Generation and characterization of a recombinant Newcastle disease virus expressing the red fluorescent protein for use in co-infection studies
© Li et al.; licensee BioMed Central Ltd. 2012
Received: 10 May 2012
Accepted: 26 September 2012
Published: 3 October 2012
Many viruses have evolved multiple strategies to prevent super infection of host cells by more than one virion. This phenomenon, known as super infection exclusion, may play an important role on virus evolution because it can affect the frequency of reassortment and/or recombination. Newcastle disease virus (NDV), a negative sense single-stranded RNA virus, is characterized by its continuous evolutionary dynamics and by a low frequency of recombination events. However, the mechanisms that contribute to the low recombination rates on NDV are still not completely understood.
In this study we assessed the ability of two NDV strains (LaSota and B1) to super infect host cells in vitro. We generated a recombinant NDV strain LaSota expressing the red fluorescent protein (RFP) and used it in co-infection assays with a related NDV strain B1 expressing the green fluorescent protein (GFP). DF-1 cells were inoculated with both viruses at the same time or at different intervals between primary infection and super infection.
When both viruses were inoculated at the same time point, a 27% co-infection rate was observed, whereas when they were inoculated at different time points the super infection rates decreased to levels as low as 1.4%.
These results indicate that although different NDV strains can co-infect host cells in vitro, the super infection rates are low, specially as the time between the primary infection and super infection increases. These results confirm the occurrence of super infection exclusion between different strains of NDV.
KeywordsNDV Co-infection Super infection Interference
Newcastle disease virus
Avian paramyxovirus type 1
- [−] ssRNA:
Non-segmented single-stranded, negative sense RNA
RNA-dependent RNA polymerase
Red fluorescent protein
Intracerebral pathogenicity index
Mean death time
Tissue culture infectious dose 50
Dulbecco’s modified eagle medium
Fetal bovine serum
Specific pathogen free
Modified vaccinia virus Ankara/T7
Open reading frame
Multiplicity of infection.
Newcastle disease (ND) is one of the most important diseases of poultry, causing significant economic losses to poultry producers around the world . The etiologic agents of ND is Newcastle disease virus (NDV), or avian paramyxovirus type 1 (APMV-1), an enveloped, non-segmented, single-stranded, negative sense RNA ([−]ssRNA) virus of the genus Avulavirus family Paramyxoviridae. The NDV genome is approximately 15.2 Kb in length and contains six genes encoding for the nucleoprotein (NP), the phosphoprotein (P), the matrix protein (M), the fusion protein (F), the hemagglutinin-neuraminidase (HN), and the RNA-dependent RNA polymerase (L). At least one additional protein (V) is produced through editing of the phosphoprotein messenger RNA (mRNA) [1, 2].
NDV is known for its continuous evolutionary dynamics, with different isolates undergoing simultaneous evolutionary changes around the world [2, 3]. This evolutionary dynamic is likely resultant from the lack of proofreading activity of the viral RNA dependent RNA polymerase [4, 5]. While mutations introduced during virus replication seem to play a major role on NDV evolution, recombination has only been rarely reported [3, 6–9]. In general, the low frequency of recombination in [−]ssRNA viruses reflect specific aspects of their life cycle, including their genome organization and the mechanisms involved in transcription and replication . Other aspects the infection cycle and virus-host interactions may also affect the recombination rates in RNA viruses . For NDV, however, the strategies/mechanisms that contribute to the low frequency of recombination are not completely understood.
Many enveloped viruses have evolved mechanisms to prevent infection of host cells by more than one virion [11–13]. This phenomenon, known as super infection exclusion, likely affects the frequency of recombination within a viral population because co-infection of a cell by related viruses is required for the exchange of genetic material. In this study we assessed the ability of two NDV strains (LaSota and B1) to super infect host cells in vitro. We generated a recombinant NDV strain LaSota, encoding the red fluorescent protein (RFP; rLS-RFP) and used it in co-infection assays with a related strain of NDV (B1) expressing the green fluorescent protein (GFP; rB1-GFP).
