Skip to main content
  • Research article
  • Open access
  • Published:

Use of live attenuated recombinant Newcastle disease virus carrying avian paramyxovirus 2 HN and F protein genes to enhance immune responses against species A rotavirus VP6 protein


Numerous infectious diseases in cattle lead to reductions in body weight, milk production, and reproductive performance. Cattle are primarily vaccinated using inactivated vaccines due to their increased safety. However, inactivated vaccines generally result in weaker immunity compared with live attenuated vaccines, which may be insufficient in certain cases. Over the last few decades, there has been extensive research on the use of the Newcastle disease virus (NDV) as a live vaccine vector for economically significant livestock diseases. A single vaccination dose of NDV can sufficiently induce immunity; therefore, a booster vaccination dose is expected to yield limited induction of further immune response. We previously developed recombinant chimeric NDV (rNDV-2F2HN), in which its hemagglutinin-neuraminidase (HN) and fusion (F) proteins were replaced with those of avian paramyxovirus 2 (APMV-2). In vitro analysis revealed that rNDV-2F2HN expressing human interferon-gamma had potential as a cancer therapeutic tool, particularly for immunized individuals. In the present study, we constructed rNDV-2F2HN expressing the bovine rotavirus antigen VP6 (rNDV-2F2HN-VP6) and evaluated its immune response in mice previously immunized with NDV. Mice primarily inoculated with recombinant wild-type NDV expressing VP6 (rNDV-WT-VP6), followed by a booster inoculation of rNDV-2F2HN-VP6, showed a significantly stronger immune response than that in mice that received rNDV-WT-VP6 as both primary and booster inoculations. Therefore, our findings suggest that robust immunity could be obtained from the effects of chimeric rNDV-2F2HN expressing the same or a different antigen of a particular pathogen as a live attenuated vaccine vector.


Emerging and re-emerging infectious diseases are a cause of concern in public health as well as in the livestock industry [1]. There are frequent epidemics in the livestock sector, which lead to substantial economic losses and require prompt countermeasures at the farm level. In cattle, infectious disease outbreaks can lead to reductions in body weight, milk production, and reproductive performance. Accordingly, vaccination represents a crucial countermeasure in this regard.

Inactivated vaccines are being extensively used for various infectious diseases in cattle, especially pregnant animals, allowing for the transfer of passive immunity to the offspring through colostrum during the neonatal period [2]. Inactivated vaccines can be administered as monovalent or bivalent formulations and sometimes even as multivalent blends with other antigens. However, the effectiveness of these vaccines can be compromised due to factors such as insufficient maternal immunity, mixed infections, and encounters with reassortant viruses [3]. The resulting inadequate immune responses may necessitate multiple rounds of vaccination. By contrast, live attenuated vaccines are robust inducers of immunity and can often establish long-term immunological memory with a single dose [4]. However, concerns about their contribution to genetic divergence and low safety, particularly in pregnant animals, limit their widespread use [5,6,7]. Therefore, there is a pressing need for safe and effective vaccine vectors capable of targeting multiple pathogens in bovines [8].

Newcastle disease virus (NDV) poses a high pathogenic threat to poultry, and live attenuated vaccines have been developed for use in chicken [9]. Additionally, NDV has been extensively applied as a vaccine vector and an anticancer therapeutic tool owing to its unique oncolytic properties [10,11,12,13,14,15]. The recombinant NDV (rNDV) vector has been developed to express various foreign genes for a wide range of applications [14, 16,17,18,19]. For example, an rNDV expressing foreign antigens such as hemagglutinin (HA) of the influenza virus has been shown to elicit immunity against HA [12, 20, 21]. Furthermore, NDV has a broad range of hosts, including humans, mice, cats, dogs, and cattle; therefore, it is an attractive vaccine vector for diverse animal pathogens, including bovine-specific pathogens [10, 17, 20, 22, 23]. However, homologous vaccines administered through primary and booster doses might have limited efficacy in generating robust immune responses. To address this issue, recombinant chimeric NDVs have been engineered through the replacement of the F or HN protein with APMV-2 or the incorporation of surface proteins comprising portions of both NDV and APMV-2 polypeptides [24,25,26]. Administration of chimeric NDVs expressing the same or different antigens during booster inoculation may be a promising strategy for fostering the development of potent immunity.

Group A bovine rotavirus (BRV) stands as a significant contributor to severe calf diarrhea worldwide, often resulting in notable economic repercussions due to treatment expenses, decreased growth rates, and decreased overall productivity [27, 28]. In light of this, we generated recombinant NDVs encompassing both wild-type (WT) and chimeric viruses, that contain the BRV VP4 or VP6 antigens, and we assessed their ability to induce immune responses in mice.

Materials and methods

Cells and rotaviruses

Human respiratory epithelial cells (HEp-2 cells), chicken embryo fibroblasts (CEF cells), bovine intestinal epithelial cells (BIE cells) [29, 30], Madin-Darby bovine kidney cells (MDBK cells), and African green monkey kidney cells (MA104 cells) were maintained in a medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Bovine rotavirus (BRV Lincoln strain (G6, P[1])) and simian rotavirus (SA11 strain (G5, P[3])) (ATCC® VR-1565™) were propagated in the MA104 cell line as previously described [31]. The viral titers were determined using the fluorescence focus units (FFU) as previously described [32]. All virus stocks were stored at -80 °C until use. The BRVs were activated using trypsin (Sigma Aldrich, MO, USA) stocked at 1 mg/mL and were diluted to a final concentration of 10 μg/mL, added to viral stock vials, and incubated for 30 min at 37 °C before in vitro infection. All experiments were performed at the Kyoto Prefectural University of Medicine under the Biosafety Level 2 conditions.

