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96 strain-implications in vaccine upscaling
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Characterization of defective interfering (DI) particles of Pestedes petitsruminants vaccine virus Sungri/96 strain-implications in vaccine upscaling Mousumi Boraa, Raja Wasim Yousufa, Pronab Dharb, M. Manub, Insha Zafira, Bina Mishraa, Kaushal Kishor Rajaka, Rabindra Prasad Singha,∗ a b
Division of Biological Products, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India
ARTICLE INFO
ABSTRACT
Keywords: PPR virus Multiplicity of infection Defective interfering particles Cell ELISA RT-PCR RT-qPCR
The present investigation deals with the characterization of defective interfering (DI) particles of Peste-des-petits ruminants (PPR) vaccine Sungri/96 strain generated as a result of high MOI in Vero cells. During the serial 10 passages, infectivity titres drastically reduced from 6.5 to 2.25 log10TCID50/ml at high MOI. Further, attenuation of CPE with high MOI indicated generation of DI particles that resulted in no/slow progression of CPE during the late passages. Monoclonal antibody based cell ELISA indicated normal protein (N & H) packaging in samples with DI activity. At genomic level, inconsistency in amplicon intensity of H gene was observed in RT-PCR, indicating a possible defect of H gene. Further analysis of copy number of PPRV by RT-qPCR indicated intermittent fluctuations of viral genomic RNA copies. The significant decline of viral RNA copies with MOI 3 (314 copies) compared to low MOI (512804 copies), proved that higher DI multiplicities cause more interference with the replication process of the standard virus. Therefore, MOI is critical for manufacturing of vaccines. These investigations will help in upscaling of PPR vaccines in view of ongoing National and Global PPR control and eradication programme.
1. Introduction Peste-des-petits ruminants (PPR) is a highly contagious and transboundary viral disease of small ruminants causing severe economic impact on the rural economies and genetic resources of around 70 countries of Africa, Middle-East and Asia [1]. The control of PPR is mainly achieved through vaccination with the available live attenuated vaccines. With the ongoing Global PPR control and eradication strategy production of quality vaccines in right quantities and their timely delivery will be necessary to eradicate the disease in near future [2].Currently, the vaccine strain, PPRV Sungri/96 is used extensively by several commercial firms and government vaccine manufacturing unit for production of PPR vaccines throughout India [3]. However, on several occasions, instances of low virus titres or insufficient infectious virus particles were often experienced by laboratory personals during PPR vaccine production which could be related to use of inappropriate multiplicity of infection (MOI) or possible formation of Defective Interfering (DI) particles. DI particles are biologically active, non-infectious forms of viruses which arise as a result of serial passages at high
∗
MOI [4,5]. They are termed as ‘defective’ as they have lost the capacity to code for the necessary proteins required for independent replication and thus are defective in absence of the parent virus [6]. Broadly, ‘interfering’ refers to the ability of DI particles to reduce titers and thereby interfere with the production of helper virus. This interference results in attenuation of cytopathic effects or symptoms caused by the helper virus [7]. The frequency of their generation is influenced by several factors such as strain of the virus, cell type, virus passage history, replication event and generation interval [5,8]. Generation of DI particles have been commonly observed in vitro during serial passages at high MOIs and often have been invoked to explain the conversion of acute in vitro infections to persistent ones [9–12]. Such non-infectious particles have been documented in Measles [8] and Rinderpest virus [13] which are antigenically similar to Pestedes-petits-ruminants virus (PPRV) suggesting that these particles may resist a desirable humoral immune response by interfering with the standard viral replication [14]. DI RNA of Paramyxovirus originates from the 5′ end of the standard genome as a result of replicative errors either by copy back mechanisms or by creating internal deletions
Corresponding author. Division of Biological Products, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243 122, Uttar Pradesh, India. E-mail addresses: [email protected], [email protected] (R.P. Singh).
https://doi.org/10.1016/j.biologicals.2019.09.008 Received 18 May 2019; Received in revised form 3 August 2019; Accepted 24 September 2019 1045-1056/ © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Mousumi Bora, et al., Biologicals, https://doi.org/10.1016/j.biologicals.2019.09.008
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resulting in formation of shorter genome [15]. Such short genomes are then transferred from one generation to the next with each passage leading to rapid accumulation of DI particles [16]. Even though, information on generation of DI particles of closely related vaccine viruses like Measles and Rinderpest is available, the significance of PPRV specific DI particles are yet to be investigated. In the present study, we explored the possible formation of DI particles by making regular passages at low as well as high MOIs using PPRV Sungri/96 vaccine virus strain. The dominance of DI particle and the effect of its interfering activity leading to loss of cytopathic effect was analyzed and discussed. The findings may have implications on upscaling of PPR vaccine in view of ongoing global PPR control and eradication program [1].
2.5. Antigenic and physical characterization of PPRV specific DI particles 2.5.1. Changes in cytopathic effects (CPE) and duration of virus harvest PPRV induced CPE were critically recorded at 24 h interval till the time of harvest. Any unusual changes in CPE and duration of virus harvest with different MOIs were recorded at regular intervals. The infected monolayers at each passage level were harvested when 70–80% of CPE was observed. The virus was aliquoted and stored at −20 °C for further use. 2.5.2. Virus infectivity assay Virus titration was carried out in 96 well cell culture plates using Vero cells. Briefly, co-cultivation of 10 fold dilution of each virus sample was carried out with 104 cells/well of the micro titre plate using three replicates per dilution. The plates were incubated at 37 °C with 5% CO2 and 100% relative humidity. The media of the plates were changed with maintenance media every 48 h and the cytopathic effects in each well were recorded. After taking the final reading, the titres were calculated using Reed and Muench method [19].
2. Materials and methods 2.1. Cells Vero cells (ATCC®, CCL-81) available at Division of Biological Products, ICAR-IVRI, were revived and routinely subcultured in Eagle Minimum Essential Media (EMEM) (Sigma Aldrich) supplemented with 10% Fetal bovine serum (Gibco, Invitrogen), 200 mM L-glutamine and 100 mM sodium pyruvate (Himedia). For maintaining the infected cells, EMEM with 2% FBS was used as maintenance medium.
2.5.3. Binding efficacy of PPRV of high MOI passages using mAb based cell ELISA Cell ELISA was employed as previously described by us [20]. Briefly, 100 μl of PPR vaccine virus propagated with different MOIs were inoculated in Vero cells in 96 well plates for titration. After visualization of PPR specific CPE on 5th day of titration, the culture fluid of each well was discarded gently. Infected cells were fixed and permeabilized by incubating with 100 μl of 80% chilled acetone per well for 30 min at −20 °C and air dried. The wells with fixed cells were blocked using 200 μl of blocking buffer for an hour after which 100 μl of primary antibody (mAb 4G6 and 4B11 against N and H protein of PPRV diluted in 1:20 and 1:24 in blocking buffer respectively) was added to the cells in duplicates and incubated for 1 h at 37 °C. After three washings, Rabbit anti-mouse IgG conjugated to HRPO (Sigma) in blocking buffer (1:1000) was added and incubated for 1 h at 37 °C. Colour reaction developed after addition of substrate was stopped using equal volume of 1 M H2SO4 and the optical density (OD) was measured at 492 nm (A492). A cut-off value with two times the absorbance (A492), compared to mock infected cells was set for declaring a well as positive.
2.2. Preparation of virus stock The vaccine virus, PPRV Sungri/96 strain (Passage 60 in Vero cells) was used in the present study [17]. The seed vaccine virus was propagated in Vero cells at a MOI of 0.01 in 300 cm2 tissue culture flasks (TPP). After 48 h of incubation, the media was removed and replenished with maintenance media. PPRV induced cytopathic effects were observed and recorded at regular intervals. The infected cells were harvested when around 70–80% of cytopathic effects were observed and stored at −20 °C for further use. 2.3. Monoclonal antibodies
2.6. Nucleic acid based characterization of PPRV specific DI particles
The PPR vaccine virus (Sungri/96 strain) neutralizing monoclonal antibody (mAb) against hemagglutinin (H) protein from hybridoma clone 4B11 and nucleocapsid (N) protein from hybridoma clone 4G6 [18], available at Division of Biological Products, ICAR-Indian Veterinary Research Institute were used for the study.
