS) virus N, P, and L proteins support replication of RS virus genomic RNA analogs and define minimal trans-acting requirements for RNA replication. J Virol 1995;69(4):2412–9. [21] Ayala AJ, Dimitrov KM, Becker CR, Goraichuk IV, Arns CW, Bolotin VI, et al. Presence of vaccine-derived newcastle disease viruses in wild birds. PLoS ONE 2016;11(9):e0162484. [22] Goldhaft TM. Historical note on the origin of the LaSota strain of Newcastle disease virus. Avian Dis 1980;24(2):297–301.
[23] Meulemans G. Control by vaccination. In: Alexander DJ, editor. Newcastle Disease. Boston, MA: Kluwer Academic Publishers; 1988. p. 318–32. [24] Bello MB, Yusoff K, Ideris A, Hair-Bejo M, Peeters BPH, Omar AR. Diagnostic and Vaccination Approaches for Newcastle Disease Virus in Poultry: The Current and Emerging Perspectives. Biomed Res Int 2018;2018:7278459. [25] Meng C, Qiu X, Yu S, Li C, Sun Y, Chen Z, et al. Evolution of newcastle disease virus quasispecies diversity and enhanced virulence after passage through chicken air sacs. J Virol 2016;90(4):2052–63. [26] Alexander DJ. Newcastle Disease Virus and other Avian Paramyxoviruses. In: Swayne D, Glisson JR, Jackwood MW, Pearson JE, Reed WM, editors. A laboratory manual for the isolation and identification of avian pathogens. 4th Edition ed. Kennett Square, PA: American Association of Avian Pathologists; 1998, p. 156-63. [27] Hu H, Roth JP, Estevez CN, Zsak L, Liu B, Yu Q. Generation and evaluation of a recombinant Newcastle disease virus expressing the glycoprotein (G) of avian metapneumovirus subgroup C as a bivalent vaccine in turkeys. Vaccine 2011;29(47):8624–33. [28] Dimitrov KM, Sharma P, Volkening JD, Goraichuk IV, Wajid A, Rehmani SF, et al. A robust and cost-effective approach to sequence and analyze complete genomes of small RNA viruses. Virol J 2017;14(1):72. [29] Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 2013;30 (4):772–80. [30] Goecks J, Nekrutenko A, Taylor J, Galaxy T. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol 2010;11(8):R86. [31] Lee SW, Markham PF, Coppo MJ, Legione AR, Markham JF, Noormohammadi AH, et al. Attenuated vaccines can recombine to form virulent field viruses. Science 2012;337(6091):188. [32] Afonso CL. Not so fast on recombination analysis of Newcastle disease virus. J Virol 2008;82(18):9303. [33] Barr JN, Fearns R. How RNA viruses maintain their genome integrity. J Gen Virol 2010;91(Pt 6):1373–87. [34] Drake JW. Rates of spontaneous mutation among RNA viruses. Proc Natl Acad Sci USA 1993;90(9):4171–5. [35] Crotty S, Andino R. Implications of high RNA virus mutation rates: lethal mutagenesis and the antiviral drug ribavirin. Microb Infect 2002;4 (13):1301–7. [36] Calain P, Roux L. The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA. J Virol 1993;67(8):4822–30. [37] Kolakofsky D, Roux L, Garcin D, Ruigrok RW. Paramyxovirus mRNA editing, the ‘‘rule of six” and error catastrophe: a hypothesis. J Gen Virol 2005;86(Pt 7):1869–77. [38] Phan SI, Adam CM, Chen Z, Citron M, Liang X, Espeseth AS. Genetic stability of parainfluenza virus 5-vectored human respiratory syncytial virus vaccine candidates after in vitro and in vivo passage. J. Virol. 2017;91(19). https://doi. org/10.1128/JVI.00559-17. [39] Collins PL, Fearns R, Graham BS. Respiratory syncytial virus: virology, reverse genetics, and pathogenesis of disease. Curr Top Microbiol Immunol 2013;372:3–38. [40] Fuchs W, Veits J, Helferich D, Granzow H, Teifke JP, Mettenleiter TC. Molecular biology of avian infectious laryngotracheitis virus. Vet Res 2007;38(2):261–79. [41] Pavlova S, Veits J, Mettenleiter TC, Fuchs W. Identification and functional analysis of membrane proteins gD, gE, gI, and pUS9 of Infectious laryngotracheitis virus. Avian Dis 2013;57(2 Suppl):416–26.
Please cite this article as: Q. Yu, Y. Li, K. Dimitrov et al., Genetic stability of a Newcastle disease virus vectored infectious laryngotracheitis virus vaccine after serial passages in chicken embryos, Vaccine, https://doi.org/10.1016/j.vaccine.2019.10.074