Recombinant chimeric NDV genome construction and rescue experiment

BRV VP4 and VP6 (accession no. AB119636.1 and JF693031.1) were amplified by reverse transcription polymerase chain reaction (RT-PCR) using specific primer sets (Additional files 5 and 6); subsequently, they were inserted between the P and M genes of the rNDV-WT or rNDV-2F2HN cDNA clones. Six recombinant cDNA with or without VP4 and VP6 were generated (Figure 1A); additionally, recombinant viruses were obtained as previously described [12]. The pathogenicity of the recombinant viruses in poultry was determined by the mean death time (MDT) assay using embryonated chicken eggs [33].

Figure 1
figure 1

Construction of recombinant NDVs expressing the VP6 gene of BRV. A Schema of constructed recombinant NDV genomes inserted with the BRV VP6 or VP4 gene. B Immunostaining of HEp-2 cells infected with each recombinant NDV. The infected cells were stained using anti-NDV antiserum (green), BRV antibody (red), and Hoechst 33342 (blue; nuclei). Scale bar, 50 µm. C Growth kinetics of recombinant viruses in different cells. The cells were infected at MOI of 0.1 for each of the viruses. The copy number from the culture supernatants was determined using real-time PCR at 24-h intervals.

To evaluate the genetic stability of VP6 within the rNDVs, the recovered viruses were serially passaged five times in MDBK cells. After the five passages, the presence and expression of VP6 were confirmed by RT-PCR and immunofluorescence (Additional file 2).

Immunofluorescence assay (IFA)

HEp-2 cells were cultured in a 96-well plate (1.0 × 104 cells/well) for 24 h and infected with rNDVs at a multiplicity of infection (MOI) of 0.5 [34]. On the next day, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. Chicken anti-NDV antisera and goat anti-BRV polyclonal antibodies (cat. ab20036; Abcam, Cambridge, UK) were used as primary antibodies for detecting NDV and BRV antigens, respectively. The secondary antibodies used were goat anti-chicken IgY-conjugated Alexa-488 (cat. A11039, Thermo Fisher Scientific, Waltham, MA, USA), donkey anti-goat IgG conjugated Alexa-555 (cat no. AP180C; Thermo Fisher Scientific) and goat anti-mouse IgG conjugated Alexa-488 (cat no. A11029 Thermo Fisher Scientific). Hoechst 33342 (DOJINDO, Kumamoto, Japan) was used for nucleus staining. The infected cells were observed under fluorescence microscopy BZ-X700 (KEYENCE, Osaka, Japan).

Viral replication assay

HEp-2, CEF, MA104, MDBK, and BIE cells were cultured in 24-well plates (1.0 × 105 cells/well) and infected with the rNDVs at an MOI of 0.1. The supernatant was collected at 24 h intervals until 96 h after infection. Total RNA was extracted from each sample using ISOGEN II (Nippon Gene, Tokyo, Japan); additionally, reverse transcription was performed using the ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). The viral copy number was determined by real-time PCR using PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) and the following forward (5′-AGTGATGTGCTCGGGCCTTC-3′) and reverse (5′-CCTGAGGAGAAGCATTTGCTA-3′) primers as previously described [26].

Animal experiments

To assess the pathogenicity of the recombinant viruses, 4-week-old female BALB/c mice were inoculated with 1 × 107 FFU of each virus via the intranasal or intraperitoneal routes. All mice were monitored daily for signs of illness, weight change, or death for 2 weeks.

To evaluate immune responses against BRV VP6, ten experimental groups (inoculation combination) were prepared as follows: (1) and (6) rNDV-WT and rNDV-WT, (2) and (7) rNDV-WT and rNDV-2F2HN, (3) and (8) rNDV-WT-VP6 and rNDV-WT-VP6, (4) and (9) rNDV-WT-VP6 and rNDV-2F2HN-VP6, and (5) and (10) PBS and PBS. The mice groups (1)–(5) and (6)–(10) were inoculated intraperitoneally and intranasally, respectively. The 4-week-old female BALB/c mice were inoculated with 1 × 107 FFU of rNDVs, while the control group received PBS. Three weeks after the primary inoculation, the mice were boosted with rNDVs. The blood samples from the different groups were collected twice (3 weeks after primary and 3 weeks after booster inoculations), and the serum was separated for serological assays. Three weeks after booster inoculations, the mouse splenocytes were harvested as previously reported [35] and stored in Bambanker (NIPPON Genetics, Tokyo, Japan) at −80 °C until use.

Neutralization assay

The sera of immunized mice were serially two-fold diluted and mixed with an equal amount of 100 FFU of BRV or 1000 FFU of NDV solution and incubated at 37 °C for 1 h. The mixture was added to the target cells in triplicate wells of a 96-well plate. At 24 post-infection hours, an immunofluorescence assay was performed, and the infected cells were counted using a fluorescence microscope. The neutralizing titers were calculated as the BRV infection inhibition ratio or the highest dilution ratio that completely inhibited the NDV infection.

Splenocyte proliferation assay

To detect the antigen-specific responses in mouse splenocytes, the cell proliferation after stimulation with each antigen was assessed. The splenocytes were seeded in 96-well plates (1 × 105 /well). Two days after stimulation, cell viability was measured using the Cell Count Reagent SF (Nacalai Tesque, Kyoto, Japan).

Statistical analysis

Data are expressed as the arithmetic mean ± standard error of the mean (SEM). All statistical analyses were performed using Student’s t-test with the GraphPad Prism 8 software (La Jolla, CA, USA). Statistical significance was set at P < 0.05.


