- Email: [email protected]
Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence
Microbes and Infection 17 (2015) 228e236 www.elsevier.com/locate/micinf
Original article
Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence a, Maja Santak *, Maja Markusic a, Maja Lang Balija b, Sandra Kec Kopac b, Renata Jug a, c € Claes Orvell , Jelena Tomac d, Dubravko Forcic a a
Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Rockefellerova 10, 10 000 Zagreb, Croatia b Quality Control Department, Institute of Immunology, Svetonedeljska Cesta 14, 10 431 Sveta Nedelja, Croatia c Department of Laboratory Medicine, Karolinska University Hospital Huddinge, 14 186 Stockholm, Sweden d Department of Histology and Embryology, School of Medicine, Brace Branchetta 20, 51000 Rijeka, Croatia Received 28 July 2014; accepted 23 November 2014 Available online 3 December 2014
Abstract Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence 97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 50 untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence. These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Mumps virus; Attenuation; Neurovirulence; Defective interfering particles; Infectious virus
1. Introduction Mumps virus is a negative stranded RNA virus, a member of the Paramyxoviridae family. The mumps virus genome is organized as a non-segmented RNA molecule with the gene order 30 NP-P-M-F-SH-HN-L-50 encoding for nine proteins: nucleoprotein (NP), phosphoprotein (P)/V protein/I protein, matrix protein (M), fusion protein (F), small hydrophobic
* Corresponding author. Laboratory for Molecular Biomedicine, Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Rockefellerova 10, HR-10 000 Zagreb, Croatia. Tel.: þ385 1 6414 401. E-mail address: [email protected] (M. Santak).
protein (SH), hemagglutinin neuraminidase (HN) and polymerase (L). The virus usually causes acute mild, self-limiting disease with parotitis as hallmark, but severe complications, such as meningitis and encephalitis, may occur indicating neurotropic nature of this virus. In the prevaccine era, mumps was a leading cause of viral meningitis [1]. Mumps is considered as a vaccine preventable disease. National immunization programs have implemented live attenuated vaccine against mumps in one-dose or two-dose schedule. Two doses have been proven to give better protection and countries which have two-dose schedule in their vaccination programs reached high level of reduction in mumps incidence of 97% (reviewed in Ref. [2]). It has been
http://dx.doi.org/10.1016/j.micinf.2014.11.006 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
already a half a century since the vaccine against mumps has been available on the market and there have been only a few vaccine strains licensed so far. The fact that some of them were abandoned due to high frequency of unwanted side effects, such as aseptic meningitis, or low efficacy urged scientists to seek for the attenuation markers in order to find an optimal degree of attenuation for live attenuated vaccine. So far the universal genetic marker of the mumps virus attenuation has not been identified. In the study presented herein, we hypothesized that noninfectious viral particles, in particular defective interfering particles (DIPs), can contribute to the neuroattenuation of the mumps virus. This hypothesis was based on the findings that DIPs have been observed to attenuate disease in infected animals (reviewed in Refs. [3] and [4]) indicating a high potential of DIPs as a novel antiviral agent. Infectious viral particles draw major attention because of their critical role in replication and pathogenesis. Other subpopulations of the biologically active particles, which are intrinsically non-infectious, may largely accompany infectious virus. They have a potential to influence the course of pathogenesis through their capacity to stimulate or suppress antiviral responses intrinsic to the innate immune system and/or by direct interference with virus replication. The best studied population of non-infectious viral particles are DIPs. The term DIP was first proposed in 1970 after molecular analysis of DI RNA [5], although von Magnus described these entities as incomplete influenza viruses in 1940s [6]. DIPs are lacking a part of the genome and are defective for replication in the absence of the complete functional virus genome to provide the missing functions. DIPs exert a negative effect on the replication of the infectious virus in the process termed interference. The exact mechanism for generation of DIPs is not known. However, it is thought that viral polymerase makes an error whereupon it slips from the template and resumes the synthesis either on the nascent chain as a new template to generate a “copy-back” form of DIP or it resumes the elongation at the position closer to the 50 end of the antigenome what generates an “internal deletion” form of DIP. What is known is that serial high multiplicity passages accumulate DIPs. Although non-infectious, there is more and more evidence that DIPs influence viral virulence and pathogenesis in various ways. The replication of DIPs may directly interfere with the infectious virus replication since DIPs carry a strong anti-genomic promoter on both genome ends [7]. A more subtle way of interference is by modulating host antiviral response. It has been described for Sendai virus and vesicular stomatitis virus that DIPs are essential for the induction of interferon beta [8e10] showing that certain strains are better inducers than others depending on the type of DIP they are contaminated with. A report by Ref. [11] shows that adaptive immunity is also susceptible to the specific influence of DIPs which enhance maturation of dendritic cells again in strain specific manner. Altogether, the role of DIPs should be considered in the neuroattenuation of the viral vaccines.
229
2. Material and methods 2.1. Cells and viruses A549 (lung carcinoma cell line), 293T (human embryonic kidney cells) and Vero cells (African green monkey kidney cells) were purchased from ATCC. The Vero cells were maintained in Minimum Essential Medium with Hank's salts (MEM-H) (AppliChem) and A549 and 293T cells were cultivated in Dulbecco's Modification of Eagle's Medium (DMEM) (AppliChem). Media were supplemented with 10% fetal calf serum (FCS) (Moregate) and 50 mL/ml neomycin (Gibco-Life Technologies). Mumps virus wild type strains 9218/Zg98 (MuVi/ Zagreb.HRV/39.98) was isolated from a single case patient in 1998 in Zagreb, Croatia, and it was used as the second passage on Vero cells. MuVi/Zagreb.HRV/28.12 was collected from a limited outbreak in 2012, also in Zagreb, Croatia, and used as described in text. The L-Zagreb mumps vaccine, lot nr. 98/1, was produced at the Institute of Immunology, according to standard operating procedure. Virus titre was determined by the plaque test as described in Ref. [12]. 2.2. A rat-based neurovirulence test LEW/SsNHsd rats (Harlan Sprague Dawley) bred at the Institute of Immunology were used. All animal work was in accordance to Croatian Law on Animal Welfare (2013) which strictly complies with EC Directive (2010/63/EU). Each sample was inoculated in 30e40 animals (4e5 litters) except for the mock inoculation where 10 animals were inoculated. The test was performed essentially as described in Ref. [13] with minor modifications in the brain processing. The brain sections were immersed in the Cryofix gel (Biognost) and frozen in liquid nitrogen. Frozen tissue was cut with the cryostat into 15 mm-thick sections which were placed on glass slides and dried. The rat neurovirulence test (RNVT) score was calculated as the ratio/percentage of the area of the ventricle and the area of the whole brain. The data were analyzed with descriptive statistics using Statistica 6.0 software (StatSoft Inc.) and shown as Box and Whiskers plots. 2.3. Plaque reduction assay for quantification of DIPs To assay DIPs, a plaque reduction assay was performed as in Refs. [14] and [15]. The standard virus was a high-titre virus produced at MOI 0.0001 in Vero cells. The infectious virus in samples to be tested for DIP titre was inactivated by UV lamp at distance of 20 cm from the lamp for 25 s according to previously determined UV-dose. Inactivation of the infectious virus was confirmed by the plaque test. For the test, A549 cells were infected in monolayer simultaneously with the standard virus at MOI 3 and increasing amounts of the sample virus (1, 2.5, 10, 20, 50 and 100 mL). Virus was attached to the cells for 1 h at 35 C, then it was removed and the cell monolayer was washed twice with PBS. A fresh medium containing 2% FCS
230
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