Cells and viruses
HEp-2 (ATCC, CCL-23), Vero (ATCC, CCL-81) and DF-1 (ATCC, CRL-12203) cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and antibiotics and antimycotic (100U/ml Penicillin, 100 μg/ml Streptomycin, 0.25 μg/ml Amphotericin B, Thermo Scientific), and maintained at 37°C with 5% CO2. DF-1 cells were supplemented with 10% allantoic fluid (AF) (from specific pathogen free [SPF] embryonated chicken eggs) during all virus infections. The NDV strain LaSota was obtained from ATCC and the recombinant NDV strain B1 expressing the green fluorescence protein (rB1-GFP) was kindly provided by Dr. Peter Palese, Department of Microbiology, Mount Sinai University . All viruses were propagated in 9-day-old SPF embryonated chicken eggs and the virus titres were determined by limiting dilution, calculated according to the Spearman and Karber’s method, and expressed as Log10 tissue culture infectious dose 50 (TCID50)/ml. The modified vaccinia virus Ankara/T7 (MVA/T7) was used in the infection/transfection experiments to rescue the recombinant virus NDV-LS/RFP.
Generation of the recombinant NDV-LaSota containing the RFP gene
Rescue of the rLS-RFP was performed in HEp-2 cells according to standard protocols . HEp-2 cells were seeded on a six-well plate (1×106 cells/well), infected with MVA/T7 at a multiplicity of infection (MOI) of 3, and one hour later transfected with the plasmid pLS-RFP (2 μg), and the helper plasmids pTM-NP (1 μg), pTM-P (0.5 μg), and pTM-L (0.1 μg) using Lipofectamine 2000™ (Invitrogen). Seventy-two hours post-infection/transfection cells were harvested and subjected to three freeze and thaw cycles. Approximately 300 μl of the cell lysate were inoculated into the allantoic cavity of 9-day-old SPF embryonated chicken eggs for amplification of the recombinant virus. Rescue of rLS-RFP was confirmed by hemagglutination (HA) and fluorescence assays.
Biological characterization of rLS-RFP
The pathogenicity of the rLS-RFP virus was assessed by standard mean death time (MDT) and intracerebral pathogenicity index (ICPI) tests . To compare the cytopathic effect (CPE) induced by rLS-RFP, the parental NDV-LaSota and the rB1-GFP, DF-1 cells were infected with each virus (MOI=0.01) and evaluated under a light and a fluorescence microscope at 24 and 48 hours post infection (pi). Growth characteristics of rLS-RFP, NDV-LaSota, and rB1-GFP were assessed in DF-1 cells. Cells were infected with rLS-RFP, NDV-LaSota, or rB1-GFP (MOI=0.01), harvested at 0, 12, 24, 36 and 48h pi and subjected to three freeze and thaw cycles. Viral titres were determined by limiting dilution, calculated according to the Spearman and Karber’s method, and expressed as Log10 TCID50/ml.
Super infection assays
The ability of rLS-RFP and rB1-GFP to super infect host cells was assessed in a chicken fibroblast cell line (DF-1). Initially, rLS-RFP and rB1-GFP were mixed (MOI=3 of each virus), and inoculated in DF-1 cells. After 1h of adsorption at 37°C the virus inoculum was removed, cells were washed 1X with DMEM, and complete culture media was added to the cells. At 12h pi cells were stained with HCS Nuclear Mask Blue (Invitrogen) and examined under a fluorescence microscope (Nikon, Eclipse Ti, Melville, NY). Pictures were taken at 20X magnification and the numbers of red-, green-, or yellow-fluorescent cells (red + green) were determined using the ImageJ (version 1.46) software (NIH, http://rsbweb.nih.gov/ij/index.html). The numbers of cells infected with rLS-RFP, and rB1-GFP, or co-infected with both viruses (yellow-fluorescent cells) were expressed as percentage of infected cells. At least three fields were randomly selected to calculate the mean infectivity and super infection percentages.
To assess the effect of the time of super infection in the ability of rLS-RFP and rB1-GFP to co-infect host cells, DF-1 cells were culture in 12-well plates and inoculated with both viruses at different intervals between the primary infection and the super infection. Cells were inoculated with rLS-RFP and rB1-GFP (MOI=3) and super infected at 1 and 3h pi with rB1-GFP or rLS-RFP (MOI=3), respectively. In a second experiment, cells were inoculated with rLS-RFP or rB1-GFP (MOI=1) and super infected at 12, and 24h pi with rB1-GFP or rLS-RFP (MOI=3), respectively. At 24h post super infection cells were stained with HCS Nuclear Mask Blue (Invitrogen), and examined under a fluorescence microscope (Nikon, Eclipse Ti, Melville, NY). The numbers of red-, green-, and yellow-fluorescent cells were determined as described above. In addition, Vero cells were inoculated with rLS-RFP or rB1-GFP (MOI=3) and super infected at 3h pi with rB1-GFP or rLS-RFP (MOI=3), respectively. At 24h post super infection cells were examined under a fluorescence microscope (Nikon, Eclipse Ti, Melville, NY). Statistical analysis was performed with the two-tailed paired Student’s t-test, and p values < 0.05 were considered statistically significant.