BRV-VP6 antigen was expressed in infected cells with recombinant NDVs

We prepared and attempted a rescue experiment to construct the infectious viral particles using six rNDV genomes (Figure 1A). All types of infectious rNDVs were recovered and confirmed to detect NDV antigens using immunofluorescence (Additional file 1B). To assess the BRV antigen expression, the HEp-2 cells were infected with each recombinant virus. As shown in Figure 1B, non-infected HEp2 cells (mock) were not stained with either chicken anti-NDV serum or goat anti-BRV antibodies. Cells infected with rNDV-WT, rNDV-WT-VP4, rNDV-2F2HN, and rNDV-2F2HN-VP4 were only detected with chicken anti-NDV serum. rNDV-WT-VP6 and rNDV-2F2HN-VP6 infected cells were detected with anti-BRV antibody and anti-NDV serum. Contrastingly, VP4 protein expression was not detected using anti-BRV antibody (Figure 1B). Similar results were obtained even using the bovine cell lines (MDBK cells) (Additional file 1C). The insertion of VP4 gene into the recombinant viral genome was confirmed using the RT-PCR (Additional File 1D). Therefore, an insufficient VP4 expression was observed in this recombinant NDV system.; Accordingly, we proceeded with the experiments using rNDV-WT-VP6 and rNDV-2F2HN-VP6.

To investigate whether the insertion of the VP6 gene affected the growth properties of the parental NDV strain, we evaluated the growth kinetics of each virus in different cell lines, including the bovine cell lines (BIE and MDBK cells). Both rNDV-WT-VP6 and rNDV-2F2HN-VP6 showed replication kinetics similar to those of the parental vectors (rNDV-WT and rNDV-2F2HN, respectively; Figure 1C). In addition, the genetic stability of the VP6 gene within rNDV-WT-VP6 or rNDV-2F2HN-VP6 was assessed by serially passaging the viruses in MDBK cells five times and confirmed by RT-PCR and immunofluorescence assays (Additional file 2). These results suggested that VP6 gene insertion does not adversely affect NDV replication and that the virus can grow even in bovine cell lines.

Low pathogenicity of recombinant NDVs expressing BRV-VP6 in poultry and mice

The pathogenicity of rNDV-WT-VP6, rNDV-2F2HN-VP6, and their parental vector viruses was determined in 9-day-old embryonated eggs using an MDT assay. NDV strain with an MDT of more than 90 h is classified as a lentogenic strain, and, notably, all the constructed recombinant viruses showed MDT values of more than 192 h. To assess the safety of these recombinant viruses in mammals, 4-week-old female BALB/c mice were inoculated with 1 × 107 FFU of each virus via the intranasal or intraperitoneal routes (Additional file 3). All mice gained weight during the experiment, with no significant among-group differences. These results indicated that a high titer of rNDV-WT-VP6 and rNDV-2F2HN-VP6 was obtained using the embryonated eggs and that these recombinant vaccine vectors did not show severe pathogenicity in mice.

VP6-specific antibodies were induced in mice immunized with rNDV-WT-VP6

The schedule of the rNDV inoculation experiments is shown in Figure 2A. The effects of primary inoculation (Figures 2B–E) and booster inoculation (Figure 3) were evaluated. To assess the induction of immune responses against the VP6 antigen, the mice were inoculated with 1 × 107 FFU of rNDV-WT or rNDV-WT-VP6 via the intraperitoneal or intranasal route (Figure 2A). After 3 weeks, the mouse sera (primary serum) were obtained. Immunofluorescence analysis revealed that both NDV and BRV antigens were detected by serum collected from mice inoculated with rNDV-WT-VP6 (Figures 2B and C). Notably, the intranasally inoculated group showed higher antigen detection levels than the intraperitoneally inoculated group. Subsequently, we evaluated the neutralizing activity (Figures 2D and E). A neutralization assay was conducted using 100 FFU of BRV to calculate the number of positive cells. Sera obtained from mice inoculated with rNDV-WT-VP6 inhibited the BRV infection in a serum dilution-dependent manner. Consistent with the immunofluorescence assay results (Figure 2B and C), the mice intranasally inoculated with rNDV-WT-VP6 showed a significantly stronger immune response against BRV than the intraperitoneally inoculated mice (Figure 2D). Furthermore, we evaluated the induction of neutralizing antibodies against NDV. Using 1000 FFU of rNDV-WT or rNDV-2F2HN, we investigated the maximum dilution ratio that completely inhibited the infection. Higher neutralizing titers were detected when rNDV-WT, with or without VP6, was used for immunization (Figure 2E). On the contrary, there was weak cross-reactivity with rNDV-2F2HN given the lack of NDV F and HN antigens, as confirmed by a lower neutralizing titer (Figure 2E). Taken together, these results suggested that specific antibodies against VP6 were produced in the mice and that a single inoculation of rNDV-WT-derived viruses elicited high levels of neutralizing antibodies against NDV, regardless of the inoculation route.

Figure 2
figure 2

Primary inoculation of rNDV-WT-VP6 induced specific immune responses against NDV and BRV-VP6 antigens. A Schematic diagram illustrating the immunization schedule and experimental group. The immunogenicity of the recombinant NDVs was evaluated in 4-week-old female BALB/c mice, which were divided into 10 groups (each n = 3, five groups for each administration route). The mice in groups 1–5 were inoculated intraperitoneally while mice in groups 6–10 were inoculated intranasally. B NDV-infected cells and C BRV-infected cells were stained using mouse sera (green) collected after primary inoculation and Hoechst 33342 (blue; nuclei). Scale bar, 50 µm. D BRV infection was performed after treatment with each dilution of antiserum. The rate of decrease from 100 FFU is shown as BRV infection inhibition ratio. E The maximum dilution ratio for complete NDV neutralization. Data represent the mean ± SEM (n = 3, *P < 0.05).