was added and the cells were incubated for 48 h at 35 C. Then the medium was harvested, centrifuged at 1200 g for 5 min to remove cellular debris and plaque assayed to determine the titre of the infectious virus. The titre of DIP was calculated as follows: number of cells exposed/(dilution yielding 63% protection adsorption volume). 2.4. Hemagglutination Hemagglutination assay was performed according to [16] with exception that 0.5% guinea pig erythrocyte suspension was used. 2.5. Amplification and sequencing of genomic and DIP RNA For the sequencing of the complete viral genomes the genome was divided in portions which were amplified and sequenced on a 3130 Genetic Analyzer (Life Technologies) as described in Ref. [17]. Amplification of the DIP RNA was performed according to the method described by Refs. [18], which was adapted for mumps virus. RNA was isolated from 5 103 PFU, the L25 primer (50 ACC AAG GGG AGA AAG TAA AAT C 30 , position 15,384e15,363 nt) was added and the mixture was denatured at 70 C for 10 min and immediately cooled on ice. Reverse transcription was performed with MuLV reverse transcriptase under conditions 42 C for 89 min and 95 C for 5 min. Amplification was done with primer pair L25 and L23 (50 CTG ATG ATT GGC CCT TTA GGA 30 , position 14,780e14,760 nt) under conditions 95 C for 5 min, then 40 cycles 95 C for 30 s, 55 C for 20 s, 72 C for 2 min and finally 72 C for 10 min. PCR products were visualized by agarose gel electrophoresis and extracted for sequencing with the L23 and L25 primers. For the genomic RNA input control the reverse transcription was performed as described above except that random hexamers were used. Genomic region 6119e6575 nt was amplified with primers LZ1 (50 TCA ATA CAA TAT CAA GTA GTG TC 30 , position 6119e6141 nt) and LZ2 (50 GGT TCT GTG TTG TAT TGT GAT CC 30 , position 6575e6553 nt). PCR was done under conditions 94 C for 4 min, then 35 cycles 94 C/30 s, 50 C/20 s and 72 C/1 min, followed by final extension at 72 C for 7 min. All alignments were performed by using Clone Manager Suite software (Scientific & Educational Software). 2.6. Cloning of the HN gene and the assessment of HN expression The full-length HN genes of p2 and p5 were amplified from viral RNA with the primer pair p2p5START (50 AAT AAT AGA TCT ATG GAGCCTTCGAAATTC 30 ) and p2STOP (50 AAT AAT AGA TCT TTT TTT CTC AAT AAT CGA 30 ) or p5STOP (50 AAT AAT AGA TCT TTT TTT CTT AAT AAT CGA 30 ) (restriction site are underlined). The amplified HN cDNAs were digested with BglII and ligated into digested pSG5 expression vector (Agilent Technologies). The vector expressing the HN
gene of p2 was named p2_HN and the vector expressing the HN gene of p5 was named p5_HN. Vectors were electroporated into 293T cells which were lysed at different time points with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% Nadeoxycholate, 1% Igepal (Sigma)) with the addition of 0.5 mM DTT and cocktail of protease inhibitors Complete (Roche). Cell lysates (50 mg of proteins) were analysed by western blotting using mouse monoclonal antibody m1173 and secondary goat anti-mouse-AP antibody (Santa Cruz). 2.7. RNA isolation and quantitative RT-PCR The total cellular RNA was isolated with RNeasy Kit (Qiagen). Reverse transcription was performed with 2 mg of total RNA with MuLV reverse transcriptase and random hexamers. The mRNA abundance of the genes tested was measured by quantitative PCR performed on a 7500 Real-Time PCR System (Life Technologies). Primer pairs used for the quantification were: for LGP2 50 CTT TGA CTT CCT GCA GCA TT 30 and 50 CAA TGA GGT GGT CAG TCC AG 30 , for RIG-I 50 CTC TGC AGA AAG TGC AAA GC 30 and 50 GGC TTG GGATGT GGT CTA CT 30 , for MDA5 50 CGT CTT GGATAA GTG CAT GG 30 and 50 CCA CTT GAG GAC CAT CAA CTT G 30 , for ISG56 50 ACG GCT GCC TAA TTT ACA GC 30 and 50 GCT CTT CAG GGC TTC CTC AT 30 and for MxA 50 CAC GAC CAT CGG AAT CTT GA 30 and 50 CCA GCA GAT CCC TGA AATATG G 30 . The amount of the transcript was normalized against the GAPDH gene transcript amplified with the primer pair 50 AGA ACA TCA TCC CTG CCT CTA CTG 30 and 50 TGT CGC TGT TGA AGT CAG AGG AGA 30 . Final PCR mix contained: 1 Power SYBR Green PCR Master Mix (Life Technologies), 0.15 mM of each primer and 3 mL of cDNA. Thermal cycling conditions were: 95 C/10 min, 40 cycles 95 C/15 s, 60 C/ 1 min. The data were collected with the 7500 System SDS Software. All samples were done in triplicate. 2.8. Nucleotide sequence accession number Sequence data for MuVi/Zagreb.HRV/28.12 was submitted to GenBank under accession number KF481689. 2.9. Statistical analysis Statistical analysis was performed by using STATISTICA 7.0 software (StatSoft Inc., USA) and Prism 6 for Windows software Version 6.04 (GraphPad Software Inc., USA). Differences between groups in neurovirulence testing were assessed using Kruskall-Wallis One-Way ANOVA followed by Dunn's multiple comparison test. 3. Results 3.1. Non-infectious particles of the mumps vaccine attenuate wild type mumps virus neurovirulence Neurovirulence of mumps virus was assessed for many strains and variants in a newborn rat model (reviewed in
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
Ref. [19]) proving this model to be a highly sensitive indicator of the mumps virus neurovirulence. In the work presented here, we used this model to test a wild type mumps virus 9218/ Zg98 which showed high neurovirulence (median RNVT score 8.51, Fig. 1A). In contrast to that, mumps virus vaccine strain L-Zagreb, vaccine lot nr. 98/1, showed very low neurovirulence (median RNVT score 0.44, Fig. 1A). We further investigated if there are non-infectious particles present in 98/ 1 which are, at least partially, responsible for such a low RNVT score and which are able to decrease the
231
neurovirulence of 9218/Zg98 virus. As shown in Fig. 1A, UVinactivated 98/1 strongly decreased neurovirulence of 9218/ Zg98 (median RNVT score 0.17, Fig. 1A). This finding indicates that non-infectious particles present in 98/1 are very potent attenuating factor. The best studied non-infectious viral particles are defective interfering particles (DIPs) which occur as a by-product of the viral replication. We therefore used plaque reduction assay to quantify DIPs in both viral suspensions. Fig. 1B and Table 1 show that 98/1 used in the neurovirulence test had more than 3 log higher titre of DIPs
Fig. 1. Comparison of the wild type mumps virus 9218/Zg98 and the vaccine mumps virus L-Zagreb, lot nr. 98/1. (A) RNVT scores for the wild type mumps virus 9218/Zg98, the L-Zagreb vaccine lot nr. 98/1, the mixture of the wild type 9218/Zg98 and UV-inactivated L-Zagreb vaccine lot nr. 98/1 (98/1 UV) and the 98/1 UV only were tested in the newborn rat model. Dilution medium MEM-H(N) þ 2% FCS was used as mock inoculation. Significant differences between groups are shown with ** for p < 0.01, *** for p < 0.001 or **** for p < 0.0001. (B) Plaque reduction assay used to quantify DIPs in 9218/Zg98 and L-Zagreb vaccine. For the test, A549 cells were infected simultaneously with standard virus at MOI 3 and increasing amounts of sample virus being tested in which the infectious virus was UV-inactivated. Virus was attached for 1 h at 35 C, then it was removed and the cell monolayer was washed two times with PBS. After 48 h at 35 C the medium was harvested, and plaque assayed. According to a Poisson distribution of DIP, the dilution of the virus that resulted in 37% survival of plaque forming virus contained multiplicity of 1 DIP per cell. (C) RT-PCR for detection of DIP RNA for the viruses 9218/Zg98 and L-Zagreb vaccine and scheme of the DIP RNA structures. RNA was isolated from the viral solutions equivalent to 5 103 PFU, split in two and denatured together with either specific primer L25 for DIP amplification or with random hexamers for the genomic RNA input control. Amplification was done with the primers specific for DIPs or the complete region of the gene for the small hydrophobic protein. The sizes of the PCR products and DIP RNA as well as the nucleotide positions where the RNA polymerase switches (sw) the template and resumes (res) the synthesis of the new genomic RNA are indicated.
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
232
Table 1 Titres of infectious (IP) and defective interfering (DIP) particles in the 9218/ Zg98 and 98/1. Virus
IP titre (PFU/ml)
DIP titre (DIP/ml)
Number of DIPs per 104 PFU/ml
9218/Zg98 98/1
4.00 106 9.34 103
<104 8.69 104
<25 8.69 104
compared to 9218/Zg98 virus implying that DIPs might be indeed responsible for attenuation of the neurovirulence. Copy-back DI genomes of these two viruses amplified by RTPCR confirmed this finding (Fig. 1C), but also revealed that the dominant copy-back DI genome of 9218/Zg98 is almost twice the size of the dominant copy-back DI genome amplified from the 98/1 vaccine (1344 nt vs. 828 nt) where total of four major DI genomes were detected and sequenced (Fig. 1C). Attempts to detect internal deletion DIP genomes failed (data not shown) suggesting that there are no or very low levels of this type of DIP in the mumps virus suspensions analysed herein. 3.2. Deliberate attenuation of MuVi/Zagreb.HRV/28.12 in Vero cells In order to confirm active involvement of DIPs in the attenuation of the neurovirulence the mumps virus, we performed serial passages of a recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells. For the first passage (p1), Vero cells were overlayed with 0.5 ml of sterile filtered urine of the mumps patient. One week later, there was no visible cytopathic effect (CPE) in infected cells. Therefore, a complete supernatant of the first passage was transferred to a fresh Vero culture (p2). As soon as two days after the supernatant was transferred, there was a massive CPE in the cell layer (Fig. 2A). The supernatant was collected and the titre was determined with the plaque test. The next passages were done at MOI 0.05 in order to enrich viral suspension with DIPs. Fig. 2B shows the titres of viral suspensions during 16 passages (p2-p17) in Vero cells. Viruses grown at MOI 0.05 showed cyclic pattern with the lowest titre at p6 (3.78 log PFU/ml) and the highest titre at p13 (7.48 log PFU/ ml). At the level of the fifth passage (p5) very distinct type of CPE was developed when compared to the p2. The p5 viruses did not form any syncytia, indicating a persistent infection induced by DIPs, while the p2 virus formed large multinuclear syncytia (Fig. 2A).