Construction and characterization of recombinant NDV LaSota expressing the red fluorescent protein (rLS-RFP)
The recombinant NDV LaSota encoding the red fluorescent protein (RFP) was constructed using reverse genetic approaches. The cDNA clone pLS/aMPV-C G , containing the complete genome sequences of NDV strain LaSota and the coding sequences of the G protein of avian metapneumovirus type C, was used as backbone to generate the rLS-RFP virus. The open reading frame of the aMPV G protein (1,758 nt) was replaced by the coding sequence of the RFP (678 nt) using the In-Fusion cloning strategy. The rLS-RFP was rescued in HEp-2 cells and propagated in 9-days-old embryonated chicken eggs. DNA sequencing of regions flanking the inserted gene confirmed the integrity of NDV LaSota and of RFP sequences in the recombinant virus (data not shown).
To determine whether the resultant recombinant virus expresses the RFP, DF-1 cells were infected and examined under a fluorescence microscope. Expression of RFP was observed in cells infected with the rLS-RFP at 24h and 48h post-infection (pi), demonstrating efficient expression of RFP by the recombinant virus (Figure 1). The rB1-GFP virus was used as a positive control for the expression of GFP (Figure 1).
Biological characterization of the rLS-RFP virus
Biological characterization of the rLS-RFP
Super infection with rLS-RFP and rB1-GFP results in low rates of co-infection in vitro
In the present study we assessed the ability of two NDV strains (LaSota and B1) to co-infect host cells in vitro. Results here revealed that NDV strains LaSota and B1 are able to co-infect one host cell simultaneously. However, the super infection rates are low and they decrease as the time between the primary infection and super infection increases. These findings suggest the occurrence of super infection exclusion during NDV infection.
Super infection exclusion is a phenomenon by which an established virus infection blocks co-infection by a homologous super infecting virus . Here we have shown that primary infection of DF-1 cells with either rLS-RFP or rB1-GFP decreases the co-infection rates by the heterologous strain. Interestingly, the co-infection rates decreased as the time between the primary infection and the super infection increased, corroborating with findings from early studies . Super infection exclusion can occur at three different steps of the virus life cycle, including attachment, entry and/or intracellular replication [11–13]. It has been shown that for the paramyxovirus human parainfluenza type 3 the neuraminidase activity of the HN protein is responsible for blocking super infection by a heterologous strain . Whether NDV HN exerts a similar function in blocking super infection or whether the virus has evolved additional mechanisms to prevent super infection remains to be determined.
Although the biological significance of super infection exclusion for NDV and other paramyxoviruses is unknown, this phenomenon can contribute to the low frequency of recombination observed for these viruses. The limited number of cells that are co-infected during NDV infection may negatively affect recombination rates. As shown here, super infection rates were low even when a high multiplicity of infection (MOI=3 for each virus) was used (Figure 3A and B), and they significantly decreased as the time interval between each infecting virus increased (Figure 4A,B, and C; Figure 5A,B and C). Results from the super infection assays on Vero cells (Additional file 1: Figure S1A and B), known to be interferon defective , indicate that the low co-infection rates observed in DF1 cells are due to super infection exclusion between NDV strains LaSota and B1 and not due to an antiviral response of the host cells generated against the primary infection. It is important to note that in addition to specific viral mechanisms that may contribute to super infection exclusion (i.e. neuraminidase activity of the HN protein), the co-infection rates are likely affected by other factors in vivo, including strain-specific cell and tissue tropism, the time interval between infection with different strains and the presence of predisposing immunity.
In the future, it will be interesting to use the recombinant virus generated here (rLS-RFP) to determine the mechanism responsible for super infection exclusion in NDV and to assess the potential for recombination among different NDV strains. Complete genome sequencing following super infection experiments will allow us to determine if recombination can indeed occur in NDV and how frequent it may be.
In summary, the present study describes the characterization of a recombinant NDV strain encoding a red fluorescent protein that was used in co-infection studies. We have shown that NDV strains LaSota and B1 are able to co-infect host cells in vitro, however the co-infection rates are low and they decrease as the time between primary infection and super infection increases.
We thank Tim Olivier and Xiuqin Xia for technical assistance, the SEPRL sequencing facility personnel for nucleotide sequencing, and Dr. Peter Palese (Department of Microbiology, Mount Sinai School of Medicine) for providing the recombinant virus rB1-GFP used in this study. J. Li and H. Hu were sponsored by a scholarship from China Scholarship Council. This work was supported by USDA funding, CRIS no. 6612-32000-064-00D.
- Alexander DJ, Senne DA: Newcastle disease, other avian paramyxoviruses, and pneumovirus infections. In Diseases of Poultry. 12th edition. Edited by: Saif YM, Fadly AM, Glisson JR, McDougald LR, Nolan LK, Swayne DE. Iowa State University Press, Ames; 2008:75–116.
- Miller PJ, Decanini EL, Afonso CL: Newcastle disease: evolution of genotypes and the related diagnostic challenges. Infect Genet Evol 2010, 10:26–35.PubMedView Article
- Diel DG, da Silva LH, Liu H, Wang Z, Miller PJ, Afonso CL: Genetic diversity of avian paramyxovirus type 1: Proposal for a unified nomenclature and classification system of Newcastle disease virus genotypes. Infect Genet Evol 2012, 12:1770–1779.PubMedView Article
- Holland J, Spindler K, Horodyski F, Grabau E, Nichol S, VandePol S: Rapid evolution of RNA genomes. Science 1982, 215:1577–1585.PubMedView Article
- Chare ER, Gould EA, Holmes EC: Phylogenetic analysis reveals a low rate of homologous recombination in negative-sense RNA viruses. J Gen Virol 2003, 84:2691–2703.PubMedView Article
- Song Q, Cao Y, Li Q, Gu M, Zhong L, Hu S, Wan H, Liu X: Artificial recombination may influence the evolutionary analysis of Newcastle disease virus. J Virol 2011, 85:10409–10414.PubMedView Article
- Qin Z, Sun L, Ma B, Cui Z, Zhu Y, Kitamura Y, Liu W: F gene recombination between genotype II and VII Newcastle disease virus. Virus Res 2008, 131:299–303.PubMedView Article
- Han GZ, He CQ, Ding NZ, Ma LY: Identification of a natural multi-recombinant of Newcastle disease virus. Virology 2008, 371:54–60.PubMedView Article
- Zhang R, Wang X, Su J, Zhao J, Zhang G: Isolation and analysis of two naturally-occurring multi-recombination Newcastle disease viruses in China. Virus Res 2010, 151:45–53.PubMedView Article
- Simon-Loriere E, Holmes EC: Why do RNA viruses recombine? Nat Rev Microbiol 2011, 9:617–626.PubMedView Article
- Walters KA, Joyce MA, Addison WR, Fischer KP, Tyrrell DL: Super infection exclusion in duck hepatitis B virus infection is mediated by the large surface antigen. J Virol 2004, 78:7925–7937.PubMedView Article
- Lee YM, Tscherne DM, Yun SI, Frolov I, Rice CM: Dual mechanisms of pestiviral super infection exclusion at entry and RNA replication. J Virol 2005, 79:3231–3242.PubMedView Article
- Horga MA, Gusella GL, Greengard O, Poltoratskaia N, Porotto M, Moscona A: Mechanism of interference mediated by human parainfluenza virus type 3 infection. J Virol 2000, 74:11792–11799.PubMedView Article
- Park MS, Shaw ML, Munoz-Jordan J, Cros JF, Nakaya T, Bouvier N, Palese P, Garcia-Sastre A, Basler CF: Newcastle disease virus (NDV)-based assay demonstrates interferon-antagonist activity for the NDV V protein and the Nipah virus V, W, and C proteins. J Virol 2003, 77:1501–1511.PubMedView Article
- Hu H, Roth JP, Estevez CN, Zsak L, Liu B, Yu Q: Generation and evaluation of a recombinant Newcastle disease virus expressing the glycoprotein (G) of avian metapneumovirus subgroup C as a bivalent vaccine in turkeys. Vaccine 2011, 29:8624–8633.PubMedView Article
- Alexander DJ: Newcastle disease and other avian paramyxoviruses. Rev Sci Tech 2000, 19:443–462.PubMed
- Bratt MA, Rubin H: Specific interference among strains of Newcastle disease virus. I. Demonstration and measurement of the interference. Virology 1967, 33:598–608.PubMedView Article
- Emeny JM, Morgan MJ: Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production. J Gen Virol 1979, 43:247–252.PubMedView Article
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