Chimeric rNDV-2F2HN-VP6 booster inoculation induced a higher immune response in mice compared to homologous rNDV-WT-VP6 booster inoculation

Three weeks after the booster inoculation, mouse sera were collected for analysis (Figure 2A). To validate the effects of the booster inoculation, neutralizing activities against BRV were compared between single and booster inoculations using mouse sera inoculated with rNDV-WT-VP6 (Figure 3A). Intraperitoneal inoculation resulted in a booster effect; however, intranasal inoculation did not enhance the neutralizing antibody titer compared with the single inoculation. Subsequently, similar to the primary inoculation (Figure 2), we performed antigen detection in infected cells and compared the inhibition of BRV infection between the rNDV-WT-VP6 and rNDV-2F2HN-VP6 booster inoculation groups. (Figures 3B and C). As shown in Figure 3B, combinational inoculations of rNDV-WT-VP6 priming followed by rNDV-2F2HN-VP6 boosting induced a relatively higher anti-BRV antibody level for both the intraperitoneal and intranasal inoculation groups. The detection rate was correlated with the neutralizing activity within the same inoculation group. Notably, mice that received rNDV-2F2HN-VP6 as a booster inoculation exhibited significantly higher anti-BRV neutralizing antibody levels than those that received a homologous booster inoculation of rNDV-WT-VP6 (Figure 3C).

Figure 3
figure 3

Combination inoculation elicited higher induction of immune responses compared with homologous booster inoculation. A Comparison of BRV neutralization between primary and booster sera. B BRV-infected cells were stained with mouse sera (green) collected after booster inoculation and Hoechst 33342 (blue; nuclei). Scale bar, 50 µm. C The BRV infection inhibition ratio was obtained using mouse sera collected after booster inoculation. D Assessment of the specific cell proliferation of the mice splenocytes after booster inoculation using MTT assay. (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).

Enhanced splenocyte proliferation in response to specific antigens with combination inoculation

To comprehensively evaluate the immune response to BRV, we conducted a splenocyte proliferation assay (Additional file 4C). Splenocytes obtained from mice inoculated with VP6-expressing rNDVs exhibited robust proliferation upon BRV stimulation (Figure 3D). Notably, the cell proliferation was more pronounced with combination booster inoculation than with homologous booster inoculation (Figure 3D).

Cross-reactivity with another rotavirus strain

We further confirmed that the induced anti-BRV VP6 antibodies exhibited cross-reactivity with a closely related, yet heterologous, Group A rotavirus strain, i.e., the simian rotavirus SA11 strain (Additional file 4A). This cross-reactivity could be attributed to the substantial conservation of VP6 antigenicity among Group A rotaviruses [36,37,38]. Further, we confirmed neutralizing activity against the simian rotavirus using sera collected from mice inoculated with rNDV-WT-VP6 (Additional files 4B–D). Finally, we confirmed that the simian rotavirus SA11 strain could induce proliferation of mouse splenocytes.


The Group A BRV is a globally distributed virus that causes calf diarrhea and leads to serious problems in newborn calves, including high mortality rates, increased treatment expenses, and impaired growth [39]. Currently, inactivated rotavirus vaccines are administrated to pregnant dams [2]. Compared with live attenuated vaccines, inactivated vaccines are generally less potent at inducing immunity. However, live attenuated vaccines carry the risk of revertance to virulence [8]. Previously, we developed a recombinant chimeric NDV (rNDV-2F2HN) that demonstrated potential as a cancer therapy tool even in the presence of anti-NDV antibodies [26]. The potential of NDV as a vaccine vector has been demonstrated in a range of species, including humans and animals such as cattle, dogs, African green monkeys, rhesus monkeys, pigs, minks, and chickens [40]. In the present study, we generated both wild-type and chimeric rNDVs to express the BRV VP6 antigen and investigated their suitability as vaccine vectors for eliciting strong immunity against BRV in a mouse model. Additionally, we attempted to express another BRV outer capsid protein, VP4, with rNDV-2F2HN. However, VP4 expression was not detected even in bovine cell lines. Previous studies have demonstrated that the expression level of the foreign gene inserted into a full-length clone of NDV is largely affected by the size and sequence of the insert, and the expression of larger-sized genes would be challenging [41, 42]. Accordingly, we measured the expression level of the foreign antigens using flow cytometry (data not shown). It was observed that VP4 protein was expressed, albeit at substantially lower levels compared to the VP6 protein. As a result, there was no detectable level of expression observed via immunostaining, and it was concluded that rNDV-WT-VP4 and rNDV-2F2HN-VP4 were not viable candidates for vaccination due to the lack of VP4 antigenicity. Consequently, our focus turned to rNDV-2F2HN-VP6 as a potential vaccine candidate for BRV.