All these findings indicated that the p5 virus should contain large amounts of DIPs and the p2 virus should have low level of DIPs. Fig. 2C and Table 2 show amounts of DIPs for the p2 and p5 viruses. The p5 virus had more than 10-fold higher titre of DIPs and the abundance of the DIPs in the dose used for the neurovirulence testing in the newborn rat model was 923.4 fold higher than for the p2 virus (Table 2). Additionally, copyback DI genomes amplified by RT-PCR (Fig. 2D) showed different size and amount of DIP for the two viruses. The p2 virus had very faint PCR band of 646 bp while the p5 virus had strong PCR band at 500 bp. The sequencing of the PCR bands showed different structure of DIPs: the p2 virus had DI genome of 1266 nt and the p5 virus had DI genome of 1104 nt. These results are a strong indicator that the p5 virus contains DIPs accumulated during only three passages on Vero cells. To further characterize the two viral suspensions, we investigated the interplay of the two viral suspensions and different segments of the interferon pathway. Fig. 2E shows the induction of the LGP2, RIG-I, MDA5, ISG56 and MxA genes in A549 cells upon infection with either p2, p5 or p2 with the addition of UV-inactivated p5. The p5 virus strongly induced mRNA of all five genes while the p2 virus was a very poor inducer of antiviral response in these cells. The addition of the UV-inactivated p5 to the p2 virus induced the expression of the analysed gene up to the same level as noninactivated p5 what indicates that DIPs act as the major inductor of the interferon pathway. Given that p5 was a better inducer of interferon pathway, we investigated the replication capacity of the p2 and p5 in interferon-deficient cells (Vero cells) and interferon-competent cells (A549 cells). As shown in Fig. 2F the titres of both viral suspensions are almost equal in Vero cells. In contrast to that, in A549 cells the p2 viral suspension had 0.9 log PFU/ml higher titre than the p5 at day 6. On the basis of this observation we conclude that the p5 virus mainly contains viral particles with emphasized immunostimulatory activity what has previously been proven to be a very intense characteristic of some DIPs. 3.3. Fifth passage of MuVi/Zagreb.HRV/28.12 in Vero cells shows attenuated neurovirulence in a newborn rat model The two MuVi/Zagreb.HRV/28.12 viruses (p2 and p5) were tested for neurovirulence in the newborn rat model. As shown
Fig. 2. Attenuation of the wild type mumps virus MuVi/Zagreb.HRV/28.12 on Vero cells and analysis of the DIP content in passage 2 (p2) and passage 5 (p5). (A) The appearance of the cytopathic effect in the p2 and p5 on Vero cells. (B) Titre of the virus from the second to seventeenth passage on Vero cells. Limit of the test is 2 logPFU/ ml. (C) Plaque reduction assay used to quantify DIPs in p2 and p5. For the test, A549 cells were infected simultaneously with standard virus at MOI 3 and increasing amounts of sample virus being tested in which the infectious virus was UV-inactivated. Virus was attached for 1 h at 35 C, then it was removed and the cell monolayer was washed two times with PBS. After 48 h at 35 C the medium was harvested, and plaque assayed. According to a Poisson distribution of DIP, the dilution of the virus that resulted in 37% survival of plaque forming virus contained multiplicity of 1 DIP per cell. (D) RT-PCR for detection of DIP RNA and scheme of the DIP RNA structures of p2 and p5. RNA was isolated from the viral solutions equivalent to 5 103 PFU, split in two and denatured together with either specific primer L25 for DIP amplification or with random hexamers for the genomic RNA input control. Amplification was done with the primers specific for DIPs or the complete region of the gene for the small hydrophobic protein. The sizes of the PCR products and DIP RNA as well as the nucleotide positions where the RNA polymerase switches (sw) the template and resumes (res) the synthesis of the new genomic RNA are indicated. (E) Induction of genes involved in interferon pathway (LGP2, MDA5, RIG-I, ISG56 and MxA) in A549 cells infected with the p2, p5 and the mixture of the p2 and UV inactivated p5 (p2 þ p5 UV) viruses at MOI 0.5 for 24 h. RNA abundance was measured by quantitative PCR. (F) Titre of the infectious viruses p2 and p5 grown in the IFN-deficient Vero cells or IFN-competent A549 cells measured by the plaque test.
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
233
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
234
Table 2 Titres of infectious (IP) and defective interfering (DIP) particles in the passage 2 (p2) and passage 5 (p5) of the MuVi/Zagreb.HRV/28.12. Virus
IP titre (PFU/ml)
DIP titre (DIP/ml)
Number of DIPs per 104 PFU/ml
p2 p5
8.24 105 1.05 104
3.25 104 3.80 105
3.92 102 3.62 105
in Fig. 3 the p2 virus behaved as expected for the wild type virus. The incidence and severity of hydrocephalus in this group resulted with median RNVT score 8.53. The p5 virus showed significantly decreased neurovirulence with median RNVT score 0.35 what was slightly higher than mock injected animals which had median RNVT score 0.13. This shows that neuroattenuated virus was obtained in only three additional passages in Vero cells. To investigate if the neuroattenuation of the p5 virus was caused by the action of non-infectious viral particles present in the suspension, the infectious virus in the p5 was UV-inactivated and added to the p2 virus. The severity of the hydrocephalus was drastically decreased from median RNVT score 8.53 for the p2 virus to 1.82 for the p2 with added UV-inactivated p5 virus (Fig. 3). Altogether, this finding suggests that the respectable difference in the amount of DIPs per dose for the two viral suspensions used in neurovirulence testing (approx. 3 logs, Table 2) is responsible for the attenuation of the neurovirulence of the p5 virus.
Fig. 3. The phenotype of the passaged MuVi/Zagreb.HRV/28.12 in the newborn rat model. RNVT scores for the p2 and p5 viruses. Additionally, RNVT scores for p2 with the added UV inactivated p5 virus are shown. Dilution medium MEM-H(N) þ 2% FCS was used as mock inoculation. Significant differences between groups are shown with * for p < 0.05, ** for p < 0.01 or **** for p < 0.0001.
4. Discussion 3.4. Sequence analyses of MuVi/Zagreb.HRV/28.12 at different passage levels Since the results presented above could be the result of the changes in the viral genome, we were urged to perform a complete nucleotide sequence analysis of the p2 and p5 viruses. A nucleotide sequence analysis did not reveal any difference except at the nucleotide position 8420 which is positioned in the 50 untranslated region (UTR) of the HN gene. As shown in Fig. 4A both the p2 and p5 viruses have a heterogeneous position 8420 with G prevailing over A and vice versa, respectively. 3.5. Analysis of the influence of mutation in the 50 UTR of the HN gene on its expression The mutations in non-coding regions of viral genes can strongly influence the regulation of gene expression and thus virus replication and virulence. Therefore, the both variants of the HN gene (p2_HN and p5_HN) containing the HN gene coding region and 50 UTR were cloned in the mammalian expression vector, electroporated in Vero cells and the expression was analysed posttransfection every 24 h for four days (Fig. 4B). No difference in the expression of the HN gene was observed at any of the analysed time points indicating that the nucleotide change in the 50 UTR of the HN gene does not affect the abundance of the HN protein during viral replication.
Mumps virus is highly neurotropic virus and wild infections often cause meningitis or encephalitis. Vaccination programs have decreased the number of mumps virus infections in the past three decades. Recently, a number of reports were published reporting a resurgence of mumps even in highly vaccinated populations. For this there are two possible explanations which may play their roles simultaneously: (a) the evolution of the virus resulted with viruses which are able to escape from the immunity developed upon vaccination and (b) the waning immunity. Whatever the root of the mumps resurgence is, things about mumps vaccine need to be changed. Most of the current vaccines against mumps were developed more than forty years ago and maybe the time has come to bring up a more up-to-date vaccine. Development of a new vaccine is a challenging process with highly uncertain outcome. So far, the development was empirical and based on the number of passages in the cell culture or embryonated eggs. Thus the vaccine viruses passed rarely through less than 15 passages without really knowing when to cease: 10 passages for Urabe AM9 [20], 17 passages for Jeryl Lynn [21], 14 passages for S-12 [22], 22 passages for Leningrad L-3 [23], 29 passages for L-Zagreb [23]and 30 passages for Rubini [24]. Knowing the mechanisms behind the mumps virus attenuation would enable a rational design of the vaccine devoid from the empirical approach and thus it would increase the certainty of the outcome.
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
Fig. 4. Analysis of the HN gene sequence and the influence of the difference on the level of expression of hemagglutinin neuraminidase. (A) Part of the electropherograms of the p2 and p5 viruses are shown. The heterogenic position 8420 is indicated by arrow. (B) Western blot analysis of the expression of the complete HN gene of p2 (p2_HN) or p5 (p5_HN) cloned in mammalian expression vector pSG5. Mock (M) transfection was performed with the same amount of empty vector. Actin was used as an input control. Time points posttransfection (p.t) are indicated.
In spite of the efforts in the past two decades to identify a universal genetic marker of the neurovirulence in the genome of mumps virus, it has not been identified so far. The probable reason for that is that a single position in a 15 kb genome might be responsible for the attenuation of a particular virus but it can hardly be applied to other mumps viruses because of other differences throughout the genome. The most studied mumps virus is Urabe AM9 vaccine strain because it was causally associated with postvaccinal aseptic meningitis what led to its withdrawal from the vaccination programs in several countries. There have been many studies trying to elucidate and understand what the marker of the neurovirulence of this virus is. A variant carrying mutation K335E in the HN gene was isolated from vaccinees with postvaccinal aseptic meningitis [25e27]. However, K at this position is also present in Jeryl Lynn vaccine, a widely accepted as non-neurovirulent strain. Also, as reported in Ref. [28] recombinant viruses containing either K or E at position 335 showed similar neurotoxicity in the newborn rat model. Furthermore, Shah et al. [29], identified fifteen additional genetic differences which might be responsible for difference in virulence. Sauder et al. [30], argued that searching for markers of virulence based on the consensus sequence only might be misleading and showed that the level of genetic heterogeneity at specific
235
genome sites can have profound neurovirulence phenotypic consequences. We took a different approach which may lead to a more general mechanism applicable to different mumps viruses. Our hypothesis was that non-infectious virus particles present in viral suspension may attenuate the infectious virus. This was based on the prophylactic effect of DIPs in animal models shown for VSV [31e34], reovirus [35], Semliki Forest virus [36] and influenza virus [37e39]. DIPs are produced during replication of many, if not all, RNA viruses [5,40]. Their presence in the live attenuated measles vaccine was confirmed [41,42] but their relevance for the attenuation of the vaccine virus has not been further studied. Our initial experiment with UV-inactivated L-Zagreb vaccine added to the wild virus 9218/Zg98 (Fig. 1) indicated that DIPs present in the vaccine may be responsible for the neuroattenuation of the wild type, neurovirulent virus even if the difference in the nucleotide sequence of these two viruses is 3.7%. To confirm that, we simulated the development of the mumps vaccine from the recent mumps virus isolate in Vero cells at MOI 0.05 in order to yield high levels of DIPs. The neurovirulence of this virus was substantially decreased and we ascribed this phenotype to the action of DIPs due to their overwhelmed amount (Fig. 2C and D, and Table 2) and the ability of the virus suspension to trigger interferon pathway (Fig. 2E), as the induction of interferon beta correlated with the presence of copy-back DIPs [10]. It is possible that certain virus strain or cell type will support the generation of DIPs more than others. It has been shown that some cell lines fail to generate DIPs even after 200 passages at high MOI [43]. In conclusion, the findings of this study reveal DIPs as a very potent and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine. We show that only three passages in cell culture were required to obtain neuroattenuated phenotype, although this may depend on the cell type, virus strain and the passaging protocol. This study also suggests that in a search for the attenuation markers in the mumps virus genome it is important to have in mind that as few as three passages in vitro can make a difference in the content of the non-infectious particles what may strongly affect neurovirulent phenotype. Conflict of interest Authors declare no conflict of interest. Acknowledgments This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia, grant #021-0212432-3123 (to M.S). References [1] Bang OH, Bang J. Involvement of central nervous system in mumps. Acta Med Scan 1943;113:487e505. [2] Galazka AM, Robertson SE, Kraigher A. Mumps and mumps vaccine: a global review. Bull World Health Organ 1999;77:3e14.