2.6.1. Samples, RNA extraction and cDNA synthesis PPRV aliquots, from low and high MOI passages were processed for total RNA extraction using Trizol method described by Chomczynski and Sacchi [21]. The RNA was reverse transcribed using random hexamer and oligo (dT) primers and M-MLV reverse transcriptase (First Strand cDNA Synthesis Kit, Promega, Madison) as per manufacturer's protocol. The cDNA thus prepared was stored at −20 °C until use.
2.4. Serial passage of PPR virus through co-cultivation method The virus stock having an initial titre of 6.5 log10 TCID50/ml was inoculated in Vero cells with three different MOIs viz. 0.5, 1 and 3 using method of co-cultivation including the low MOI 0.01 used for PPR vaccine propagation. The experiment involved 48 h old Vero cell monolayer grown in 25 cm2 tissue culture flasks. Before infection, two 25 cm2 tissue culture flasks were trypinized, pooled and split in the ratio of 1:5 with an initial cell density of 0.6 × 106 cells per flask. The infected cells were harvested upon manifestation of 70–80% cytopathic effects. The first fixed MOI passaged viruses during passage 1 & 2, were then followed by a series of infection for which the infected cells from the preceding passage served as the inoculums for the subsequent infection. Between passage 3 to 10, only relative dilutions of inoculums were taken in to account and virus titers were not known at the time of inoculation. However all the virus inoculums were titrated subsequently post infection. Healthy Vero cells were kept as control in each passage level.
2.6.2. Primers and probe The primers sets NP3/NP4 against the nucleocapsid (N) and PPRVHF1/HR1 against hemagglutinin (H) protein gene were custom synthesized from IDT (USA) for conventional RT-PCR. The TaqMan® probe previously designed by Yousuf [22] targeting the nucleocapsid (N) protein gene of PPRV was used for quantitative PCR. The primer sets and probe used in the present study are tabulated (Table 1). 2.6.3. Reverse transcription polymerase chain reaction (RT-PCR) RT-PCR was carried out in 25 μl of total reaction volume containing 12.5 μl of 2X PCR master mix (Sapphire), 1 μl of forward and reverse primers (10pM), 8 μl of nuclease free water (NFW) and 2.5 μl of cDNA template. For each PCR run, positive and negative controls were also prepared using known positive cDNA and NFW respectively as template. The PCR components were mixed properly and thermal cycled as per the conditions mentioned in Table 2. 2
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Table 1 Details of primers and probe used in the present study. Target gene
Primer/probe
Oligonucleotide sequence (5′-3′)
Bases
Amplicon size
References
N
NP3 NP4 PPRV-HF1 PPRV-HR1 PPR_F PPR_R PPR_M
TCT CGG AAA TCG CCT CAC AGA CTC CCT CCT CCT GGT CCT CCA GAA TCT ACG CAC GAA ACC CAG CCT CC GGC ATC GGA TCT CCA GAC GG CAC ACA ACA AAG GAG AGT GAT TG ATG AAC CGC CGA AGT GAT AG FAM-AAT TTC GCC TTG ACA AAG GGT GGC-MGBNFQ
24 24 20 20 23 20 24
351 bp
[23]
851 bp
[24]
102 bp
[22]
H N
2.6.4. Agarose gel electrophoresis Agarose gel (1.5% for N gene and 1% for H gene) was prepared in 1X TAE buffer and was cast containing ethidium bromide (0.5 μg/ml). After casting, gel was completely immersed in electrophoresis tank containing 1X TAE. Then the PCR products of samples, positive and negative control sample were loaded in wells alongside the DNA marker (Fermentas, USA). The gel was electrophoresed at 80 V for 1 h for resolving PCR products and visualized under Gel DocTM XR + System (Bio-Rad, USA) for documentation of results.
MOIs 1 and 3 showed atypical cytopathic changes, such as extensive granulation, cording and heaping of cells (Fig. 1B). No syncytia formation was observed after P6 with MOIs 1 and 3 and areas of cell degeneration were progressively filled in by surrounding healthy intact cells. Similar atypical changes were observed in monolayer infected with MOI 0.5 by P8. By P10, no characteristic CPE could be detected upto 10 dpi with all the three high MOI passages (0.5, 1 and 3). The infected cells at P10 with the high MOI inoculums were harvested at 10 dpi with around 5–10% CPE clearly indicating the interference of the standard virus replication with the generation of PPRV specific DI particles. At passage 10, a declining trend of CPE was also observed with the time (Fig. 1C). The percentage of CPE observed in the serial passages (1–10) at different dpi of PPR vaccine virus with various MOIs is represented in Table 3.
2.6.5. Quantitative polymerase chain reaction (RT-qPCR) The two step RT-qPCR was carried out as described by Yousuf (2018) [22]. Briefly, 10 μL reaction volume containing 9 pmol of each primer, 2.5 pmol of TaqMan® hydrolysis probe, 1 μL of cDNA, 5 μL of 2× TaqMan® fast advanced master mix containing Hotstart Taq DNA polymerase, dNTPs and MgCl2 was used. The optimized cycling conditions included Uracil-DNA Glycoylase (UDG) incubation at 50 °C for 2 min and 1 s followed by 20 s hold at 50 °C and then 40 cycles of denaturation at 95 °C for 5 s with combined primer annealing and extension step at 60 °C for 30 s (2-step cycling) in a real time thermal cycler (MxAria, Agilent Technologies, USA). For calculation of copy number of samples, PPR vaccine virus at low MOI (0.01) was taken as reference control. The copy numbers were deduced from the standard curve Y (Cq or Δ Rn) = −3.296* log (x) + 34.98 designed for PPRV using nucleocapsid plasmid (Yousuf, 2018) [22].
3.2. Infectivity titres at different MOIs The infectivity titre of stock virus sample having an initial titre of 6.5 log10 TCID50/ml was rapidly dropped to 2.25–2.75 log 10TCID50/ml by the end of passage 10 on serial passages with the higher MOIs. The infectivity titres of the vaccine virus propagated with MOI 3 dropped significantly from an initial stock titre of 6.5 log10TCID50/ml to 3.5log10TCID50/ml by P6 and by the end of P10 the infectivity titre was found to be 2.25 log10TCID50/ml. Similar trend of infectivity titres were observed with MOI 1 as evidenced by drop of titres to 3.5 log10 TCID50/ ml by P7 to 2.5 log10TCID50/ml by P10. However, in contrast, low MOI (0.01) resulted in higher infectious virus titres with a constant infectivity titre of 5.75–6.5 log10TCID50/ml throughout the ten serial passages in Vero cells. The trends of virus infectivity titre obtained with various MOIs at each passage level are represented in Table 4.
3. Results 3.1. Changes due to cytopathic effects (CPE) and duration of virus harvest Cells infected with the low MOI (0.01) of PPR vaccine virus showed characteristic CPE of cell rounding, ballooning and formation of syncytia after 2 days post inoculation (dpi) and attained maximum CPE (~70–80%) at around 5–6 dpi (Fig. 1A) which could be observed throughout 1–10 passages. However, in the initial four serial passages with all the higher MOIs (0.5, 1 and 3), onset of earlier CPE was observed 18 h post infection and the cells were harvested by 3–5 dpi with 80–90% CPE. By the sixth undiluted passage (P6), virus inoculums with
3.3. Binding efficacy of PPRV with high MOI passages using mAb based cell ELISA To detect the reactivity of PPRV Sungri/96 propagated with high MOIs, Cell ELISA was done using mAbs targeting N and H protein of PPRV. The binding efficacy of anti-N and anti-H mAbs with PPRV inoculums of low (0.01) and high MOIs (0.5, 1 and 3) were compared and assessed in terms of OD492 values. Based on the OD values, anti-N mAb
Table 2 Thermal cycling conditions for amplifying nucleocapsid (N) and hemagglutinin (H) protein gene using RT-PCR.
3
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Fig. 1. Cytopathic effects induced by PPRV/Sungri 96 virus at Passage 1 (1A), Passage 6 (1B) and Passage 10 (1C) with inoculums at various MOIs.
showed low reactivity with MOI 1 and 3 from the initial to 5th serial passage. However, with the same MOIs, an increase in OD values was observed from P6 to P10. In contrast, PPRV passaged with MOI 0.01 and 0.5 showed similar and consistent reactivity pattern with anti-N mAb based on the OD values using cell ELISA (Fig. 2A). The binding efficacy of anti-H mAb revealed reduced and more variable reactivity with PPRV Sungri/96 propagated with MOI 1 and 3 which can be observed with the comparable difference in OD values of inoculums with low MOI (0.01). Analysis of OD values of PPRV propagated with MOI 0.5 showed OD values relatively similar to low MOI throughout the series of 10 passages indicating less DI activity in the standard virus population. Reduced reactivity pattern of anti-H mAb was apparent from P1 to P7 with MOI 1 and P1 to P8 with MOI 3. However with both the higher MOIs (1 and 3), a fluctuating pattern of reactivity (based on OD values) was obtained in every consecutive passage till P7 and P8 (Fig. 2B).
compared to that of low MOI (Fig. 3B). Partial length amplification of the H protein gene from the low MOI passages or the diluted series resulted in consistent and uniform amplicon intensities with an amplicon size of 851 bp throughout the series of 10 passages (Fig. 3C). Comparison of band intensities of the serial passages propagated with MOI 0.5 (data not presented) revealed fluctuation in levels of amplicon intensity after P6 indicating defective nucleic acids as formation of DI particles. Inconsistent amplification of H gene was observed after P5 with MOI 1 (data not presented) and this phenomenon was observed from the initial passage itself (P2) with MOI 3 (Fig. 3D). 3.4.2. Estimation of viral RNA copies using RT-qPCR The copy number of PPRV from the alternate passages viz. P2, P4, P6, P8 and P10 passaged with MOI 0.5, 1 and 3 were analyzed in terms of copy number and compared with the reference control (Table 5). During the series of undiluted passages in Vero cells, cycling between maximum and minimum copy numbers of viral RNA occurred with every alternate passage which was apparent with MOI 1 and 3. The first significant decline in viral RNA copies was observed in P2 with MOI 3, (1023 copies) following a rise in P4 (305800 copies) which subsequently descends again in P6 (314 copies) following a cyclical pattern. Similar pattern was observed in passages with MOI 0.5 and 1. During the series of passages with MOI 1, a drop in copy numbers was detected in P6, whereas similar pattern of decline in copy numbers was observed in P4 passaged with MOI 0.5. The magnitude of difference of copy
3.4. Nucleic acid based characterization using RT-PCR and RT-qPCR 3.4.1. Detection of viral nucleic acid of PPRV using RT-PCR from low and high MOI passages In N-gene based RT-PCR, uniform and consistent amplicon intensities producing a specific amplicon of 351 bp was observed throughout all the passages (P1–P10) with low MOI 0.01 (Fig. 3A). However, with the same reaction conditions and concentration, inconsistency in amplicon intensity was observed with MOI 1 and 3 when 4
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Table 3 Percentage of CPE observed in the serial passages at different dpi of PPR vaccine virus with various MOIs (0.01, 0.5, 1 and 3).
A = Atypical CPE like heaping and cording of cells; no cell rounding or ballooning or formation of foci of infected cells were observed. S= Slow progression of CPE. H= Harvested.
number was lowest in P6 with MOI 3 which was found to be 314 as compared to 512804 copies of the standard reference control propagated with MOI 0.01.
virus, which are antigenically closely related to PPR virus. PPRV Sungri/96 strain is presently being extensively used as a vaccine candidate to control the disease in the Indian subcontinent and some of the countries in the Middle-East and South-Asia [3,28]. Instances of low vaccine titres of PPR virus were often experienced in several biological firms (unpublished data), which may be due to certain reasons such as use of lower MOI than the recommended MOI used for preparation of PPR vaccines or possible evolution of DI particles during batch cultivation of vaccines due to higher MOIs. Both could be a limiting factor for rapid large scale vaccine production [29]. Therefore, identification of critical process parameters such as cell concentration, multiplicity of infection and time point of harvest is crucial in developing effective and quality vaccines [30]. In order to generate defective interfering particles of PPR virus in Vero cells, PPRV Sungri/96 with an initial titre of 6.5 log10 TCID50/ml was serially passaged at high MOIs viz. 0.5, 1 and 3 including the low
4. Discussion Defective interfering (DI) particles arise spontaneously as a result of high multiplicities of infection during virus growth and replicate at the expense of standard helper virus during a co-infection [25]. Generation of DI particles during the process of batch cultivation of vaccines remains challenging to commercial vaccine manufacturers [26] and veterinary biological units to ensure the quality status of the manufactured vaccines. Further, evolution of mutant population of DI particles may adapt to circumvent the resistance of interference produced by the standard virus [27]. Evidence of generation of DI particles has been observed in vaccine strains of Measles [8] and Rinderpest [13] 5
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the replication before infecting the remaining surrounding healthy cells during second replication cycle. These observations were concomitant with our finding that PPRV Sungri/96 has an eclipse period of 12 h at MOI 1 and can be harvested by 72 h (3 dpi) with 70–80% of CPE as per the analysis of one step growth curve [31]. Two such replication cycles would be required if low MOIs (0.01) are used for virus propagation and therefore may take 5–6 days to induce 70–80% of CPE. Moreover, the eclipse period may change for Paramyxoviruses depending upon the multiplicity of infection [32]. The infectivity titre of stock vaccine virus (6.5 log10 TCID50/ml) dropped drastically to 2.25–2.75 log10 TCID50/ml by the end of P10 in case of all the high virus inoculums. The delayed cytopathic effects with the increase in serial undiluted virus passages was correlated with the reduction of virus yields of each passage by at least 0.5–1 log10 TCID50/ ml, with intermittent increase and decrease of virus yield, a phenomenon characteristic of formation of DI particles. Similar findings were observed during generation of DI particles of antigenically related Measles vaccine virus [8] and Rinderpest vaccine virus [13]. Absence of characteristic CPE by serial undiluted passage 5 with high MOI 1 and 3 might be due to effects of DI genomes as reported earlier for rinderpest virus [13]. Absence or no progression of CPE with all the higher MOIs (0.5, 1 and 3) at P10 might be due to interference with the standard virus replication thereby attenuating the typical cytopathic changes leading to a persistent infection as also reported for Measles [6], Sendai [10] and Murray Valley Encephalitis virus [11]. Further characterization of generated DI particles was done by mAb based cell ELISA using mAbs against N and H protein of PPRV. The vaccine virus propagated with low MOI 0.01 showed specific reactivity
Table 4 Infectivity titres after serial passage of PPRV Sungri/96 at different MOIs in Vero cells. Passage number
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
Multiplicity of infection (MOI) 0.01
0.5
1
3
6.5 5.5 6.2 6.25 6.5 6.25 6.5 6.25 6.25 5.75
6.5 7.2 5.5 5.5 5.5 5.5 5.5 3.75 4.25 2.5
6.5 6.5 5.75 5.5 5.25 4 3.5 3.5 2.75 2.5
6.5 6.5 6.25 5.75 5.5 3.5 4.75 3.75 2.75 2.25
The titres are represented in log10 TCID50/ml.
MOI 0.01 used for vaccine preparation. Visible CPE after 2 dpi with MOI 0.01 of PPRV Sungri/96 vaccine virus includes cell rounding, ballooning and formation of syncytia [31]. Similar observation was made by us while investigating the growth of PPRV Sungri/96 in Vero cells infected with a low MOI 0.01. The speed of cytopathic effect was relatively slow and initiation of characteristic CPE was observed after 2 dpi. The infected cells were harvested at 5 dpi when 70–80% of CPE was manifested. The early onset of CPE in the initial four passages with high MOIs (0.5, 1 and 3) was due to infection of majority of the cells simultaneously producing 80–90% CPE at 3–5 dpi. Whereas, the infected cells with low MOI of PPR vaccine virus took time to complete
Fig. 2. Reactivity of anti-nucleocapsid mAb 4G6 (2A) and anti-hemagglutinin mAb 4B11 (2B) with PPRV Sungri/96 passaged with variable MOIs 0.01, 0.5, 1 and 3 in cell ELISA. 6
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Fig. 3. Partial length amplification of nucleocapsid (351 bp) and hemagglutinin (851 bp) protein gene with MOI 0.01 (A & C) and MOI 3 (B & D) respectively. Lane M-DNA marker (100 bp; 1 kb), Lane P1–P10-serial passage 1–10.
observed with both the MOIs 1 and 3. Similar fluctuations were noticed with the band intensities of H gene of PPRV with MOI 0.5, 1 and 3. Lower intensities of bands observed after P6 with MOI 1 and as early as P2 with MOI 3 correspond to low copies of H gene. This finding indicated that the reason for the lower yield of PPR virus at high MOI passages than the passages with low MOI was reduction of total number of RNA genomes synthesized due to simultaneous generation of interfering particles that inhibited the normal replication process. Similar fluctuations in band intensities were observed while studying DI particles of polio and influenza virus using RT-PCR, rendering the DI genomes non-infectious [34,35]. However, with an amplification cycle of 35, no differences in amplicon intensities were observed with both the low and high MOIs. As the amplification cycle has been increased the viral RNA copies are exponentially amplified due to which the difference in copy numbers cannot be estimated. Analysis of copy number of PPRV Sungri/96 at P2, P4, P6, P8 and P10 by RT-qPCR targeting the nucleocapsid protein gene indicated significant decline and fluctuation in viral RNA copies with MOI 3, which is in support with the finding of Thompson et al. suggesting that as the dilution of virus inoculums decreases the virus yield dropped to maximum levels [25]. Fluctuations in infectivity titres and viral copies numbers were observed during the series of 10 undiluted passages with all the higher MOIs (0.5, 1 and 3) which could be due to uncontrolled MOI in successive passages as also reported by Whistler and coworkers [8]. Increase in the copy numbers in the later passages (P8–P10) as detected by RT-qPCR was because of abundant generation of DI particles which dominated the parent PPRV population and hence attenuated the characteristics CPE in the later passages. Our finding on PPR also supports the model higher DI particle multiplicities cause more interference of the parent virus population [25].
Table 5 Analysis of copy number of PPRV Sungri/96 vaccine virus of high MOI (0.5, 1 and 3) passages using RT-qPCR targeting the nucleocapsid (N) gene of PPRV. MOI
Passage no
Ct
log copy number
copy no
0.01 0.5
Control 2 4 6 8 10 2 4 6 8 10 2 4 6 8 10
16.16 18.53 24.67 17.72 17.1 18.32 19.1 17.3 25.89 16.95 22.08 25.06 16.9 26.75 18.36 25.19
5.71 4.99 3.13 5.24 5.42 5.05 4.82 5.36 2.76 5.47 3.91 3.01 5.49 2.50 5.04 2.97
512804 97926 1343 172445 265924 113400 65760 231248 573 295302 8200 1023 305800 314 110275 934
1
3
based on the OD values expressing sufficient antigen throughout 1–10 passages. Analysis of OD values of undiluted virus samples with both anti-N and anti-H mAb showed slightly lower and variable reactivity during initial passages, following an increase of OD values up to P10. These findings suggested that accumulation of DI particles during the serial undiluted passages might have lead to a persistent infection attenuating PPRV specific CPE, yet continuously expressing the viral proteins and normal packaging of virus, which could be detected by cell ELISA. Variation in viral protein expression in high MOI inoculums was more evident in H protein as compared to N protein, which indicates that H gene may get affected more as compared to N gene. Our findings are in agreement with the findings of Katayama et al. which described a method of quantitative detection of nucleoprotein (N) of both infective and defective interfering particles of Rabies virus using ELISA [33]. During the present investigation, 25 numbers of amplification cycles were used for partial length detection of N and H gene of PPRV passaged with various MOIs to find out the differences in viral RNA copies by reducing the exponential amplification. The band intensity obtained with both the genes using 25 cycles of amplification was also compared with amplicon intensity of PPRV obtained with RT-PCR with 35 amplification cycles (data not shown). Comparing the band intensities of PPR virus passaged with low MOI to that of the high MOIs in RT-PCR targeting the N gene, a fluctuating trend of band intensities were
5. Conclusion The findings demonstrated the evidence of generation of defective interfering particles while passaging PPRV Sungri/96 vaccine virus with higher MOIs viz 0.5, 1 and 3. In the present study, PPRV specific DI particles participated in attenuation impeding the standard virus replication. In view of this, use of recommended MOI is crucial during batch cultivation of vaccines to avoid the generation of such defective particles. The initial virus sample containing DI particles or generated during high MOI passages will lead to depletion of standard virus in a longer run. As a result, vaccine viruses will be developed containing DI particles in high concentration with interfering activity. Efforts should 7
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be made to obtain DI free vaccine preparations during upscaling of vaccine and a better understanding of how DI particles quantitatively impact the growth of virus is required in order to get uniform, consistent and quality vaccine batches.
[15] Lamb RA, Kolakofsky D. Fields BN, Knipe DM, Howley PM, editors. Paramyxoviridae: the viruses and their replication. Fields Virology. third ed.vol. I. Philadelphia-New York: Lippincott-Raven; 1996. p. 1176–204. [16] Frensing T, Pflugmacher A, Bachmann M, Peschel B, Reichl U. Impact of defective interfering particles on virus replication and antiviral host response in cell culturebased influenza vaccine production. Appl. Microbial.Biotechnol. 2014;98(21):8999–9008. [17] Sreenivasa BP, Dhar P, Singh RP, Bandyopadhyay SK. Evaluation of an indigenously developed homologous live attenuated cell culture vaccine against Peste-des-petitsruminants infection of small ruminants. Proceedings of XX Annual Conference of Indian Association of veterinary microbiologists, immunologists and specialists in infectious diseases (IAVMI), pantnagar, Uttaranchal, India, vol. 84. 2000. [18] Singh RP, Bandyopadhyay SK, Sreenivasa BP, Dhar P. Production and characterization of monoclonal antibodies to peste des petits ruminants (PPR) virus. Vet Res Commun 2004;28(7):623–39. [19] Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Hyg 1938;27:493–7. [20] Sarkar J, Belaineh G, Sreenivasa BP, Singh RP. Development of a cell-elisa using anti-nucleocapsid protein monoclonal antibody for the titration of peste des petits ruminants (PPR) vaccine virus. Indian J Comp Microbiol Immunol Infect Dis 2012;33(12):18–20. [21] Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162(1):156–9. [22] Yousuf RW. Application of combinatorial Lateral Flow Assay-qRT-PCR and RT-PCRElisa for confirmatory diagnosis of Peste des petits ruminants PhD Thesis IVRI: Division of Virology; 2018 [23] Couacy-Hymann E, Roger F, Hurard C, Guillou JP, Libeau G, Diallo A. Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay. J Virol Methods 2002;100(1–2):17–25. [24] Haq AA. Evaluation of monoclonal antibody resistant mutant of PPR vaccine virus as a candidate marker vaccine and application of biosensor based technique for PPR disease diagnosis PhD Thesis IVRI: Division of Virology; 2016 [25] Thompson KA, Rempala GA, Yin J. Multiple-hit inhibition of infection by defective interfering particles. J Gen Virol 2009;90(4):888–99. [26] Frensing T, Heldt FS, Pflugmacher A, Behrendt I, Jordan I, Flockerzi D, et al. Continuous influenza virus production in cell culture shows a periodic accumulation of defective interfering particles. PLoS One 2013;8(9):e72288. [27] DePolo NJ, Giachetti C, Holland JJ. Continuing co-evolution of virus and defective interfering particles and of viral genome sequences during undiluted passages: virus mutants exhibiting nearly complete resistance to formerly dominant defective interfering particles. J Virol 1987;61:454–64. [28] Parida S, Muniraju M, Mahapatra M, Muthuchelvan D, Buczkowski H, Banyard AC. Peste des petits ruminants. Vet Microbiol 2015;181(1):90–106. [29] Aggarwal K, Jing F, Maranga L, Liu J. Bioprocess optimization for cell culture based influenza vaccine production. Vaccine 2011;29:3320–8. [30] Bora M, Yousuf RW, Dhar P, Singh RP. An overview of process intensification and thermo stabilization for upscaling of Peste des petits ruminants vaccines in view of global control and eradication. VirusDisease 2018;29(3):285–96. [31] Singh RP, De UK, Pandey KD. Virological and antigenic characterization of two peste des petits ruminants (PPR) vaccine viruses of Indian origin. Comp Immunol Microbiol InfectDis 2010;33(4):343–53. [32] Fenner FJ, Gibbs EPJ, Murphy FA, Rott R, Studdert MJ, White DO. Virus replication. Veterinary virology. second ed. Academic Press Inc. (London) Ltd; 1993. [33] Katayama S, Yamanaka M, Ota S, Shimizu Y. A new quantitative method for Rabies virus by detection of nucleocapsid in virion using ELISA. J Vet Med Sci 1999;61(4):411–6. [34] Song Y, Paul AV, Wimmer E. Evolution of poliovirus defective interfering particles expressing Gaussia luciferase. J Virol 2012;86(4):1999–2010. [35] Wasik MA, Eichwald L, Genzel Y, Reichl U. Cell culture-based production of defective interfering particles for influenza antiviral therapy. Appl Microbiol Biotechnol 2018;102(3):1167–77.
Declaration of competing interest The authors declare no conflict of interest. Acknowledgements The authors would like to acknowledge the Director, ICAR-IVRI, Joint Director, Research and Joint Director, Academic, for the facilities to carry out this work. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biologicals.2019.09.008. References [1] FAO, OIE. Global strategy for the control and eradication of PPR. FAO and OIE; 2015. [2] FAO. Producing the right vaccine for the PPR global control and eradication programme. http://www.fao.org/ag/againfo/programmes/en/empres/news_181214b. html; 2014. [3] Singh RP, Bandyopadhyay SK. Peste des petits ruminants vaccine and vaccination in India: sharing experience with disease endemic countries. VirusDisease 2015;26(4):215–24. [4] Von Magnus P. Propagation of the PR8 strain of influenza A virus in chick embryos. IIL Properties of the incomplete virus produced in serial passages of undiluted virus. Acta Pathol Microbiol Scand 1951;29:157–81. [5] Thompson KAS, Yin J. Population dynamics of an RNA virus and its defective interfering particles in passage cultures. Virology 2010. 7-257. [6] Rima BK, Davidson WB, Martin SJ. The role of defective interfering particles in persistent infection of Vero cells by measles virus. J Gen Virol 1977;35(1):89–97. [7] Pathak KB, Nagy PD. Defective interfering RNAs: foes of viruses and friends of virologists. Viruses 2009;1(3):895–919. [8] Whistler T, Bellini WJ, Rota PA. Generation of defective interfering particles by two vaccine strains of measles virus. Virology 1996;220:480–4. [9] Brinton MA. Characterization of West Nile virus persistent infections in genetically resistant and susceptible mouse cells. I. Generation of defective nonplaquing virus particles. Virology 1982;116:84–98. [10] Roux L, Holland JJ. Role of defective interfering particles of Sendai virus in persistent infections. Virology 1979;93(1):91–103. [11] Lancaster MU, Hodgetts SI, Mackenzie JS, Urosevic N. Characterization of defective viral RNA produced during persistent infection of Vero cells with Murray Valley encephalitis virus. J Virol 1998;72:2474–82. [12] Ebner PD, Kim SK, O'Callaghan DJ. Biological and genotypic properties of defective interfering particles of equine herpesvirus 1 that mediate persistent infection. Virology 2008;381:98–105. [13] Dhar P. Studies on defective interfering particles of rinderpest virus PhD Thesis Izatnagar, Uttar Pradesh, India: Indian Veterinary Research Institute; 1997 [14] Dhar P, Bandyopadhyay SK. Defective interfering particles of morbillivirus: a review. Indian J Virol 1999;15(1):1–5.
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Characterization of defective interfering (DI) particles of Pestedes petitsruminants vaccine virus Sungri/96 strain-implications in vaccine upscaling Mousumi Boraa, Raja Wasim Yousufa, Pronab Dharb, M. Manub, Insha Zafira, Bina Mishraa, Kaushal Kishor Rajaka, Rabindra Prasad Singha,∗ a b
Division of Biological Products, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India Division of Biological Standardization, ICAR-Indian Veterinary Research Institute, Uttar Pradesh, India
ARTICLE INFO
ABSTRACT
Keywords: PPR virus Multiplicity of infection Defective interfering particles Cell ELISA RT-PCR RT-qPCR
The present investigation deals with the characterization of defective interfering (DI) particles of Peste-des-petits ruminants (PPR) vaccine Sungri/96 strain generated as a result of high MOI in Vero cells. During the serial 10 passages, infectivity titres drastically reduced from 6.5 to 2.25 log10TCID50/ml at high MOI. Further, attenuation of CPE with high MOI indicated generation of DI particles that resulted in no/slow progression of CPE during the late passages. Monoclonal antibody based cell ELISA indicated normal protein (N & H) packaging in samples with DI activity. At genomic level, inconsistency in amplicon intensity of H gene was observed in RT-PCR, indicating a possible defect of H gene. Further analysis of copy number of PPRV by RT-qPCR indicated intermittent fluctuations of viral genomic RNA copies. The significant decline of viral RNA copies with MOI 3 (314 copies) compared to low MOI (512804 copies), proved that higher DI multiplicities cause more interference with the replication process of the standard virus. Therefore, MOI is critical for manufacturing of vaccines. These investigations will help in upscaling of PPR vaccines in view of ongoing National and Global PPR control and eradication programme.
1. Introduction Peste-des-petits ruminants (PPR) is a highly contagious and transboundary viral disease of small ruminants causing severe economic impact on the rural economies and genetic resources of around 70 countries of Africa, Middle-East and Asia [1]. The control of PPR is mainly achieved through vaccination with the available live attenuated vaccines. With the ongoing Global PPR control and eradication strategy production of quality vaccines in right quantities and their timely delivery will be necessary to eradicate the disease in near future [2].Currently, the vaccine strain, PPRV Sungri/96 is used extensively by several commercial firms and government vaccine manufacturing unit for production of PPR vaccines throughout India [3]. However, on several occasions, instances of low virus titres or insufficient infectious virus particles were often experienced by laboratory personals during PPR vaccine production which could be related to use of inappropriate multiplicity of infection (MOI) or possible formation of Defective Interfering (DI) particles. DI particles are biologically active, non-infectious forms of viruses which arise as a result of serial passages at high
∗
MOI [4,5]. They are termed as ‘defective’ as they have lost the capacity to code for the necessary proteins required for independent replication and thus are defective in absence of the parent virus [6]. Broadly, ‘interfering’ refers to the ability of DI particles to reduce titers and thereby interfere with the production of helper virus. This interference results in attenuation of cytopathic effects or symptoms caused by the helper virus [7]. The frequency of their generation is influenced by several factors such as strain of the virus, cell type, virus passage history, replication event and generation interval [5,8]. Generation of DI particles have been commonly observed in vitro during serial passages at high MOIs and often have been invoked to explain the conversion of acute in vitro infections to persistent ones [9–12]. Such non-infectious particles have been documented in Measles [8] and Rinderpest virus [13] which are antigenically similar to Pestedes-petits-ruminants virus (PPRV) suggesting that these particles may resist a desirable humoral immune response by interfering with the standard viral replication [14]. DI RNA of Paramyxovirus originates from the 5′ end of the standard genome as a result of replicative errors either by copy back mechanisms or by creating internal deletions
Corresponding author. Division of Biological Products, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243 122, Uttar Pradesh, India. E-mail addresses: [email protected], [email protected] (R.P. Singh).
https://doi.org/10.1016/j.biologicals.2019.09.008 Received 18 May 2019; Received in revised form 3 August 2019; Accepted 24 September 2019 1045-1056/ © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Mousumi Bora, et al., Biologicals, https://doi.org/10.1016/j.biologicals.2019.09.008
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resulting in formation of shorter genome [15]. Such short genomes are then transferred from one generation to the next with each passage leading to rapid accumulation of DI particles [16]. Even though, information on generation of DI particles of closely related vaccine viruses like Measles and Rinderpest is available, the significance of PPRV specific DI particles are yet to be investigated. In the present study, we explored the possible formation of DI particles by making regular passages at low as well as high MOIs using PPRV Sungri/96 vaccine virus strain. The dominance of DI particle and the effect of its interfering activity leading to loss of cytopathic effect was analyzed and discussed. The findings may have implications on upscaling of PPR vaccine in view of ongoing global PPR control and eradication program [1].
2.5. Antigenic and physical characterization of PPRV specific DI particles 2.5.1. Changes in cytopathic effects (CPE) and duration of virus harvest PPRV induced CPE were critically recorded at 24 h interval till the time of harvest. Any unusual changes in CPE and duration of virus harvest with different MOIs were recorded at regular intervals. The infected monolayers at each passage level were harvested when 70–80% of CPE was observed. The virus was aliquoted and stored at −20 °C for further use. 2.5.2. Virus infectivity assay Virus titration was carried out in 96 well cell culture plates using Vero cells. Briefly, co-cultivation of 10 fold dilution of each virus sample was carried out with 104 cells/well of the micro titre plate using three replicates per dilution. The plates were incubated at 37 °C with 5% CO2 and 100% relative humidity. The media of the plates were changed with maintenance media every 48 h and the cytopathic effects in each well were recorded. After taking the final reading, the titres were calculated using Reed and Muench method [19].
2. Materials and methods 2.1. Cells Vero cells (ATCC®, CCL-81) available at Division of Biological Products, ICAR-IVRI, were revived and routinely subcultured in Eagle Minimum Essential Media (EMEM) (Sigma Aldrich) supplemented with 10% Fetal bovine serum (Gibco, Invitrogen), 200 mM L-glutamine and 100 mM sodium pyruvate (Himedia). For maintaining the infected cells, EMEM with 2% FBS was used as maintenance medium.
2.5.3. Binding efficacy of PPRV of high MOI passages using mAb based cell ELISA Cell ELISA was employed as previously described by us [20]. Briefly, 100 μl of PPR vaccine virus propagated with different MOIs were inoculated in Vero cells in 96 well plates for titration. After visualization of PPR specific CPE on 5th day of titration, the culture fluid of each well was discarded gently. Infected cells were fixed and permeabilized by incubating with 100 μl of 80% chilled acetone per well for 30 min at −20 °C and air dried. The wells with fixed cells were blocked using 200 μl of blocking buffer for an hour after which 100 μl of primary antibody (mAb 4G6 and 4B11 against N and H protein of PPRV diluted in 1:20 and 1:24 in blocking buffer respectively) was added to the cells in duplicates and incubated for 1 h at 37 °C. After three washings, Rabbit anti-mouse IgG conjugated to HRPO (Sigma) in blocking buffer (1:1000) was added and incubated for 1 h at 37 °C. Colour reaction developed after addition of substrate was stopped using equal volume of 1 M H2SO4 and the optical density (OD) was measured at 492 nm (A492). A cut-off value with two times the absorbance (A492), compared to mock infected cells was set for declaring a well as positive.
2.2. Preparation of virus stock The vaccine virus, PPRV Sungri/96 strain (Passage 60 in Vero cells) was used in the present study [17]. The seed vaccine virus was propagated in Vero cells at a MOI of 0.01 in 300 cm2 tissue culture flasks (TPP). After 48 h of incubation, the media was removed and replenished with maintenance media. PPRV induced cytopathic effects were observed and recorded at regular intervals. The infected cells were harvested when around 70–80% of cytopathic effects were observed and stored at −20 °C for further use. 2.3. Monoclonal antibodies
2.6. Nucleic acid based characterization of PPRV specific DI particles
The PPR vaccine virus (Sungri/96 strain) neutralizing monoclonal antibody (mAb) against hemagglutinin (H) protein from hybridoma clone 4B11 and nucleocapsid (N) protein from hybridoma clone 4G6 [18], available at Division of Biological Products, ICAR-Indian Veterinary Research Institute were used for the study.
2.6.1. Samples, RNA extraction and cDNA synthesis PPRV aliquots, from low and high MOI passages were processed for total RNA extraction using Trizol method described by Chomczynski and Sacchi [21]. The RNA was reverse transcribed using random hexamer and oligo (dT) primers and M-MLV reverse transcriptase (First Strand cDNA Synthesis Kit, Promega, Madison) as per manufacturer's protocol. The cDNA thus prepared was stored at −20 °C until use.
2.4. Serial passage of PPR virus through co-cultivation method The virus stock having an initial titre of 6.5 log10 TCID50/ml was inoculated in Vero cells with three different MOIs viz. 0.5, 1 and 3 using method of co-cultivation including the low MOI 0.01 used for PPR vaccine propagation. The experiment involved 48 h old Vero cell monolayer grown in 25 cm2 tissue culture flasks. Before infection, two 25 cm2 tissue culture flasks were trypinized, pooled and split in the ratio of 1:5 with an initial cell density of 0.6 × 106 cells per flask. The infected cells were harvested upon manifestation of 70–80% cytopathic effects. The first fixed MOI passaged viruses during passage 1 & 2, were then followed by a series of infection for which the infected cells from the preceding passage served as the inoculums for the subsequent infection. Between passage 3 to 10, only relative dilutions of inoculums were taken in to account and virus titers were not known at the time of inoculation. However all the virus inoculums were titrated subsequently post infection. Healthy Vero cells were kept as control in each passage level.
2.6.2. Primers and probe The primers sets NP3/NP4 against the nucleocapsid (N) and PPRVHF1/HR1 against hemagglutinin (H) protein gene were custom synthesized from IDT (USA) for conventional RT-PCR. The TaqMan® probe previously designed by Yousuf [22] targeting the nucleocapsid (N) protein gene of PPRV was used for quantitative PCR. The primer sets and probe used in the present study are tabulated (Table 1). 2.6.3. Reverse transcription polymerase chain reaction (RT-PCR) RT-PCR was carried out in 25 μl of total reaction volume containing 12.5 μl of 2X PCR master mix (Sapphire), 1 μl of forward and reverse primers (10pM), 8 μl of nuclease free water (NFW) and 2.5 μl of cDNA template. For each PCR run, positive and negative controls were also prepared using known positive cDNA and NFW respectively as template. The PCR components were mixed properly and thermal cycled as per the conditions mentioned in Table 2. 2
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Table 1 Details of primers and probe used in the present study. Target gene
Primer/probe
Oligonucleotide sequence (5′-3′)
Bases
Amplicon size
References
N
NP3 NP4 PPRV-HF1 PPRV-HR1 PPR_F PPR_R PPR_M
TCT CGG AAA TCG CCT CAC AGA CTC CCT CCT CCT GGT CCT CCA GAA TCT ACG CAC GAA ACC CAG CCT CC GGC ATC GGA TCT CCA GAC GG CAC ACA ACA AAG GAG AGT GAT TG ATG AAC CGC CGA AGT GAT AG FAM-AAT TTC GCC TTG ACA AAG GGT GGC-MGBNFQ
24 24 20 20 23 20 24
351 bp
[23]
851 bp
[24]
102 bp
[22]
H N
2.6.4. Agarose gel electrophoresis Agarose gel (1.5% for N gene and 1% for H gene) was prepared in 1X TAE buffer and was cast containing ethidium bromide (0.5 μg/ml). After casting, gel was completely immersed in electrophoresis tank containing 1X TAE. Then the PCR products of samples, positive and negative control sample were loaded in wells alongside the DNA marker (Fermentas, USA). The gel was electrophoresed at 80 V for 1 h for resolving PCR products and visualized under Gel DocTM XR + System (Bio-Rad, USA) for documentation of results.
MOIs 1 and 3 showed atypical cytopathic changes, such as extensive granulation, cording and heaping of cells (Fig. 1B). No syncytia formation was observed after P6 with MOIs 1 and 3 and areas of cell degeneration were progressively filled in by surrounding healthy intact cells. Similar atypical changes were observed in monolayer infected with MOI 0.5 by P8. By P10, no characteristic CPE could be detected upto 10 dpi with all the three high MOI passages (0.5, 1 and 3). The infected cells at P10 with the high MOI inoculums were harvested at 10 dpi with around 5–10% CPE clearly indicating the interference of the standard virus replication with the generation of PPRV specific DI particles. At passage 10, a declining trend of CPE was also observed with the time (Fig. 1C). The percentage of CPE observed in the serial passages (1–10) at different dpi of PPR vaccine virus with various MOIs is represented in Table 3.
2.6.5. Quantitative polymerase chain reaction (RT-qPCR) The two step RT-qPCR was carried out as described by Yousuf (2018) [22]. Briefly, 10 μL reaction volume containing 9 pmol of each primer, 2.5 pmol of TaqMan® hydrolysis probe, 1 μL of cDNA, 5 μL of 2× TaqMan® fast advanced master mix containing Hotstart Taq DNA polymerase, dNTPs and MgCl2 was used. The optimized cycling conditions included Uracil-DNA Glycoylase (UDG) incubation at 50 °C for 2 min and 1 s followed by 20 s hold at 50 °C and then 40 cycles of denaturation at 95 °C for 5 s with combined primer annealing and extension step at 60 °C for 30 s (2-step cycling) in a real time thermal cycler (MxAria, Agilent Technologies, USA). For calculation of copy number of samples, PPR vaccine virus at low MOI (0.01) was taken as reference control. The copy numbers were deduced from the standard curve Y (Cq or Δ Rn) = −3.296* log (x) + 34.98 designed for PPRV using nucleocapsid plasmid (Yousuf, 2018) [22].
3.2. Infectivity titres at different MOIs The infectivity titre of stock virus sample having an initial titre of 6.5 log10 TCID50/ml was rapidly dropped to 2.25–2.75 log 10TCID50/ml by the end of passage 10 on serial passages with the higher MOIs. The infectivity titres of the vaccine virus propagated with MOI 3 dropped significantly from an initial stock titre of 6.5 log10TCID50/ml to 3.5log10TCID50/ml by P6 and by the end of P10 the infectivity titre was found to be 2.25 log10TCID50/ml. Similar trend of infectivity titres were observed with MOI 1 as evidenced by drop of titres to 3.5 log10 TCID50/ ml by P7 to 2.5 log10TCID50/ml by P10. However, in contrast, low MOI (0.01) resulted in higher infectious virus titres with a constant infectivity titre of 5.75–6.5 log10TCID50/ml throughout the ten serial passages in Vero cells. The trends of virus infectivity titre obtained with various MOIs at each passage level are represented in Table 4.
3. Results 3.1. Changes due to cytopathic effects (CPE) and duration of virus harvest Cells infected with the low MOI (0.01) of PPR vaccine virus showed characteristic CPE of cell rounding, ballooning and formation of syncytia after 2 days post inoculation (dpi) and attained maximum CPE (~70–80%) at around 5–6 dpi (Fig. 1A) which could be observed throughout 1–10 passages. However, in the initial four serial passages with all the higher MOIs (0.5, 1 and 3), onset of earlier CPE was observed 18 h post infection and the cells were harvested by 3–5 dpi with 80–90% CPE. By the sixth undiluted passage (P6), virus inoculums with
3.3. Binding efficacy of PPRV with high MOI passages using mAb based cell ELISA To detect the reactivity of PPRV Sungri/96 propagated with high MOIs, Cell ELISA was done using mAbs targeting N and H protein of PPRV. The binding efficacy of anti-N and anti-H mAbs with PPRV inoculums of low (0.01) and high MOIs (0.5, 1 and 3) were compared and assessed in terms of OD492 values. Based on the OD values, anti-N mAb
Table 2 Thermal cycling conditions for amplifying nucleocapsid (N) and hemagglutinin (H) protein gene using RT-PCR.
3
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Fig. 1. Cytopathic effects induced by PPRV/Sungri 96 virus at Passage 1 (1A), Passage 6 (1B) and Passage 10 (1C) with inoculums at various MOIs.
showed low reactivity with MOI 1 and 3 from the initial to 5th serial passage. However, with the same MOIs, an increase in OD values was observed from P6 to P10. In contrast, PPRV passaged with MOI 0.01 and 0.5 showed similar and consistent reactivity pattern with anti-N mAb based on the OD values using cell ELISA (Fig. 2A). The binding efficacy of anti-H mAb revealed reduced and more variable reactivity with PPRV Sungri/96 propagated with MOI 1 and 3 which can be observed with the comparable difference in OD values of inoculums with low MOI (0.01). Analysis of OD values of PPRV propagated with MOI 0.5 showed OD values relatively similar to low MOI throughout the series of 10 passages indicating less DI activity in the standard virus population. Reduced reactivity pattern of anti-H mAb was apparent from P1 to P7 with MOI 1 and P1 to P8 with MOI 3. However with both the higher MOIs (1 and 3), a fluctuating pattern of reactivity (based on OD values) was obtained in every consecutive passage till P7 and P8 (Fig. 2B).
compared to that of low MOI (Fig. 3B). Partial length amplification of the H protein gene from the low MOI passages or the diluted series resulted in consistent and uniform amplicon intensities with an amplicon size of 851 bp throughout the series of 10 passages (Fig. 3C). Comparison of band intensities of the serial passages propagated with MOI 0.5 (data not presented) revealed fluctuation in levels of amplicon intensity after P6 indicating defective nucleic acids as formation of DI particles. Inconsistent amplification of H gene was observed after P5 with MOI 1 (data not presented) and this phenomenon was observed from the initial passage itself (P2) with MOI 3 (Fig. 3D). 3.4.2. Estimation of viral RNA copies using RT-qPCR The copy number of PPRV from the alternate passages viz. P2, P4, P6, P8 and P10 passaged with MOI 0.5, 1 and 3 were analyzed in terms of copy number and compared with the reference control (Table 5). During the series of undiluted passages in Vero cells, cycling between maximum and minimum copy numbers of viral RNA occurred with every alternate passage which was apparent with MOI 1 and 3. The first significant decline in viral RNA copies was observed in P2 with MOI 3, (1023 copies) following a rise in P4 (305800 copies) which subsequently descends again in P6 (314 copies) following a cyclical pattern. Similar pattern was observed in passages with MOI 0.5 and 1. During the series of passages with MOI 1, a drop in copy numbers was detected in P6, whereas similar pattern of decline in copy numbers was observed in P4 passaged with MOI 0.5. The magnitude of difference of copy
3.4. Nucleic acid based characterization using RT-PCR and RT-qPCR 3.4.1. Detection of viral nucleic acid of PPRV using RT-PCR from low and high MOI passages In N-gene based RT-PCR, uniform and consistent amplicon intensities producing a specific amplicon of 351 bp was observed throughout all the passages (P1–P10) with low MOI 0.01 (Fig. 3A). However, with the same reaction conditions and concentration, inconsistency in amplicon intensity was observed with MOI 1 and 3 when 4
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Table 3 Percentage of CPE observed in the serial passages at different dpi of PPR vaccine virus with various MOIs (0.01, 0.5, 1 and 3).
A = Atypical CPE like heaping and cording of cells; no cell rounding or ballooning or formation of foci of infected cells were observed. S= Slow progression of CPE. H= Harvested.
number was lowest in P6 with MOI 3 which was found to be 314 as compared to 512804 copies of the standard reference control propagated with MOI 0.01.
virus, which are antigenically closely related to PPR virus. PPRV Sungri/96 strain is presently being extensively used as a vaccine candidate to control the disease in the Indian subcontinent and some of the countries in the Middle-East and South-Asia [3,28]. Instances of low vaccine titres of PPR virus were often experienced in several biological firms (unpublished data), which may be due to certain reasons such as use of lower MOI than the recommended MOI used for preparation of PPR vaccines or possible evolution of DI particles during batch cultivation of vaccines due to higher MOIs. Both could be a limiting factor for rapid large scale vaccine production [29]. Therefore, identification of critical process parameters such as cell concentration, multiplicity of infection and time point of harvest is crucial in developing effective and quality vaccines [30]. In order to generate defective interfering particles of PPR virus in Vero cells, PPRV Sungri/96 with an initial titre of 6.5 log10 TCID50/ml was serially passaged at high MOIs viz. 0.5, 1 and 3 including the low
4. Discussion Defective interfering (DI) particles arise spontaneously as a result of high multiplicities of infection during virus growth and replicate at the expense of standard helper virus during a co-infection [25]. Generation of DI particles during the process of batch cultivation of vaccines remains challenging to commercial vaccine manufacturers [26] and veterinary biological units to ensure the quality status of the manufactured vaccines. Further, evolution of mutant population of DI particles may adapt to circumvent the resistance of interference produced by the standard virus [27]. Evidence of generation of DI particles has been observed in vaccine strains of Measles [8] and Rinderpest [13] 5
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the replication before infecting the remaining surrounding healthy cells during second replication cycle. These observations were concomitant with our finding that PPRV Sungri/96 has an eclipse period of 12 h at MOI 1 and can be harvested by 72 h (3 dpi) with 70–80% of CPE as per the analysis of one step growth curve [31]. Two such replication cycles would be required if low MOIs (0.01) are used for virus propagation and therefore may take 5–6 days to induce 70–80% of CPE. Moreover, the eclipse period may change for Paramyxoviruses depending upon the multiplicity of infection [32]. The infectivity titre of stock vaccine virus (6.5 log10 TCID50/ml) dropped drastically to 2.25–2.75 log10 TCID50/ml by the end of P10 in case of all the high virus inoculums. The delayed cytopathic effects with the increase in serial undiluted virus passages was correlated with the reduction of virus yields of each passage by at least 0.5–1 log10 TCID50/ ml, with intermittent increase and decrease of virus yield, a phenomenon characteristic of formation of DI particles. Similar findings were observed during generation of DI particles of antigenically related Measles vaccine virus [8] and Rinderpest vaccine virus [13]. Absence of characteristic CPE by serial undiluted passage 5 with high MOI 1 and 3 might be due to effects of DI genomes as reported earlier for rinderpest virus [13]. Absence or no progression of CPE with all the higher MOIs (0.5, 1 and 3) at P10 might be due to interference with the standard virus replication thereby attenuating the typical cytopathic changes leading to a persistent infection as also reported for Measles [6], Sendai [10] and Murray Valley Encephalitis virus [11]. Further characterization of generated DI particles was done by mAb based cell ELISA using mAbs against N and H protein of PPRV. The vaccine virus propagated with low MOI 0.01 showed specific reactivity
Table 4 Infectivity titres after serial passage of PPRV Sungri/96 at different MOIs in Vero cells. Passage number
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
Multiplicity of infection (MOI) 0.01
0.5
1
3
6.5 5.5 6.2 6.25 6.5 6.25 6.5 6.25 6.25 5.75
6.5 7.2 5.5 5.5 5.5 5.5 5.5 3.75 4.25 2.5
6.5 6.5 5.75 5.5 5.25 4 3.5 3.5 2.75 2.5
6.5 6.5 6.25 5.75 5.5 3.5 4.75 3.75 2.75 2.25
The titres are represented in log10 TCID50/ml.
MOI 0.01 used for vaccine preparation. Visible CPE after 2 dpi with MOI 0.01 of PPRV Sungri/96 vaccine virus includes cell rounding, ballooning and formation of syncytia [31]. Similar observation was made by us while investigating the growth of PPRV Sungri/96 in Vero cells infected with a low MOI 0.01. The speed of cytopathic effect was relatively slow and initiation of characteristic CPE was observed after 2 dpi. The infected cells were harvested at 5 dpi when 70–80% of CPE was manifested. The early onset of CPE in the initial four passages with high MOIs (0.5, 1 and 3) was due to infection of majority of the cells simultaneously producing 80–90% CPE at 3–5 dpi. Whereas, the infected cells with low MOI of PPR vaccine virus took time to complete
Fig. 2. Reactivity of anti-nucleocapsid mAb 4G6 (2A) and anti-hemagglutinin mAb 4B11 (2B) with PPRV Sungri/96 passaged with variable MOIs 0.01, 0.5, 1 and 3 in cell ELISA. 6
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Fig. 3. Partial length amplification of nucleocapsid (351 bp) and hemagglutinin (851 bp) protein gene with MOI 0.01 (A & C) and MOI 3 (B & D) respectively. Lane M-DNA marker (100 bp; 1 kb), Lane P1–P10-serial passage 1–10.
observed with both the MOIs 1 and 3. Similar fluctuations were noticed with the band intensities of H gene of PPRV with MOI 0.5, 1 and 3. Lower intensities of bands observed after P6 with MOI 1 and as early as P2 with MOI 3 correspond to low copies of H gene. This finding indicated that the reason for the lower yield of PPR virus at high MOI passages than the passages with low MOI was reduction of total number of RNA genomes synthesized due to simultaneous generation of interfering particles that inhibited the normal replication process. Similar fluctuations in band intensities were observed while studying DI particles of polio and influenza virus using RT-PCR, rendering the DI genomes non-infectious [34,35]. However, with an amplification cycle of 35, no differences in amplicon intensities were observed with both the low and high MOIs. As the amplification cycle has been increased the viral RNA copies are exponentially amplified due to which the difference in copy numbers cannot be estimated. Analysis of copy number of PPRV Sungri/96 at P2, P4, P6, P8 and P10 by RT-qPCR targeting the nucleocapsid protein gene indicated significant decline and fluctuation in viral RNA copies with MOI 3, which is in support with the finding of Thompson et al. suggesting that as the dilution of virus inoculums decreases the virus yield dropped to maximum levels [25]. Fluctuations in infectivity titres and viral copies numbers were observed during the series of 10 undiluted passages with all the higher MOIs (0.5, 1 and 3) which could be due to uncontrolled MOI in successive passages as also reported by Whistler and coworkers [8]. Increase in the copy numbers in the later passages (P8–P10) as detected by RT-qPCR was because of abundant generation of DI particles which dominated the parent PPRV population and hence attenuated the characteristics CPE in the later passages. Our finding on PPR also supports the model higher DI particle multiplicities cause more interference of the parent virus population [25].
Table 5 Analysis of copy number of PPRV Sungri/96 vaccine virus of high MOI (0.5, 1 and 3) passages using RT-qPCR targeting the nucleocapsid (N) gene of PPRV. MOI
Passage no
Ct
log copy number
copy no
0.01 0.5
Control 2 4 6 8 10 2 4 6 8 10 2 4 6 8 10
16.16 18.53 24.67 17.72 17.1 18.32 19.1 17.3 25.89 16.95 22.08 25.06 16.9 26.75 18.36 25.19
5.71 4.99 3.13 5.24 5.42 5.05 4.82 5.36 2.76 5.47 3.91 3.01 5.49 2.50 5.04 2.97
512804 97926 1343 172445 265924 113400 65760 231248 573 295302 8200 1023 305800 314 110275 934
1
3
based on the OD values expressing sufficient antigen throughout 1–10 passages. Analysis of OD values of undiluted virus samples with both anti-N and anti-H mAb showed slightly lower and variable reactivity during initial passages, following an increase of OD values up to P10. These findings suggested that accumulation of DI particles during the serial undiluted passages might have lead to a persistent infection attenuating PPRV specific CPE, yet continuously expressing the viral proteins and normal packaging of virus, which could be detected by cell ELISA. Variation in viral protein expression in high MOI inoculums was more evident in H protein as compared to N protein, which indicates that H gene may get affected more as compared to N gene. Our findings are in agreement with the findings of Katayama et al. which described a method of quantitative detection of nucleoprotein (N) of both infective and defective interfering particles of Rabies virus using ELISA [33]. During the present investigation, 25 numbers of amplification cycles were used for partial length detection of N and H gene of PPRV passaged with various MOIs to find out the differences in viral RNA copies by reducing the exponential amplification. The band intensity obtained with both the genes using 25 cycles of amplification was also compared with amplicon intensity of PPRV obtained with RT-PCR with 35 amplification cycles (data not shown). Comparing the band intensities of PPR virus passaged with low MOI to that of the high MOIs in RT-PCR targeting the N gene, a fluctuating trend of band intensities were
5. Conclusion The findings demonstrated the evidence of generation of defective interfering particles while passaging PPRV Sungri/96 vaccine virus with higher MOIs viz 0.5, 1 and 3. In the present study, PPRV specific DI particles participated in attenuation impeding the standard virus replication. In view of this, use of recommended MOI is crucial during batch cultivation of vaccines to avoid the generation of such defective particles. The initial virus sample containing DI particles or generated during high MOI passages will lead to depletion of standard virus in a longer run. As a result, vaccine viruses will be developed containing DI particles in high concentration with interfering activity. Efforts should 7
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be made to obtain DI free vaccine preparations during upscaling of vaccine and a better understanding of how DI particles quantitatively impact the growth of virus is required in order to get uniform, consistent and quality vaccine batches.
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