We demonstrated the efficient propagation of both rNDV-WT-VP6 and rNDV-2F2HN-VP6 in embryonated chicken eggs, which maintained consistently high titers even after multiple passages. These findings highlight the advantages of both recombinant vaccine vectors in yielding high quantities at a low cost, which is consistent with previous reports [43]. To evaluate the safety of rNDV-2F2HN-VP6, we used a mouse model with intraperitoneal and intranasal inoculations. Regardless of the administration route, all mice exhibited post-inoculation weight gain. To assess the immunogenicity of the constructed vaccine candidates, mice were inoculated twice via intranasal or intraperitoneal route. Interestingly, the results of the prime inoculation showed a higher induction of immune responses in the serum of the intranasally inoculated group than in the intraperitoneally inoculated group. This finding is in agreement with a previous work that used rNDV expressing simian immunodeficiency virus antigens for evaluation in mice [44]. They reported that intranasal inoculation induced the strongest immune response among all other routes. This may be attributed to that NDV infects naturally via the respiratory tract and can induce humoral and cellular immune responses both at the mucosal and systemic levels. While earlier reports suggested that antibodies elicited against rotavirus inner capsid VP6 are non-neutralizing in classical neutralization assays [45, 46], our neutralization assay results revealed that VP6-specific antibodies exhibited an inhibitory effect of the BRV infection, varying with serum dilution. Comparable findings were noted in a prior study examining the effects of the VP6 vaccine candidate in calves, where they also detected VP6-specific neutralizing antibodies at lower titers, utilizing the same method as in our investigation [47]. We hypothesized that the potential mechanism involving extracellular neutralization of the virus might have occurred, albeit to a limited extent. However, other potential mechanisms involving VP6 IgG and IgA would also be possible, consisting of intracellular disruption of the viral replication cycle (In classical NT tests, VP6-specific antibodies are not neutralizing) [48]. Although rNDV-WT-VP6 inoculation induced specific immune responses against the VP6 antigen in mice, it also resulted in the production of neutralizing antibodies against NDV. Accordingly, using rNDV-WT-VP6 as a booster did not significantly enhance the immunity induced by the primary inoculation, especially in the intranasally inoculated group. By contrast, the chimeric rNDV-2F2HN-VP6 vaccine vector, in which NDV HN and F proteins were replaced with those of APMV-2, has shown the ability to evade preexisting anti-NDV immunity. The antigenicity of NDV is determined by the surface antigens of F and HN proteins [49]. We have reported in vitro that rNDV-2F2HN escapes neutralization by NDV antiserum [26], and this study indicated that it also escaped in vivo. Therefore, it is considered that the immune system recognized rNDV-2F2HN-VP6 as a new antigen in the booster inoculation. As a result, booster inoculation with rNDV-2F2HN-VP6 induced a significantly enhanced immune response in mice. Furthermore, splenocytes obtained from mice immunized with rNDV-WT-VP6 and rNDV-2F2HN-VP6, but not control rNDVs, showed specific proliferation when stimulated with BRV (Figure 3D). Notably, rNDV-2F2HN-VP6 booster inoculation led to a higher cell proliferation rate than rNDV-WT-VP6 booster inoculation, regardless of the administration route.

Given the high degree of antigenic identity among VP6 antigens of all Group A rotaviruses, eliciting an immune response against VP6 may allow heterotypic protection against other various rotavirus infections [36, 37]. Numerous studies have assessed the intracellular neutralization capacity of VP6 antibodies, revealing their ability to inhibit viral replication in the sera [45, 46, 50]. Therefore, it is believed that VP6 antibodies could play a significant role in alleviating symptoms. In addition, there have been many reports that highlighted the protective impact of VP6 antibodies against rotavirus infection [36, 47, 51,52,53]. These antibodies have been shown to be effective in several animals, such as calves and mice. Hence, these protective mechanisms are anticipated to be operative across a broad spectrum of animal species. We demonstrated that sera and splenocytes obtained from mice immunized with VP6-expressing rNDVs displayed reactivity with simian rotavirus-infected cells (Additional file 4). These findings indicate the potential utility of VP6-based vaccines against other species of Group A Rotavirus, which concurs with a previous report [52]. Rotaviruses exhibit wide genetic diversity, in particular of the VP7 and VP4 (and NSP1) encoding genes, and possess the ability to reassort and mutate, resulting in the emergence of new strains that can evade preexisting immune responses [54]. Since VP6 proteins are highly immunogenic and possess the remarkable ability to induce potent homologous and cross-reactive immune responses, therefore, a live vaccine vector targeting the BRV VP6 might offer effective protection against other Group A rotaviruses. Our data suggest that the immune cells recognized the chimeric NDV as a novel antigen, which enhanced immune cell proliferation and boosted the immune reactions. Studies using large animals (models) are warranted to elucidate the vaccination efficacy of these recombinant viruses.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its supplementary information files.


  1. Gummow B (2010) Challenges posed by new and re-emerging infectious diseases in livestock production, wildlife and humans. Livest Sci 130:41–46

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Maier GU, Breitenbuecher J, Gomez JP, Samah F, Fausak E, Van Noord M (2022) Vaccination for the prevention of neonatal calf diarrhea in cow-calf operations: a scoping review. Vet Anim Sci 15:100238

    Article  PubMed  PubMed Central  Google Scholar 

  3. Patel JR (2005) Relative efficacy of inactivated bovine herpesvirus-1 (BHV-1) vaccines. Vaccine 23:4054–4061

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Platt R, Widel PW, Kesl LD, Roth JA (2009) Comparison of humoral and cellular immune responses to a pentavalent modified live virus vaccine in three age groups of calves with maternal antibodies, before and after BVDV type 2 challenge. Vaccine 27:4508–4519

    Article  CAS  PubMed  Google Scholar 

  5. Greiser-Wilke I, Grummer B, Moennig V (2003) Bovine viral diarrhoea eradication and control programmes in Europe. Biologicals 31:113–118

    Article  PubMed  Google Scholar 

  6. Vlasova AN, Deol P, Sircar S, Ghosh S, Jakab S, Bányai K, Dhama K, Amimo JO, Saif LJ, Malik YS (2020) Animal rotaviruses. Animal-Origin Viral Zoonoses 2020:163–202

    Article  Google Scholar 

  7. Guo Z, Chen XX, Li R, Qiao S, Zhang G (2018) The prevalent status and genetic diversity of porcine reproductive and respiratory syndrome virus in China: a molecular epidemiological perspective. Virol J 15:2