236
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
[3] Barrett AD, Dimmock NJ. Defective interfering viruses and infections of animals. Curr Top Microbiol Immunol 1986;128:55e84. [4] Marriott AC, Dimmock NJ. Defective interfering viruses and their potential as antiviral agents. Rev Med Virol 2010;20:51e62. [5] Huang AS, Baltimore D. Defective viral particles and viral disease processes. Nature 1970;226:325e7. [6] Von Magnus P. Incomplete forms of influenza virus. Adv Virus Res 1954;2:59e79. [7] Whelan SP, Wertz GW. The 50 terminal trailer region of vesicular stomatitis virus contains a position-dependent cis-acting signal for assembly of RNA into infectious particles. J Virol 1999;73:307e15. [8] Marcus PI, Sekellick MJ. Defective interfering particles with covalently linked [þ/]RNA induce interferon. Nature 1977;266:815e9. [9] Sekellick MJ, Marcus PI. Interferon induction by viruses. VIII. Vesicular stomatitis virus: [þ/]DI-011 particles induce interferon in the absence of standard virions. Virology 1982;117:280e5. [10] Strahle L, Garcin D, Kolakofsky D. Sendai virus defective-interfering genomes and the activation of interferon-beta. Virology 2006;351:101e11. [11] Yount JS, Kraus TA, Horvath CM, Moran TM, Lopez CB. A novel role for viral-defective interfering particles in enhancing dendritic cell maturation. J Immunol 2006;177:4503e13. [12] Forcic D, Kosutic-Gulija T, Santak M, Jug R, Ivancic-Jelecki J, Markusic M, et al. Comparisons of mumps virus potency estimates obtained by 50% cell culture infective dose assay and plaque assay. Vaccine 2010;28:1887e92. [13] Rubin SA, Afzal MA, Powell CL, Bentley ML, Auda GR, Taffs RE, et al. The rat-based neurovirulence safety test for the assessment of mumps virus neurovirulence in humans: an international collaborative study. J Infect Dis 2005;191:1123e8. [14] Treuhaft MW. A colorimetric assay for quantification of defective interfering particles of respiratory syncytial virus. J Gen Virol 1983;64:1301e9. [15] Marcus PI, Ngunjiri JM, Sekellick MJ. Dynamics of biologically active subpopulations of influenza virus: plaque-forming, noninfectious cellkilling, and defective interfering particles. J Virol 2009;83:8122e30. [16] Mahy BWJ, Kangro HO. Haemagglutination. In: Mahy BWJ, Kangro HO, editors. Virology methods manuals. London: Academic Press Ltd.; 1995. p. 41e3. [17] Ivancic J, Gulija TK, Forcic D, Baricevic M, Jug R, Mesko-Prejac M, et al. Genetic characterization of L-Zagreb mumps vaccine strain. Virus Res 2005;109:95e105. [18] Calain P, Curran J, Kolakofsky D, Roux L. Molecular cloning of natural copy-back defective interfering RNAs and their expression from DNA. Virol 1992;191:62e71. [19] Rubin SA, Afzal MA. Neurovirulence safety testing of mumps vaccineshistorical perspective and current status. Vaccine 2011;29:2850e5. [20] Yamanishi K, Takahashi M, Ueda S, Minekawa Y, Ogino T. Studies on live mumps virus vaccine. V. Development of a new mumps vaccine “AM 9” by plaque cloning. Biken J 1973;16:161e6. [21] WHO. The mumps vaccine. http://web.archive.org/web/ 20060423093835/http://www.who.int/vaccines-diseases/diseases/ mumps_vaccine.shtml. [22] Sassani A, Mirchamsy H, Shafyi A, Ahourai P, Razavi J, Gholami MR, et al. Development of a new live attenuated mumps virus vaccine in human diploid cells. Biologicals 1991;19:203e11. [23] Beck M, Welsz-Malecek R, Mesko-Prejac M, Radman V, Juzbasic M, Rajninger-Miholic M, et al. Mumps vaccine L-Zagreb, prepared in chick fibroblasts. I. Production and field trials. J Biol Stand 1989;17:85e90.
[24] Glu¨ck R, Hoskins JM, Wegmann A, Just M, Germanier R. Rubini, a new live attenuated mumps vaccine virus strain for human diploid cells. Dev Biol Stand 1986;65:29e35. [25] Brown EG, Dimock K, Wright KE. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid 335 of the hemagglutininneuraminidase gene with one form associated with disease. J Infect Dis 1996;174:619e22. [26] Afzal MA, Yates PJ, Minor PD. Nucleotide sequence at position 1081 of the hemagglutinin-neuraminidase gene in the mumps Urabe vaccine strain. J Infect Dis 1998;177:265e6. [27] Wright KE, Dimock K, Brown EG. Biological characteristics of genetic variants of Urabe AM9 mumps vaccine virus. Virus Res 2000;67:49e57. [28] Sauder CJ, Zhang CX, Link MA, Duprex WP, Carbone KM, Rubin SA. Presence of lysine at aa 335 of the hemagglutinin-neuraminidase protein of mumps virus vaccine strain Urabe AM9 is not a requirement for neurovirulence. Vaccine 2009;27:5822e9. [29] Shah D, Vidal S, Link MA, Rubin SA, Wright KE. Identification of genetic mutations associated with attenuation and changes in tropism of Urabe mumps virus. J Med Virol 2009;81:130e8. [30] Sauder CJ, Vandenburgh KM, Iskow RC, Malik T, Carbone KM, Rubin SA. Changes in mumps virus neurovirulence phenotype associated with quasispecies heterogeneity. Virology 2006;350:48e57. [31] Doyle M, Holland JJ. Prophylaxis and immunization in mice by use of virus-free defective T particles to protect against intracerebral infection by vesicular stomatitis virus. Proc Natl Acad Sci U S A 1973;70:2105e8. [32] Rabinowitz SG, Dal Canto MC, Johnson TC. Infection of the central nervous system produced by mixtures of defective-interfering particles and wild-type vesicular stomatitis virus in mice. J Infect Dis 1977;136:59e74. [33] Jones CL, Holland JJ. Requirements for DI particle prophylaxis against vesicular stomatitis virus infection in vivo. J Gen Virol 1980;49:215e20. [34] Cave DR, Hendrickson FM, Huang AS. Defective interfering virus particles modulate virulence. J Virol 1985;55:366e73. [35] Spandidos DA, Graham AF. Generation of defective virus after infection of newborn rats with reovirus. J Virol 1976;20:234e7. [36] Barrett AD, Guest AR, Mackenzie A, Dimmock NJ. Protection of mice infected with a lethal dose of Semliki Forest virus by defective interfering virus: modulation of virus multiplication. J Gen Virol 1984;65:1909e20. [37] Rabinowitz SG, Huprikar J. The influence of defective-interfering particles of the PR-8 strain of influenza A virus on the pathogenesis of pulmonary infection in mice. J Infect Dis 1979;140:305e15. [38] Dimmock NJ, Beck S, McLain L. Protection of mice from lethal influenza: evidence that defective interfering virus modulates the immune response and not virus multiplication. J Gen Virol 1986;67:839e50. [39] Mann A, Marriott AC, Balasingam S, Lambkin R, Oxford JS, Dimmock NJ. Interfering vaccine (defective interfering influenza A virus) protects ferrets from influenza, and allows them to develop solid immunity to reinfection. Vaccine 2006;24:4290e6. [40] Perrault J. Origin and replication of defective interfering particles. Curr Top Microbiol Immun 1981;93:151e207. [41] Calain P, Roux L. Generation of measles virus defective interfering particles and their presence in a preparation of attenuated live-virus vaccine. J Virol 1988;62:2859e66. [42] Bellocq C, Mottet G, Roux L. Wide occurrence of measles virus subgenomic RNAs in attenuated live-virus vaccines. Biologicals 1990;18:337e43. [43] Stark C, Kennedy SI. The generation and propagation of defectiveinterfering particles of Semliki Forest virus in different cell types. Virology 1978;89:285e9.