    Article  PubMed  PubMed Central  Google Scholar 

  8. Jorge S, Dellagostin OA (2017) The development of veterinary vaccines: a review of traditional methods and modern biotechnology approaches. Biotechnol Res Innov 1:6–13

    Article  Google Scholar 

  9. Mayers J, Mansfield KL, Brown IH (2017) The role of vaccination in risk mitigation and control of Newcastle disease in poultry. Vaccine 35:5974–5980

    Article  PubMed  Google Scholar 

  10. Ge J, Wang X, Tian M, Gao Y, Wen Z, Yu G, Zhou W, Zu S, Bu Z (2015) Recombinant Newcastle disease viral vector expressing hemagglutinin or fusion of canine distemper virus is safe and immunogenic in minks. Vaccine 33:2457–2462

    Article  CAS  PubMed  Google Scholar 

  11. Ren G, Tian G, Liu Y, He J, Gao X, Yu Y, Liu X, Zhang X, Sun T, Liu S, Yin J, Li D (2016) Recombinant Newcastle disease virus encoding IL-12 and/or IL-2 as potential candidate for hepatoma carcinoma therapy. Technol Cancer Res Treat 15:NP83-94

    Article  CAS  PubMed  Google Scholar 

  12. Nakaya T, Cros J, Park MS, Nakaya Y, Zheng H, Sagrera A, Villar E, Garcia-Sastre A, Palese P (2001) Recombinant Newcastle disease virus as a vaccine vector. J Virol 75:11868–11873

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wu Y, He J, Geng J, An Y, Ye X, Yan S, Yu Q, Yin J, Zhang Z, Li D (2017) Recombinant Newcastle disease virus expressing human TRAIL as a potential candidate for hepatoma therapy. Eur J Pharmacol 802:85–92

    Article  CAS  PubMed  Google Scholar 

  14. Zhang M, Ge J, Li X, Chen W, Wang X, Wen Z, Bu Z (2016) Protective efficacy of a recombinant Newcastle disease virus expressing glycoprotein of vesicular stomatitis virus in mice. Virol J 13:31

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zamarin D, Palese P (2012) Oncolytic Newcastle disease virus for cancer therapy: old challenges and new directions. Future Microbiol 7:347–367

    Article  CAS  PubMed  Google Scholar 

  16. Khattar SK, Collins PL, Samal SK (2010) Immunization of cattle with recombinant Newcastle disease virus expressing bovine herpesvirus-1 (BHV-1) glycoprotein D induces mucosal and serum antibody responses and provides partial protection against BHV-1. Vaccine 28:3159–3170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhang M, Ge J, Wen Z, Chen W, Wang X, Liu R, Bu Z (2017) Characterization of a recombinant Newcastle disease virus expressing the glycoprotein of bovine ephemeral fever virus. Arch Virol 162:359–367

    Article  CAS  PubMed  Google Scholar 

  18. Sun W, Liu Y, Amanat F, Gonzalez-Dominguez I, McCroskery S, Slamanig S, Coughlan L, Rosado V, Lemus N, Jangra S, Rathnasinghe R, Schotsaert M, Martinez JL, Sano K, Mena I, Innis BL, Wirachwong P, Thai DH, Oliveira RDN, Scharf R, Hjorth R, Raghunandan R, Krammer F, Garcia-Sastre A, Palese P (2021) A Newcastle disease virus expressing a stabilized spike protein of SARS-CoV-2 induces protective immune responses. Nat Commun 12:6197

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  19. Gonzalez-Dominguez I, Martinez JL, Slamanig S, Lemus N, Liu Y, Lai TY, Carreno JM, Singh G, Singh G, Schotsaert M, Mena I, McCroskery S, Coughlan L, Krammer F, García-Sastre A, Palese P, Sun W (2022) Trivalent NDV-HXP-S vaccine protects against phylogenetically distant SARS-CoV-2 variants of concern in mice. Microbiol Spectr 10:e0153822

    Article  PubMed  Google Scholar 

  20. Ge J, Deng G, Wen Z, Tian G, Wang Y, Shi J, Wang X, Li Y, Hu S, Jiang Y, Yang C, Yu K, Bu Z, Chen H (2007) Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses. J Virol 81:150–158

    Article  CAS  PubMed  Google Scholar 

  21. DiNapoli JM, Nayak B, Yang L, Finneyfrock BW, Cook A, Andersen H, Torres-Velez F, Murphy BR, Samal SK, Collins PL, Bukreyev A (2010) Newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of H5N1 highly pathogenic avian influenza virus protect against virus challenge in monkeys. J Virol 84:1489–1503

    Article  CAS  PubMed  Google Scholar 

  22. Ge J, Wang X, Tao L, Wen Z, Feng N, Yang S, Xia X, Yang C, Chen H, Bu Z (2011) Newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats. J Virol 85:8241–8252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Kong D, Wen Z, Su H, Ge J, Chen W, Wang X, Wu C, Yang C, Chen H, Bu Z (2012) Newcastle disease virus-vectored Nipah encephalitis vaccines induce B and T cell responses in mice and long-lasting neutralizing antibodies in pigs. Virology 432:327–335

    Article  CAS  PubMed  Google Scholar 

  24. Kim SH, Subbiah M, Samuel AS, Collins PL, Samal SK (2011) Roles of the fusion and hemagglutinin-neuraminidase proteins in replication, tropism, and pathogenicity of avian paramyxoviruses. J Virol 85:8582–8596

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kim SH, Paldurai A, Samal SK (2017) A novel chimeric Newcastle disease virus vectored vaccine against highly pathogenic avian influenza virus. Virology 503:31–36

    Article  CAS  PubMed  Google Scholar 

  26. Soliman RM, Nishioka K, Daidoji T, Noyori O, Nakaya T (2023) Chimeric Newcastle disease virus vectors expressing human IFN-gamma mediate target immune responses and enable multifaceted treatments. Biomedicines 11:455

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cho YI, Yoon KJ (2014) An overview of calf diarrhea—infectious etiology, diagnosis, and intervention. J Vet Sci 15:1–17

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  28. Brunauer M, Roch FF, Conrady B (2021) Prevalence of worldwide neonatal calf diarrhoea caused by bovine rotavirus in combination with bovine coronavirus, Escherichia coli K99 and Cryptosporidium spp.: a meta-analysis. Animals (Basel) 11:1014

    Article  PubMed  Google Scholar 

  29. Miyazawa K, Hondo T, Kanaya T, Tanaka S, Takakura I, Itani W, Rose MT, Kitazawa H, Yamaguchi T, Aso H (2010) Characterization of newly established bovine intestinal epithelial cell line. Histochem Cell Biol 133:125–134

    Article  CAS  PubMed  Google Scholar 

  30. Miyazawa K, Kanaya T, Takakura I, Tanaka S, Hondo T, Watanabe H, Rose MT, Kitazawa H, Yamaguchi T, Katamine S, Nishida N, Aso H (2010) Transcytosis of murine-adapted bovine spongiform encephalopathy agents in an in vitro bovine M cell model. J Virol 84:12285–12291

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Arnold M, Patton JT, McDonald SM (2009) Culturing, storage, and quantification of rotaviruses. Curr Protoc Microbiol Chapter 15:Unit 15C.3

    Google Scholar 

  32. Payne AF, Binduga-Gajewska I, Kauffman EB, Kramer LD (2006) Quantitation of flaviviruses by fluorescent focus assay. J Virol Methods 134:183–189

    Article  CAS  PubMed  Google Scholar 

  33. Krishnamurthy S, Huang Z, Samal SK (2000) Recovery of a virulent strain of Newcastle disease virus from cloned cDNA: expression of a foreign gene results in growth retardation and attenuation. Virology 278:168–182

    Article  CAS  PubMed  Google Scholar 

  34. Nishioka K, Daidoji T, Nakaya T (2022) Downregulation of calcium-regulated heat stable protein 1 expression by low-temperature stimulation causes reduction of interferon-beta expression and sensitivity to influenza viral infection. Virus Res 309:198659

    Article  CAS  PubMed  Google Scholar 

  35. Zhao P, Cao J, Zhao LJ, Qin ZL, Ke JS, Pan W, Ren H, Yu JG, Qi ZT (2005) Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine. Virology 331:128–135

    Article  CAS  PubMed  Google Scholar 

  36. Lee S, Belitsky BR, Brinker JP, Kerstein KO, Brown DW, Clements JD, Keusch GT, Tzipori S, Sonenshein AL, Herrmann JE (2010) Development of a Bacillus subtilis-based rotavirus vaccine. Clin Vaccine Immunol 17:1647–1655

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Lappalainen S, Pastor AR, Malm M, Lopez-Guerrero V, Esquivel-Guadarrama F, Palomares LA, Vesikari T, Blazevic V (2015) Protection against live rotavirus challenge in mice induced by parenteral and mucosal delivery of VP6 subunit rotavirus vaccine. Arch Virol 160:2075–2078

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Shoja Z, Jalilvand S, Latifi T, Roohvand F (2022) Rotavirus VP6: involvement in immunogenicity, adjuvant activity, and use as a vector for heterologous peptides, drug delivery, and production of nano-biomaterials. Arch Virol 167:1013–1023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Luchs A, Timenetsky MdCST (2016) Group A rotavirus gastroenteritis: post-vaccine era, genotypes and zoonotic transmission. Einstein (São Paulo) 14:278–287

    Article  PubMed  Google Scholar 

  40. Kim SH, Samal SK (2016) Newcastle disease virus as a vaccine vector for development of human and veterinary vaccines. Viruses 8:183

    Article  PubMed  PubMed Central  Google Scholar 

  41. He L, Zhang Z, Yu Q (2020) Expression of two foreign genes by a Newcastle disease virus vector from the optimal insertion sites through a combination of the ITU and IRES-dependent expression approaches. Front Microbiol 11:769

    Article  PubMed  PubMed Central  Google Scholar 

  42. Hu Z, Ni J, Cao Y, Liu X (2020) Newcastle disease virus as a vaccine vector for 20 years: A focus on maternally derived antibody interference. Vaccines (Basel) 8:222

    Article  CAS  PubMed  Google Scholar 

  43. Bello MB, Yusoff K, Ideris A, Hair-Bejo M, Jibril AH, Peeters BPH, Omar AR (2020) Exploring the prospects of engineered Newcastle disease virus in modern vaccinology. Viruses 12:451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Nakaya Y, Nakaya T, Park MS, Cros J, Imanishi J, Palese P, Garcia-Sastre A (2004) Induction of cellular immune responses to simian immunodeficiency virus gag by two recombinant negative-strand RNA virus vectors. J Virol 78:9366–9375

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Caddy SL, Vaysburd M, Wing M, Foss S, Andersen JT, O’Connell K, Mayes K, Higginson K, Iturriza-Gomara M, Desselberger U, James LC (2020) Intracellular neutralisation of rotavirus by VP6-specific IgG. PLoS Pathog 16:e1008732

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Bottermann M, Caddy SL (2022) Virus neutralisation by intracellular antibodies. Semin Cell Dev Biol 126:108–116

    Article  CAS  PubMed  Google Scholar 

  47. Gonzalez DD, Mozgovoj MV, Bellido D, Rodriguez DV, Fernandez FM, Wigdorovitz A, Parreno VG, Dus Santos MJ (2010) Evaluation of a bovine rotavirus VP6 vaccine efficacy in the calf model of infection and disease. Vet Immunol Immunopathol 137:155–160