Original article
Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence a, Maja Santak *, Maja Markusic a, Maja Lang Balija b, Sandra Kec Kopac b, Renata Jug a, c € Claes Orvell , Jelena Tomac d, Dubravko Forcic a a
Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Rockefellerova 10, 10 000 Zagreb, Croatia b Quality Control Department, Institute of Immunology, Svetonedeljska Cesta 14, 10 431 Sveta Nedelja, Croatia c Department of Laboratory Medicine, Karolinska University Hospital Huddinge, 14 186 Stockholm, Sweden d Department of Histology and Embryology, School of Medicine, Brace Branchetta 20, 51000 Rijeka, Croatia Received 28 July 2014; accepted 23 November 2014 Available online 3 December 2014
Abstract Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence 97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 50 untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence. These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Mumps virus; Attenuation; Neurovirulence; Defective interfering particles; Infectious virus
1. Introduction Mumps virus is a negative stranded RNA virus, a member of the Paramyxoviridae family. The mumps virus genome is organized as a non-segmented RNA molecule with the gene order 30 NP-P-M-F-SH-HN-L-50 encoding for nine proteins: nucleoprotein (NP), phosphoprotein (P)/V protein/I protein, matrix protein (M), fusion protein (F), small hydrophobic
* Corresponding author. Laboratory for Molecular Biomedicine, Centre for Research and Knowledge Transfer in Biotechnology, University of Zagreb, Rockefellerova 10, HR-10 000 Zagreb, Croatia. Tel.: þ385 1 6414 401. E-mail address: [email protected] (M. Santak).
protein (SH), hemagglutinin neuraminidase (HN) and polymerase (L). The virus usually causes acute mild, self-limiting disease with parotitis as hallmark, but severe complications, such as meningitis and encephalitis, may occur indicating neurotropic nature of this virus. In the prevaccine era, mumps was a leading cause of viral meningitis [1]. Mumps is considered as a vaccine preventable disease. National immunization programs have implemented live attenuated vaccine against mumps in one-dose or two-dose schedule. Two doses have been proven to give better protection and countries which have two-dose schedule in their vaccination programs reached high level of reduction in mumps incidence of 97% (reviewed in Ref. [2]). It has been
http://dx.doi.org/10.1016/j.micinf.2014.11.006 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
already a half a century since the vaccine against mumps has been available on the market and there have been only a few vaccine strains licensed so far. The fact that some of them were abandoned due to high frequency of unwanted side effects, such as aseptic meningitis, or low efficacy urged scientists to seek for the attenuation markers in order to find an optimal degree of attenuation for live attenuated vaccine. So far the universal genetic marker of the mumps virus attenuation has not been identified. In the study presented herein, we hypothesized that noninfectious viral particles, in particular defective interfering particles (DIPs), can contribute to the neuroattenuation of the mumps virus. This hypothesis was based on the findings that DIPs have been observed to attenuate disease in infected animals (reviewed in Refs. [3] and [4]) indicating a high potential of DIPs as a novel antiviral agent. Infectious viral particles draw major attention because of their critical role in replication and pathogenesis. Other subpopulations of the biologically active particles, which are intrinsically non-infectious, may largely accompany infectious virus. They have a potential to influence the course of pathogenesis through their capacity to stimulate or suppress antiviral responses intrinsic to the innate immune system and/or by direct interference with virus replication. The best studied population of non-infectious viral particles are DIPs. The term DIP was first proposed in 1970 after molecular analysis of DI RNA [5], although von Magnus described these entities as incomplete influenza viruses in 1940s [6]. DIPs are lacking a part of the genome and are defective for replication in the absence of the complete functional virus genome to provide the missing functions. DIPs exert a negative effect on the replication of the infectious virus in the process termed interference. The exact mechanism for generation of DIPs is not known. However, it is thought that viral polymerase makes an error whereupon it slips from the template and resumes the synthesis either on the nascent chain as a new template to generate a “copy-back” form of DIP or it resumes the elongation at the position closer to the 50 end of the antigenome what generates an “internal deletion” form of DIP. What is known is that serial high multiplicity passages accumulate DIPs. Although non-infectious, there is more and more evidence that DIPs influence viral virulence and pathogenesis in various ways. The replication of DIPs may directly interfere with the infectious virus replication since DIPs carry a strong anti-genomic promoter on both genome ends [7]. A more subtle way of interference is by modulating host antiviral response. It has been described for Sendai virus and vesicular stomatitis virus that DIPs are essential for the induction of interferon beta [8e10] showing that certain strains are better inducers than others depending on the type of DIP they are contaminated with. A report by Ref. [11] shows that adaptive immunity is also susceptible to the specific influence of DIPs which enhance maturation of dendritic cells again in strain specific manner. Altogether, the role of DIPs should be considered in the neuroattenuation of the viral vaccines.
229
2. Material and methods 2.1. Cells and viruses A549 (lung carcinoma cell line), 293T (human embryonic kidney cells) and Vero cells (African green monkey kidney cells) were purchased from ATCC. The Vero cells were maintained in Minimum Essential Medium with Hank's salts (MEM-H) (AppliChem) and A549 and 293T cells were cultivated in Dulbecco's Modification of Eagle's Medium (DMEM) (AppliChem). Media were supplemented with 10% fetal calf serum (FCS) (Moregate) and 50 mL/ml neomycin (Gibco-Life Technologies). Mumps virus wild type strains 9218/Zg98 (MuVi/ Zagreb.HRV/39.98) was isolated from a single case patient in 1998 in Zagreb, Croatia, and it was used as the second passage on Vero cells. MuVi/Zagreb.HRV/28.12 was collected from a limited outbreak in 2012, also in Zagreb, Croatia, and used as described in text. The L-Zagreb mumps vaccine, lot nr. 98/1, was produced at the Institute of Immunology, according to standard operating procedure. Virus titre was determined by the plaque test as described in Ref. [12]. 2.2. A rat-based neurovirulence test LEW/SsNHsd rats (Harlan Sprague Dawley) bred at the Institute of Immunology were used. All animal work was in accordance to Croatian Law on Animal Welfare (2013) which strictly complies with EC Directive (2010/63/EU). Each sample was inoculated in 30e40 animals (4e5 litters) except for the mock inoculation where 10 animals were inoculated. The test was performed essentially as described in Ref. [13] with minor modifications in the brain processing. The brain sections were immersed in the Cryofix gel (Biognost) and frozen in liquid nitrogen. Frozen tissue was cut with the cryostat into 15 mm-thick sections which were placed on glass slides and dried. The rat neurovirulence test (RNVT) score was calculated as the ratio/percentage of the area of the ventricle and the area of the whole brain. The data were analyzed with descriptive statistics using Statistica 6.0 software (StatSoft Inc.) and shown as Box and Whiskers plots. 2.3. Plaque reduction assay for quantification of DIPs To assay DIPs, a plaque reduction assay was performed as in Refs. [14] and [15]. The standard virus was a high-titre virus produced at MOI 0.0001 in Vero cells. The infectious virus in samples to be tested for DIP titre was inactivated by UV lamp at distance of 20 cm from the lamp for 25 s according to previously determined UV-dose. Inactivation of the infectious virus was confirmed by the plaque test. For the test, A549 cells were infected in monolayer simultaneously with the standard virus at MOI 3 and increasing amounts of the sample virus (1, 2.5, 10, 20, 50 and 100 mL). Virus was attached to the cells for 1 h at 35 C, then it was removed and the cell monolayer was washed twice with PBS. A fresh medium containing 2% FCS
230
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
was added and the cells were incubated for 48 h at 35 C. Then the medium was harvested, centrifuged at 1200 g for 5 min to remove cellular debris and plaque assayed to determine the titre of the infectious virus. The titre of DIP was calculated as follows: number of cells exposed/(dilution yielding 63% protection adsorption volume). 2.4. Hemagglutination Hemagglutination assay was performed according to [16] with exception that 0.5% guinea pig erythrocyte suspension was used. 2.5. Amplification and sequencing of genomic and DIP RNA For the sequencing of the complete viral genomes the genome was divided in portions which were amplified and sequenced on a 3130 Genetic Analyzer (Life Technologies) as described in Ref. [17]. Amplification of the DIP RNA was performed according to the method described by Refs. [18], which was adapted for mumps virus. RNA was isolated from 5 103 PFU, the L25 primer (50 ACC AAG GGG AGA AAG TAA AAT C 30 , position 15,384e15,363 nt) was added and the mixture was denatured at 70 C for 10 min and immediately cooled on ice. Reverse transcription was performed with MuLV reverse transcriptase under conditions 42 C for 89 min and 95 C for 5 min. Amplification was done with primer pair L25 and L23 (50 CTG ATG ATT GGC CCT TTA GGA 30 , position 14,780e14,760 nt) under conditions 95 C for 5 min, then 40 cycles 95 C for 30 s, 55 C for 20 s, 72 C for 2 min and finally 72 C for 10 min. PCR products were visualized by agarose gel electrophoresis and extracted for sequencing with the L23 and L25 primers. For the genomic RNA input control the reverse transcription was performed as described above except that random hexamers were used. Genomic region 6119e6575 nt was amplified with primers LZ1 (50 TCA ATA CAA TAT CAA GTA GTG TC 30 , position 6119e6141 nt) and LZ2 (50 GGT TCT GTG TTG TAT TGT GAT CC 30 , position 6575e6553 nt). PCR was done under conditions 94 C for 4 min, then 35 cycles 94 C/30 s, 50 C/20 s and 72 C/1 min, followed by final extension at 72 C for 7 min. All alignments were performed by using Clone Manager Suite software (Scientific & Educational Software). 2.6. Cloning of the HN gene and the assessment of HN expression The full-length HN genes of p2 and p5 were amplified from viral RNA with the primer pair p2p5START (50 AAT AAT AGA TCT ATG GAGCCTTCGAAATTC 30 ) and p2STOP (50 AAT AAT AGA TCT TTT TTT CTC AAT AAT CGA 30 ) or p5STOP (50 AAT AAT AGA TCT TTT TTT CTT AAT AAT CGA 30 ) (restriction site are underlined). The amplified HN cDNAs were digested with BglII and ligated into digested pSG5 expression vector (Agilent Technologies). The vector expressing the HN
gene of p2 was named p2_HN and the vector expressing the HN gene of p5 was named p5_HN. Vectors were electroporated into 293T cells which were lysed at different time points with RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% Nadeoxycholate, 1% Igepal (Sigma)) with the addition of 0.5 mM DTT and cocktail of protease inhibitors Complete (Roche). Cell lysates (50 mg of proteins) were analysed by western blotting using mouse monoclonal antibody m1173 and secondary goat anti-mouse-AP antibody (Santa Cruz). 2.7. RNA isolation and quantitative RT-PCR The total cellular RNA was isolated with RNeasy Kit (Qiagen). Reverse transcription was performed with 2 mg of total RNA with MuLV reverse transcriptase and random hexamers. The mRNA abundance of the genes tested was measured by quantitative PCR performed on a 7500 Real-Time PCR System (Life Technologies). Primer pairs used for the quantification were: for LGP2 50 CTT TGA CTT CCT GCA GCA TT 30 and 50 CAA TGA GGT GGT CAG TCC AG 30 , for RIG-I 50 CTC TGC AGA AAG TGC AAA GC 30 and 50 GGC TTG GGATGT GGT CTA CT 30 , for MDA5 50 CGT CTT GGATAA GTG CAT GG 30 and 50 CCA CTT GAG GAC CAT CAA CTT G 30 , for ISG56 50 ACG GCT GCC TAA TTT ACA GC 30 and 50 GCT CTT CAG GGC TTC CTC AT 30 and for MxA 50 CAC GAC CAT CGG AAT CTT GA 30 and 50 CCA GCA GAT CCC TGA AATATG G 30 . The amount of the transcript was normalized against the GAPDH gene transcript amplified with the primer pair 50 AGA ACA TCA TCC CTG CCT CTA CTG 30 and 50 TGT CGC TGT TGA AGT CAG AGG AGA 30 . Final PCR mix contained: 1 Power SYBR Green PCR Master Mix (Life Technologies), 0.15 mM of each primer and 3 mL of cDNA. Thermal cycling conditions were: 95 C/10 min, 40 cycles 95 C/15 s, 60 C/ 1 min. The data were collected with the 7500 System SDS Software. All samples were done in triplicate. 2.8. Nucleotide sequence accession number Sequence data for MuVi/Zagreb.HRV/28.12 was submitted to GenBank under accession number KF481689. 2.9. Statistical analysis Statistical analysis was performed by using STATISTICA 7.0 software (StatSoft Inc., USA) and Prism 6 for Windows software Version 6.04 (GraphPad Software Inc., USA). Differences between groups in neurovirulence testing were assessed using Kruskall-Wallis One-Way ANOVA followed by Dunn's multiple comparison test. 3. Results 3.1. Non-infectious particles of the mumps vaccine attenuate wild type mumps virus neurovirulence Neurovirulence of mumps virus was assessed for many strains and variants in a newborn rat model (reviewed in
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
Ref. [19]) proving this model to be a highly sensitive indicator of the mumps virus neurovirulence. In the work presented here, we used this model to test a wild type mumps virus 9218/ Zg98 which showed high neurovirulence (median RNVT score 8.51, Fig. 1A). In contrast to that, mumps virus vaccine strain L-Zagreb, vaccine lot nr. 98/1, showed very low neurovirulence (median RNVT score 0.44, Fig. 1A). We further investigated if there are non-infectious particles present in 98/ 1 which are, at least partially, responsible for such a low RNVT score and which are able to decrease the
231
neurovirulence of 9218/Zg98 virus. As shown in Fig. 1A, UVinactivated 98/1 strongly decreased neurovirulence of 9218/ Zg98 (median RNVT score 0.17, Fig. 1A). This finding indicates that non-infectious particles present in 98/1 are very potent attenuating factor. The best studied non-infectious viral particles are defective interfering particles (DIPs) which occur as a by-product of the viral replication. We therefore used plaque reduction assay to quantify DIPs in both viral suspensions. Fig. 1B and Table 1 show that 98/1 used in the neurovirulence test had more than 3 log higher titre of DIPs
Fig. 1. Comparison of the wild type mumps virus 9218/Zg98 and the vaccine mumps virus L-Zagreb, lot nr. 98/1. (A) RNVT scores for the wild type mumps virus 9218/Zg98, the L-Zagreb vaccine lot nr. 98/1, the mixture of the wild type 9218/Zg98 and UV-inactivated L-Zagreb vaccine lot nr. 98/1 (98/1 UV) and the 98/1 UV only were tested in the newborn rat model. Dilution medium MEM-H(N) þ 2% FCS was used as mock inoculation. Significant differences between groups are shown with ** for p < 0.01, *** for p < 0.001 or **** for p < 0.0001. (B) Plaque reduction assay used to quantify DIPs in 9218/Zg98 and L-Zagreb vaccine. For the test, A549 cells were infected simultaneously with standard virus at MOI 3 and increasing amounts of sample virus being tested in which the infectious virus was UV-inactivated. Virus was attached for 1 h at 35 C, then it was removed and the cell monolayer was washed two times with PBS. After 48 h at 35 C the medium was harvested, and plaque assayed. According to a Poisson distribution of DIP, the dilution of the virus that resulted in 37% survival of plaque forming virus contained multiplicity of 1 DIP per cell. (C) RT-PCR for detection of DIP RNA for the viruses 9218/Zg98 and L-Zagreb vaccine and scheme of the DIP RNA structures. RNA was isolated from the viral solutions equivalent to 5 103 PFU, split in two and denatured together with either specific primer L25 for DIP amplification or with random hexamers for the genomic RNA input control. Amplification was done with the primers specific for DIPs or the complete region of the gene for the small hydrophobic protein. The sizes of the PCR products and DIP RNA as well as the nucleotide positions where the RNA polymerase switches (sw) the template and resumes (res) the synthesis of the new genomic RNA are indicated.
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
232
Table 1 Titres of infectious (IP) and defective interfering (DIP) particles in the 9218/ Zg98 and 98/1. Virus
IP titre (PFU/ml)
DIP titre (DIP/ml)
Number of DIPs per 104 PFU/ml
9218/Zg98 98/1
4.00 106 9.34 103
<104 8.69 104
<25 8.69 104
compared to 9218/Zg98 virus implying that DIPs might be indeed responsible for attenuation of the neurovirulence. Copy-back DI genomes of these two viruses amplified by RTPCR confirmed this finding (Fig. 1C), but also revealed that the dominant copy-back DI genome of 9218/Zg98 is almost twice the size of the dominant copy-back DI genome amplified from the 98/1 vaccine (1344 nt vs. 828 nt) where total of four major DI genomes were detected and sequenced (Fig. 1C). Attempts to detect internal deletion DIP genomes failed (data not shown) suggesting that there are no or very low levels of this type of DIP in the mumps virus suspensions analysed herein. 3.2. Deliberate attenuation of MuVi/Zagreb.HRV/28.12 in Vero cells In order to confirm active involvement of DIPs in the attenuation of the neurovirulence the mumps virus, we performed serial passages of a recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells. For the first passage (p1), Vero cells were overlayed with 0.5 ml of sterile filtered urine of the mumps patient. One week later, there was no visible cytopathic effect (CPE) in infected cells. Therefore, a complete supernatant of the first passage was transferred to a fresh Vero culture (p2). As soon as two days after the supernatant was transferred, there was a massive CPE in the cell layer (Fig. 2A). The supernatant was collected and the titre was determined with the plaque test. The next passages were done at MOI 0.05 in order to enrich viral suspension with DIPs. Fig. 2B shows the titres of viral suspensions during 16 passages (p2-p17) in Vero cells. Viruses grown at MOI 0.05 showed cyclic pattern with the lowest titre at p6 (3.78 log PFU/ml) and the highest titre at p13 (7.48 log PFU/ ml). At the level of the fifth passage (p5) very distinct type of CPE was developed when compared to the p2. The p5 viruses did not form any syncytia, indicating a persistent infection induced by DIPs, while the p2 virus formed large multinuclear syncytia (Fig. 2A).
All these findings indicated that the p5 virus should contain large amounts of DIPs and the p2 virus should have low level of DIPs. Fig. 2C and Table 2 show amounts of DIPs for the p2 and p5 viruses. The p5 virus had more than 10-fold higher titre of DIPs and the abundance of the DIPs in the dose used for the neurovirulence testing in the newborn rat model was 923.4 fold higher than for the p2 virus (Table 2). Additionally, copyback DI genomes amplified by RT-PCR (Fig. 2D) showed different size and amount of DIP for the two viruses. The p2 virus had very faint PCR band of 646 bp while the p5 virus had strong PCR band at 500 bp. The sequencing of the PCR bands showed different structure of DIPs: the p2 virus had DI genome of 1266 nt and the p5 virus had DI genome of 1104 nt. These results are a strong indicator that the p5 virus contains DIPs accumulated during only three passages on Vero cells. To further characterize the two viral suspensions, we investigated the interplay of the two viral suspensions and different segments of the interferon pathway. Fig. 2E shows the induction of the LGP2, RIG-I, MDA5, ISG56 and MxA genes in A549 cells upon infection with either p2, p5 or p2 with the addition of UV-inactivated p5. The p5 virus strongly induced mRNA of all five genes while the p2 virus was a very poor inducer of antiviral response in these cells. The addition of the UV-inactivated p5 to the p2 virus induced the expression of the analysed gene up to the same level as noninactivated p5 what indicates that DIPs act as the major inductor of the interferon pathway. Given that p5 was a better inducer of interferon pathway, we investigated the replication capacity of the p2 and p5 in interferon-deficient cells (Vero cells) and interferon-competent cells (A549 cells). As shown in Fig. 2F the titres of both viral suspensions are almost equal in Vero cells. In contrast to that, in A549 cells the p2 viral suspension had 0.9 log PFU/ml higher titre than the p5 at day 6. On the basis of this observation we conclude that the p5 virus mainly contains viral particles with emphasized immunostimulatory activity what has previously been proven to be a very intense characteristic of some DIPs. 3.3. Fifth passage of MuVi/Zagreb.HRV/28.12 in Vero cells shows attenuated neurovirulence in a newborn rat model The two MuVi/Zagreb.HRV/28.12 viruses (p2 and p5) were tested for neurovirulence in the newborn rat model. As shown
Fig. 2. Attenuation of the wild type mumps virus MuVi/Zagreb.HRV/28.12 on Vero cells and analysis of the DIP content in passage 2 (p2) and passage 5 (p5). (A) The appearance of the cytopathic effect in the p2 and p5 on Vero cells. (B) Titre of the virus from the second to seventeenth passage on Vero cells. Limit of the test is 2 logPFU/ ml. (C) Plaque reduction assay used to quantify DIPs in p2 and p5. For the test, A549 cells were infected simultaneously with standard virus at MOI 3 and increasing amounts of sample virus being tested in which the infectious virus was UV-inactivated. Virus was attached for 1 h at 35 C, then it was removed and the cell monolayer was washed two times with PBS. After 48 h at 35 C the medium was harvested, and plaque assayed. According to a Poisson distribution of DIP, the dilution of the virus that resulted in 37% survival of plaque forming virus contained multiplicity of 1 DIP per cell. (D) RT-PCR for detection of DIP RNA and scheme of the DIP RNA structures of p2 and p5. RNA was isolated from the viral solutions equivalent to 5 103 PFU, split in two and denatured together with either specific primer L25 for DIP amplification or with random hexamers for the genomic RNA input control. Amplification was done with the primers specific for DIPs or the complete region of the gene for the small hydrophobic protein. The sizes of the PCR products and DIP RNA as well as the nucleotide positions where the RNA polymerase switches (sw) the template and resumes (res) the synthesis of the new genomic RNA are indicated. (E) Induction of genes involved in interferon pathway (LGP2, MDA5, RIG-I, ISG56 and MxA) in A549 cells infected with the p2, p5 and the mixture of the p2 and UV inactivated p5 (p2 þ p5 UV) viruses at MOI 0.5 for 24 h. RNA abundance was measured by quantitative PCR. (F) Titre of the infectious viruses p2 and p5 grown in the IFN-deficient Vero cells or IFN-competent A549 cells measured by the plaque test.
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
233
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
234
Table 2 Titres of infectious (IP) and defective interfering (DIP) particles in the passage 2 (p2) and passage 5 (p5) of the MuVi/Zagreb.HRV/28.12. Virus
IP titre (PFU/ml)
DIP titre (DIP/ml)
Number of DIPs per 104 PFU/ml
p2 p5
8.24 105 1.05 104
3.25 104 3.80 105
3.92 102 3.62 105
in Fig. 3 the p2 virus behaved as expected for the wild type virus. The incidence and severity of hydrocephalus in this group resulted with median RNVT score 8.53. The p5 virus showed significantly decreased neurovirulence with median RNVT score 0.35 what was slightly higher than mock injected animals which had median RNVT score 0.13. This shows that neuroattenuated virus was obtained in only three additional passages in Vero cells. To investigate if the neuroattenuation of the p5 virus was caused by the action of non-infectious viral particles present in the suspension, the infectious virus in the p5 was UV-inactivated and added to the p2 virus. The severity of the hydrocephalus was drastically decreased from median RNVT score 8.53 for the p2 virus to 1.82 for the p2 with added UV-inactivated p5 virus (Fig. 3). Altogether, this finding suggests that the respectable difference in the amount of DIPs per dose for the two viral suspensions used in neurovirulence testing (approx. 3 logs, Table 2) is responsible for the attenuation of the neurovirulence of the p5 virus.
Fig. 3. The phenotype of the passaged MuVi/Zagreb.HRV/28.12 in the newborn rat model. RNVT scores for the p2 and p5 viruses. Additionally, RNVT scores for p2 with the added UV inactivated p5 virus are shown. Dilution medium MEM-H(N) þ 2% FCS was used as mock inoculation. Significant differences between groups are shown with * for p < 0.05, ** for p < 0.01 or **** for p < 0.0001.
4. Discussion 3.4. Sequence analyses of MuVi/Zagreb.HRV/28.12 at different passage levels Since the results presented above could be the result of the changes in the viral genome, we were urged to perform a complete nucleotide sequence analysis of the p2 and p5 viruses. A nucleotide sequence analysis did not reveal any difference except at the nucleotide position 8420 which is positioned in the 50 untranslated region (UTR) of the HN gene. As shown in Fig. 4A both the p2 and p5 viruses have a heterogeneous position 8420 with G prevailing over A and vice versa, respectively. 3.5. Analysis of the influence of mutation in the 50 UTR of the HN gene on its expression The mutations in non-coding regions of viral genes can strongly influence the regulation of gene expression and thus virus replication and virulence. Therefore, the both variants of the HN gene (p2_HN and p5_HN) containing the HN gene coding region and 50 UTR were cloned in the mammalian expression vector, electroporated in Vero cells and the expression was analysed posttransfection every 24 h for four days (Fig. 4B). No difference in the expression of the HN gene was observed at any of the analysed time points indicating that the nucleotide change in the 50 UTR of the HN gene does not affect the abundance of the HN protein during viral replication.
Mumps virus is highly neurotropic virus and wild infections often cause meningitis or encephalitis. Vaccination programs have decreased the number of mumps virus infections in the past three decades. Recently, a number of reports were published reporting a resurgence of mumps even in highly vaccinated populations. For this there are two possible explanations which may play their roles simultaneously: (a) the evolution of the virus resulted with viruses which are able to escape from the immunity developed upon vaccination and (b) the waning immunity. Whatever the root of the mumps resurgence is, things about mumps vaccine need to be changed. Most of the current vaccines against mumps were developed more than forty years ago and maybe the time has come to bring up a more up-to-date vaccine. Development of a new vaccine is a challenging process with highly uncertain outcome. So far, the development was empirical and based on the number of passages in the cell culture or embryonated eggs. Thus the vaccine viruses passed rarely through less than 15 passages without really knowing when to cease: 10 passages for Urabe AM9 [20], 17 passages for Jeryl Lynn [21], 14 passages for S-12 [22], 22 passages for Leningrad L-3 [23], 29 passages for L-Zagreb [23]and 30 passages for Rubini [24]. Knowing the mechanisms behind the mumps virus attenuation would enable a rational design of the vaccine devoid from the empirical approach and thus it would increase the certainty of the outcome.
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
Fig. 4. Analysis of the HN gene sequence and the influence of the difference on the level of expression of hemagglutinin neuraminidase. (A) Part of the electropherograms of the p2 and p5 viruses are shown. The heterogenic position 8420 is indicated by arrow. (B) Western blot analysis of the expression of the complete HN gene of p2 (p2_HN) or p5 (p5_HN) cloned in mammalian expression vector pSG5. Mock (M) transfection was performed with the same amount of empty vector. Actin was used as an input control. Time points posttransfection (p.t) are indicated.
In spite of the efforts in the past two decades to identify a universal genetic marker of the neurovirulence in the genome of mumps virus, it has not been identified so far. The probable reason for that is that a single position in a 15 kb genome might be responsible for the attenuation of a particular virus but it can hardly be applied to other mumps viruses because of other differences throughout the genome. The most studied mumps virus is Urabe AM9 vaccine strain because it was causally associated with postvaccinal aseptic meningitis what led to its withdrawal from the vaccination programs in several countries. There have been many studies trying to elucidate and understand what the marker of the neurovirulence of this virus is. A variant carrying mutation K335E in the HN gene was isolated from vaccinees with postvaccinal aseptic meningitis [25e27]. However, K at this position is also present in Jeryl Lynn vaccine, a widely accepted as non-neurovirulent strain. Also, as reported in Ref. [28] recombinant viruses containing either K or E at position 335 showed similar neurotoxicity in the newborn rat model. Furthermore, Shah et al. [29], identified fifteen additional genetic differences which might be responsible for difference in virulence. Sauder et al. [30], argued that searching for markers of virulence based on the consensus sequence only might be misleading and showed that the level of genetic heterogeneity at specific
235
genome sites can have profound neurovirulence phenotypic consequences. We took a different approach which may lead to a more general mechanism applicable to different mumps viruses. Our hypothesis was that non-infectious virus particles present in viral suspension may attenuate the infectious virus. This was based on the prophylactic effect of DIPs in animal models shown for VSV [31e34], reovirus [35], Semliki Forest virus [36] and influenza virus [37e39]. DIPs are produced during replication of many, if not all, RNA viruses [5,40]. Their presence in the live attenuated measles vaccine was confirmed [41,42] but their relevance for the attenuation of the vaccine virus has not been further studied. Our initial experiment with UV-inactivated L-Zagreb vaccine added to the wild virus 9218/Zg98 (Fig. 1) indicated that DIPs present in the vaccine may be responsible for the neuroattenuation of the wild type, neurovirulent virus even if the difference in the nucleotide sequence of these two viruses is 3.7%. To confirm that, we simulated the development of the mumps vaccine from the recent mumps virus isolate in Vero cells at MOI 0.05 in order to yield high levels of DIPs. The neurovirulence of this virus was substantially decreased and we ascribed this phenotype to the action of DIPs due to their overwhelmed amount (Fig. 2C and D, and Table 2) and the ability of the virus suspension to trigger interferon pathway (Fig. 2E), as the induction of interferon beta correlated with the presence of copy-back DIPs [10]. It is possible that certain virus strain or cell type will support the generation of DIPs more than others. It has been shown that some cell lines fail to generate DIPs even after 200 passages at high MOI [43]. In conclusion, the findings of this study reveal DIPs as a very potent and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine. We show that only three passages in cell culture were required to obtain neuroattenuated phenotype, although this may depend on the cell type, virus strain and the passaging protocol. This study also suggests that in a search for the attenuation markers in the mumps virus genome it is important to have in mind that as few as three passages in vitro can make a difference in the content of the non-infectious particles what may strongly affect neurovirulent phenotype. Conflict of interest Authors declare no conflict of interest. Acknowledgments This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia, grant #021-0212432-3123 (to M.S). References [1] Bang OH, Bang J. Involvement of central nervous system in mumps. Acta Med Scan 1943;113:487e505. [2] Galazka AM, Robertson SE, Kraigher A. Mumps and mumps vaccine: a global review. Bull World Health Organ 1999;77:3e14.
236
M. Santak et al. / Microbes and Infection 17 (2015) 228e236
[3] Barrett AD, Dimmock NJ. Defective interfering viruses and infections of animals. Curr Top Microbiol Immunol 1986;128:55e84. [4] Marriott AC, Dimmock NJ. Defective interfering viruses and their potential as antiviral agents. Rev Med Virol 2010;20:51e62. [5] Huang AS, Baltimore D. Defective viral particles and viral disease processes. Nature 1970;226:325e7. [6] Von Magnus P. Incomplete forms of influenza virus. Adv Virus Res 1954;2:59e79. [7] Whelan SP, Wertz GW. The 50 terminal trailer region of vesicular stomatitis virus contains a position-dependent cis-acting signal for assembly of RNA into infectious particles. J Virol 1999;73:307e15. [8] Marcus PI, Sekellick MJ. Defective interfering particles with covalently linked [þ/]RNA induce interferon. Nature 1977;266:815e9. [9] Sekellick MJ, Marcus PI. Interferon induction by viruses. VIII. Vesicular stomatitis virus: [þ/]DI-011 particles induce interferon in the absence of standard virions. Virology 1982;117:280e5. [10] Strahle L, Garcin D, Kolakofsky D. Sendai virus defective-interfering genomes and the activation of interferon-beta. Virology 2006;351:101e11. [11] Yount JS, Kraus TA, Horvath CM, Moran TM, Lopez CB. A novel role for viral-defective interfering particles in enhancing dendritic cell maturation. J Immunol 2006;177:4503e13. [12] Forcic D, Kosutic-Gulija T, Santak M, Jug R, Ivancic-Jelecki J, Markusic M, et al. Comparisons of mumps virus potency estimates obtained by 50% cell culture infective dose assay and plaque assay. Vaccine 2010;28:1887e92. [13] Rubin SA, Afzal MA, Powell CL, Bentley ML, Auda GR, Taffs RE, et al. The rat-based neurovirulence safety test for the assessment of mumps virus neurovirulence in humans: an international collaborative study. J Infect Dis 2005;191:1123e8. [14] Treuhaft MW. A colorimetric assay for quantification of defective interfering particles of respiratory syncytial virus. J Gen Virol 1983;64:1301e9. [15] Marcus PI, Ngunjiri JM, Sekellick MJ. Dynamics of biologically active subpopulations of influenza virus: plaque-forming, noninfectious cellkilling, and defective interfering particles. J Virol 2009;83:8122e30. [16] Mahy BWJ, Kangro HO. Haemagglutination. In: Mahy BWJ, Kangro HO, editors. Virology methods manuals. London: Academic Press Ltd.; 1995. p. 41e3. [17] Ivancic J, Gulija TK, Forcic D, Baricevic M, Jug R, Mesko-Prejac M, et al. Genetic characterization of L-Zagreb mumps vaccine strain. Virus Res 2005;109:95e105. [18] Calain P, Curran J, Kolakofsky D, Roux L. Molecular cloning of natural copy-back defective interfering RNAs and their expression from DNA. Virol 1992;191:62e71. [19] Rubin SA, Afzal MA. Neurovirulence safety testing of mumps vaccineshistorical perspective and current status. Vaccine 2011;29:2850e5. [20] Yamanishi K, Takahashi M, Ueda S, Minekawa Y, Ogino T. Studies on live mumps virus vaccine. V. Development of a new mumps vaccine “AM 9” by plaque cloning. Biken J 1973;16:161e6. [21] WHO. The mumps vaccine. http://web.archive.org/web/ 20060423093835/http://www.who.int/vaccines-diseases/diseases/ mumps_vaccine.shtml. [22] Sassani A, Mirchamsy H, Shafyi A, Ahourai P, Razavi J, Gholami MR, et al. Development of a new live attenuated mumps virus vaccine in human diploid cells. Biologicals 1991;19:203e11. [23] Beck M, Welsz-Malecek R, Mesko-Prejac M, Radman V, Juzbasic M, Rajninger-Miholic M, et al. Mumps vaccine L-Zagreb, prepared in chick fibroblasts. I. Production and field trials. J Biol Stand 1989;17:85e90.
[24] Glu¨ck R, Hoskins JM, Wegmann A, Just M, Germanier R. Rubini, a new live attenuated mumps vaccine virus strain for human diploid cells. Dev Biol Stand 1986;65:29e35. [25] Brown EG, Dimock K, Wright KE. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid 335 of the hemagglutininneuraminidase gene with one form associated with disease. J Infect Dis 1996;174:619e22. [26] Afzal MA, Yates PJ, Minor PD. Nucleotide sequence at position 1081 of the hemagglutinin-neuraminidase gene in the mumps Urabe vaccine strain. J Infect Dis 1998;177:265e6. [27] Wright KE, Dimock K, Brown EG. Biological characteristics of genetic variants of Urabe AM9 mumps vaccine virus. Virus Res 2000;67:49e57. [28] Sauder CJ, Zhang CX, Link MA, Duprex WP, Carbone KM, Rubin SA. Presence of lysine at aa 335 of the hemagglutinin-neuraminidase protein of mumps virus vaccine strain Urabe AM9 is not a requirement for neurovirulence. Vaccine 2009;27:5822e9. [29] Shah D, Vidal S, Link MA, Rubin SA, Wright KE. Identification of genetic mutations associated with attenuation and changes in tropism of Urabe mumps virus. J Med Virol 2009;81:130e8. [30] Sauder CJ, Vandenburgh KM, Iskow RC, Malik T, Carbone KM, Rubin SA. Changes in mumps virus neurovirulence phenotype associated with quasispecies heterogeneity. Virology 2006;350:48e57. [31] Doyle M, Holland JJ. Prophylaxis and immunization in mice by use of virus-free defective T particles to protect against intracerebral infection by vesicular stomatitis virus. Proc Natl Acad Sci U S A 1973;70:2105e8. [32] Rabinowitz SG, Dal Canto MC, Johnson TC. Infection of the central nervous system produced by mixtures of defective-interfering particles and wild-type vesicular stomatitis virus in mice. J Infect Dis 1977;136:59e74. [33] Jones CL, Holland JJ. Requirements for DI particle prophylaxis against vesicular stomatitis virus infection in vivo. J Gen Virol 1980;49:215e20. [34] Cave DR, Hendrickson FM, Huang AS. Defective interfering virus particles modulate virulence. J Virol 1985;55:366e73. [35] Spandidos DA, Graham AF. Generation of defective virus after infection of newborn rats with reovirus. J Virol 1976;20:234e7. [36] Barrett AD, Guest AR, Mackenzie A, Dimmock NJ. Protection of mice infected with a lethal dose of Semliki Forest virus by defective interfering virus: modulation of virus multiplication. J Gen Virol 1984;65:1909e20. [37] Rabinowitz SG, Huprikar J. The influence of defective-interfering particles of the PR-8 strain of influenza A virus on the pathogenesis of pulmonary infection in mice. J Infect Dis 1979;140:305e15. [38] Dimmock NJ, Beck S, McLain L. Protection of mice from lethal influenza: evidence that defective interfering virus modulates the immune response and not virus multiplication. J Gen Virol 1986;67:839e50. [39] Mann A, Marriott AC, Balasingam S, Lambkin R, Oxford JS, Dimmock NJ. Interfering vaccine (defective interfering influenza A virus) protects ferrets from influenza, and allows them to develop solid immunity to reinfection. Vaccine 2006;24:4290e6. [40] Perrault J. Origin and replication of defective interfering particles. Curr Top Microbiol Immun 1981;93:151e207. [41] Calain P, Roux L. Generation of measles virus defective interfering particles and their presence in a preparation of attenuated live-virus vaccine. J Virol 1988;62:2859e66. [42] Bellocq C, Mottet G, Roux L. Wide occurrence of measles virus subgenomic RNAs in attenuated live-virus vaccines. Biologicals 1990;18:337e43. [43] Stark C, Kennedy SI. The generation and propagation of defectiveinterfering particles of Semliki Forest virus in different cell types. Virology 1978;89:285e9.