    Article  CAS  PubMed  Google Scholar 

  48. Afchangi A, Jalilvand S, Mohajel N, Marashi SM, Shoja Z (2019) Rotavirus VP6 as a potential vaccine candidate. Rev Med Virol 29:e2027

    Article  PubMed  Google Scholar 

  49. Liu J, Xue L, Hu S, Cheng H, Deng Y, Hu Z, Wang X, Liu X (2018) Chimeric Newcastle disease virus-vectored vaccine protects chickens against H9N2 avian influenza virus in the presence of pre-existing NDV immunity. Arch Virol 163:3365–3371

    Article  CAS  PubMed  Google Scholar 

  50. Chandler TL, Yang A, Otero CE, Permar SR, Caddy SL (2023) Protective mechanisms of nonneutralizing antiviral antibodies. PLoS Pathog 19:e1011670

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Feng N, Lawton JA, Gilbert J, Kuklin N, Vo P, Prasad BVV, Greenberg HB (2002) Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6–specific IgA mAb. J Clin Invest 109:1203–1213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Jalilvand S, Marashi SM, Shoja Z (2015) Rotavirus VP6 preparations as a non-replicating vaccine candidates. Vaccine 33:3281–3287

    Article  CAS  PubMed  Google Scholar 

  53. Lappalainen S, Pastor AR, Tamminen K, Lopez-Guerrero V, Esquivel-Guadarrama F, Palomares LA, Vesikari T, Blazevic V (2014) Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo. Hum Vaccin Immunother 10:2039–2047

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Geletu US, Usmael MA, Bari FD (2021) Rotavirus in calves and its zoonotic importance. Vet Med Int 2021:6639701

    Article  PubMed  PubMed Central  Google Scholar 

Download references


We thank Yoshihiko Sakoda (Graduate School of Veterinary Medicine, Hokkaido University, Japan) for providing APMV-2, Ryota Tsunekuni and Takehiko Saito (National Institute of Animal Health, National Agriculture and Food Research Organization) for providing the NDV antiserum, and Hisashi Aso (Tohoku University, Japan) for providing BIE cell line. Appreciation is extended to Professor Nabil Bkear for his support and to all members of the Department of Animal Medicine, Faculty of Veterinary Medicine, Damanhour University, Damanhour, Egypt. The African green monkey kidney epithelial cell lines, MA104 (RCB0994), were provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT/AMED, Japan. We thank the staff at the Department of Infectious Diseases, Graduate School of Medical Science, Kyoto Prefectural University of Medicine.


This study was supported by KAKENHI Grants from the Japan Society for the Promotion of Science (JSPS; Grant nos. 20K17468 and 21H03184). A full scholarship from the Ministry of Higher Education of the Arab Republic of Egypt (ID: 40437).

Author information

Authors and Affiliations



RMS, KN, and TN designed the study; RMS, KN, and FM conducted the experiments; FM provided rotavirus samples; RMS and KN drafted the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Takaaki Nakaya.

Ethics declarations

Ethics approval and consent to participate

The experiments involving mice were conducted according to the guidelines and recommendations of the Institutional Animal Ethics Committee of the Animal Center of KPUM (Approval Number: M2022-541). 

Competing interests

The authors declare that they have no competing interests.

Additional information

Handling editor: Tina Dalgaard

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Confirmation of recombinant NDVs inserted BRV VP4 or VP6.

(A) Schema of constructed recombinant NDV genomes inserted with the BRV VP6 or VP4 gene. (B) indicates viral titer of the rescued viruses grown in embryonated eggs. (C) MDBK cells infected with either rNDV-WT-VP4 or rNDV-2F2HN-VP4 were stained by anti-NDV antiserum (green), BRV antibody (red), and Hoechst 33342 (blue; nuclei). (D) Electrophoresis of the PCR products for the verification of BRV-VP6 and VP4 antigens within the constructed viruses using each primer set as indicated. Arrows indicate the expected size (VP6: 1354 bp and VP4: 2511 bp).

Additional file 2: Assessment of genetic stability of VP6 gene within rNDV-WT-VP6 and rNDV-2F2HN-VP6 viruses.

(A) The recovered viruses were serially passaged 5 times in MDBK cells. After the five passages, expression of the VP6 gene was confirmed by immunostaining using anti-BRV antibody. (B) VP6 inserted region was amplified. Electrophoresis of PCR products was performed. Arrows indicate the expected size.

Additional file 3: Pathogenicity of the recombinant viruses in mice.

Weight changes in the mice inoculated intraperitoneally or intranasally with 1 × 107 FFU of each recombinant NDV. The mice in each group (n = 3) were observed and weighed daily for 14 days.

Additional file 4: Cross-reactivity of mice serum antibodies to SA11 Rotavirus.

(A) SA11 rotavirus-infected cells were stained by mice serum (green) after primary inoculation and Hoechst 33342 (blue; nuclei). (B) indicates SA11 rotavirus infection inhibition ratio using mice serum collected after primary inoculation. (C) Schematic drawing illustrating specific cell proliferation assay of mice splenocytes. (D) Assessment of specific cell proliferation of mice splenocytes after booster inoculation using MTT assay. (n = 3, *P < 0.05, **P < 0.01, ***P < 0.001).

Additional file 5: Primer sets used in this study.

Additional file 6: Schema of the base sites for each primer set.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Soliman, R.M., Nishioka, K., Murakoshi, F. et al. Use of live attenuated recombinant Newcastle disease virus carrying avian paramyxovirus 2 HN and F protein genes to enhance immune responses against species A rotavirus VP6 protein. Vet Res 55, 16 (2024).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: