Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge

Veterinary Microbiology 174 (2014) 362–371

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Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge Feng-Xue Wang a, Shu-Qin Zhang a, Hong-Wei Zhu a, Yong Yang a, Na Sun a, Bin Tan a, Zhen-Guang Li a,b, Shi-Peng Cheng a, Zhen F. Fu b,*, Yong-Jun Wen a,* a

State Key Laboratory for Molecular Biology of Special Economic Animals, Institute of Special Economic Animals and Plants, Chinese Academy of Agricultural Sciences CAAS, Changchun 130112, Jilin, China Department of Pathology, University of Georgia, Athens, GA 30602, USA

b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 11 April 2014 Received in revised form 15 October 2014 Accepted 27 October 2014

The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection. ß 2014 Elsevier B.V. All rights reserved.

Keywords: Canine distemper virus Rabies virus Hemagglutinin gene Replicating viral vector Dog

1. Introduction Canine distemper virus (CDV) is belonging to the genus morbillivirus within the Paramyxoviridae family, which is an enveloped nonsegmented negative-strand RNA virus

* Corresponding authors at: Chinese Academy of Agricultural Sciences CAAS, State Key Laboratory for Molecular Biology of Special Economic Animals, No. 4899, Juye Street, Changchun, 130122, China. Tel.: +1 706 542 7021/+86 431 81919840; fax: +1 706 542 5828/+86 431 81919840. E-mail addresses: [email protected] (Z.F. Fu), [email protected] (Y.-J. Wen). http://dx.doi.org/10.1016/j.vetmic.2014.10.023 0378-1135/ß 2014 Elsevier B.V. All rights reserved.

(Rozenblatt et al., 1985). Canine distemper (CD) occurs worldwide, affects a broad range of mammalian host species (Barrett, 1999), and is highly infectious, particularly in young dogs, resulting in elevated morbidity and mortality (Appel et al., 1991), which is characterized by fever, rash, diarrhea, nasal discharge, conjunctivitis and occasional neurologic complications (Moss and Griffin, 2006; von Messling et al., 2003). CDV Mortality rates are 50% for domestic dogs (Ek-Kommonen et al., 2003) and 100% for ferrets (Silin et al., 2007). Currently, attenuated CDV strains are available for use worldwide as live vaccines against CDV infection in dogs, minks, and other carnivores (Blixenkrone-Moller, 1989). There is insufficient to induce

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protection when administered in the presence of maternal antibody which is undetectable within 12 weeks (Appel and Harris, 1988; Stephensen et al., 1997). In addition, the modified-live vaccine elicits robust immunity in dogs, but retains sufficient virulence to cause severe disease and death in more susceptible species (Barrett, 1999). These disadvantages have resulted in increasing interest in a safe and efficient CDV vaccine for highly susceptible species. The development of reverse genetics has provided a powerful tool to create recombinant nonsegmented negative-strand RNA virus-based vaccine (Billeter et al., 2009; Nagai, 1999). Recombinant rabies virus vaccine strain-based vectors expressing proteins of pathogens stably show exceptional promise as vaccine vehicles against other infectious diseases (McGettigan et al., 2001a,b; Siler et al., 2001), and safety concerns have recently been addressed (Wen et al., 2011). Our previous studies have shown that recombinant RV expressing chemokines/cytokines including granulocyte-macrophage colonystimulating factor (GM-CSF), macrophage-derived chemokine (MDC), and macrophage inflammatory protein (MIP-1a), can enhance RV immunogenicity via recruitment and/or activation of DCs (Wen et al., 2011; Zhou et al., 2013). As of today, CDV is the primary target for most of these vaccine vectors, and a large body of information regarding their use is available (Jones et al., 1997; Nielsen et al., 2012b). The strategy is delivering the CDV antigen to the immune system using recombinant rabies virus (LBNSE) as a vector, which is generated from an SAD L16 cDNA clone in pcDNA3.1(+) (Invitrogen, Carlsbad, CA) (Schnell et al., 1994). Previously, several studies have shown that expression of CDV H gene results in potent immune responses directed against CDV in a small animal model (Stephensen et al., 1997). The H gene plays the determinant role in viral entry, as it mediates the binding of the virus to the surface, and responsible for viral attachment and cell tropism. The H protein has been shown to induce specific humoral and cellular immune responses against CDV in mice. In this study, we generated a recombinant RV attenuated vaccine strain expressing CDV H gene which is responsible for viral attachment to host cell to initiate virus infection and play an important role in inducting the protective immunity as well (von Messling et al., 2001). The feasibility of this recombinant RV to serve as a bivalent vaccine against rabies and canine distemper was evaluated, especially, for the wild life conservation efforts. 2. Materials and methods 2.1. Cells and viruses Mouse neuroblastoma cells (NA) were maintained in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY). BSR cells, a cloned cell line derived from BHK21 cells, and MDCK (canine epithelial kidney cells), were purchased from American Type Culture Collection (ATCC) (Manassas, VA) and maintained in Dulbecco’s modified Eagle’s medium (Mediatech) containing 10% FBS. Recombinant RV strains were propagated in BSR cells. Challenge virus standard CDV-ZJ7 was propagated in MDCK cells

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(Tan et al., 2011). Fluorescein isothiocyanate (FITC)conjugated antibody against the RV N protein was purchased from FujiRab (Melvin, PA). Goat Anti-Mouse IgG H&L (DyLight1 594) preadsorbed (ab96881) was purchased from Abcam. 2.2. Plasmid construction The plasmids encoding the recombinant RV vaccine vector, pLBNSE, were described previously (Wen et al., 2011). Briefly, the recombinant RV vector pLBNSE, flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences, was generated from an SAD L16 cDNA clone in pcDNA3.1(+) (Invitrogen, Carlsbad, CA). A transcription unit with the BwsiI and NheI restriction sites was created between the G and L-coding sequences by deleting the pseudogene. To create a new RV vaccine vector expressing CDV H gene, the gene encoding the H gene was amplified from commercial vaccine strain CDV3 in china by using PfX polymerase (Biolabs, Inc.). And the primers RP40 (50 AAACTCH GENECGTACGACAATGGGTGCH GENEAGCGTCA30 ) and RP184 (50 -AAAGCTAGCTTAATCCTCATCCTGTCTAC30 ) were designed. The PCR product was digested with BsiWI-NheI and ligated to the previously BsiWI-NheIdigested plasmid pLBNSE. The resulting plasmids were designated pLBNSE and pLBNSE-CDVH (Fig. 1a) and insert was verified by restriction analysis and DNA sequencing. 2.3. Recombinant virus rescue and insert gene expressing examination The full-length chimeric cDNA clones were purified and was co-transfected with the plasmids encoding the RV N, P, G, and L proteins into BSR cells which stably expressing T7 RNA polymerase (Buchholz et al., 1999) by standard methods (McGettigan et al., 2001b), and infectious was detected in 2 out of 6 wells of transfected cells. The chimeric virus was recovered as described previously (Inoue et al., 2003). For recovery experiments with the recombinant RVs, the previously described RV recovery system was used (Inoue et al., 2003; Takayama-Ito et al., 2006). Briefly, BSR cells were transfected with 2.0 m g of the full-length infectious clone, 0.5 m g of N-, 0.25 mg of P-, 0.1 mg of L-, and 0.15 m g of G-expressing plasmids using SuperFect Transfection Reagent (Qiagen, Valencia, CA) according to the manufacture’s protocol. After incubation for 4 days, the culture medium was removed and fresh medium added to the cells. After incubation for another 3 days, the culture medium was harvested and the cells examined for the presence of rescued viruses by using FITC-conjugated antibody against the RV N protein (Fujirebio Diagnostic, Inc. DA, USA). And the transfected cells also were examined by IFA, which using mus musculus sera against CDV and, Dylight 594-conjugated second antibody against the mus musculus IgG. 2.4. The growth curve of recombinant virus BSR cells (a BHK-21 clone) were plated in 60-mmdiameter dishes. After 16 h incubation, the cells were

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Fig. 1. Construction of recombinant RV vaccine vector expressing CDV H gene. (a) Schematic diagrams of two full-length cDNA clones used to rescue recombinant virus. The construction of the individual plasmid, based on pLBNSE, is displayed. BsiwI and NheI were introduced ahead of L gene in pLBNSE. The hemagglutinin of CDV was inserted by BsiwI and NheI. (b and c) The growth curve of recombinant vaccine vectors. BSR cells (b) or mNA cells (c) were infected with LBNSE and LBNSE-CDV-H at an MOI of 5. Aliquots of tissue culture supernatants were collected and viral titers were determined in duplicate as indicated.

infected at an MOI of 5 (one-step growth) with LBNSE, LBNSE-CDV-H. After incubation at 37 8C for 1 h, inocula were removed, and cells were washed four times with PBS to remove any unabsorbed virus. Three milliliters of complete medium was added back, and 100 ml of tissue culture supernatants was removed at the indicated time points after infection. Titers of virus aliquots were determined in duplicate on BSR cells. 2.5. Virus titration We determined the virus titration by using the direct fluorescentantibody assay in NA cells. Serial 10-fold dilutions of virus were placed on 96-well tissue culture plates containing confluent BSR cells or mNA cells and incubated at 34 8C. The culture supernatant was discarded at 2 days after infection, and the cells were fixed with icecold 80% acetone for 20 min. Fixed cells were derectly incubated with an FITC-conjugated to anti-dog antibody RV for 1 h at 37 8C. Antigen-positive foci were counted under a fluorescence microscope (Leica, Germany), and viral titers were calculated as numbers of fluorescent focus units (FFU) per milliliter. All titrations were carried out in quadruplicate. 2.5.1. Real-time PCR A real-time (RT) SYBR green PCR assay was carried out in an MJ MiNi (BioRad, USA) to quantify CDV H mRNA transcription. RNA was extracted from infected cell cultures with Trizol (Invitrogen, USA) and used as a template for one-step quantitative RT PCR (qRT-PCR) (TransGen, Beijing). The primer pair 50 -CAGAGGCAGAGGTGACATA-30 and 50 -CTGAGATAGCGGTTAGCAT-30 was based on inserted gene CDV H. Amplification was carried out at

45 8C for 5 min and 94 8C for 30 s, followed by 40 cycles in three steps: 94 8C for 5 s, 48.7 8C for 15 s and 72 8C for 1 min. A without template as negative control reaction mixture were performed with the above protocol. 2.6. Vaccination and challenge experiments of dogs Twenty-fourth 5-weeks-old female dogs, whose serology is negative to CDV and RV, were purchased from Shifang Experiment Animal Corporation (Jiangshu, China) and raised by isolated cages in the Animal Facility, Division of Zoonoses, Institute of Special Economic Animal and Plant Sciences. Twenty-fourth dogs were randomly divided into 4 groups, and 6 dogs in each group were used to verify the vaccine efficacy. The test procedure and group is shown in Fig. 2. Two groups of dogs were vaccinated by i.m. route at 6 weeks of age with 106 FFU of the LBNSE and LBNSECDVH constructs with 2 ml per dose, respectively. One group of dogs received an attenuated CDV vaccine, CDV3, which has been widely used in dogs or fur animals in China. The last group remained as a not-immunized and not-challenged strict control (innoculated with medium and no challenge) during the experiment. At age of 6 weeks and thereafter, blood was collected from anesthetized dogs by venipuncture of the cephalic or saphenous vein or terminally by cardiac puncture, and blood samples were collected as given time post-innoculation. All animal work and experimental procedures were conducted with an approval of Institutional Animal Care and Use Committee of Special Economic Animals and Plants, Chinese Academy of Agricultural Sciences, China. The ZJ7 strain of CDV was grown and titered in MDCK cells essentially as described previously (Tan et al., 2011), which has been isolated from lung tissues of a dog. We

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Fig. 2. Study design of in vivo experiment. Time schedule for the vaccination and challenge period. Numbers represent sampling time points in days post vaccination, C-numbers in days post challenge infection. Sampling time points of blood and throat swabs are depicted by $ and #, respectively. The grouping was showed.

used the third virus passage after plaque purification. Four groups animals were challenged intranasally at 10 weeks of age with 103 50% tissue culture infectious doses (TCID50) of 1 ml CDV strain ZJ7 in while under anesthesia to prevent sneezing. Under anesthesia with protocol (Institute of Special Economic Animal and Plant Sciences, Jilin, China), the viral suspension was dropped into the right conjunctiva and nostril of 18 dogs using a syringe without a needle. All animal work and experimental procedures were conducted with approval of Institutional Animal Care and Use Committee of Institute of Special Economic Animals and Plants, Chinese Academy of Agricultural Sciences, China.

Animal sera were measured for RV neutralizing antibodies (NAs) using the rapid fluorescent focus inhibition test as described previously (Zavadova et al., 1996), and titers of RV NAs were normalized to international units (IU/ml). For serum CDV NA assessment, neutralizing titers were determined in Vero cells using a TCID50 format assay based on the method of Appel and Robson (1973). Briefly, serial two-fold dilutions of serum were prepared in microtiter plates and mixed with an equal volume of CDV antigen, inoculated in to Vero cells and examined for a CPE after six days. Subjective evaluation of clinical signs was record blinded by identifying animals, without reference to vaccine group.

2.7. Monitoring clinical course and Serological tests After challenge, dogs were monitored daily by body temperature and clinical examination until they were euthanized with Nembutal (Solabio Pharmaceutical Co. Ltd., China) at the end of the experiment. The nasal, rectal swabs were collected before inoculation of virus and each day post-infection from all infected dogs until the end of experiment. All samples for isolation were stored at 80 8C until being used. Following inoculation, the clinical condition of the animals was evaluated daily. To assess clinical disease severity during the experimental phase, a score system was used. The score standard has been figured out in Table 1 (normal, (0) loss of appetite or depressed, (1) diarrhea and bloody stool or eye nose secretions increase or epidermal keratinization, (3) neural symptoms, (4) and dead, (5)).

2.8. Gross pathology and histopathology The necropsy, lung, spleen, kidney, liver, heart, tonsil, lymph nodes, and brain were visually inspected, and the lungs and brains were harvested for histopathology following hematoxylin and eosin (H&E) staining. 2.9. Statistical analysis The statistical significance of the differences between group values was determined using one-way analysis of variance (ANOVA) or Fisher’s exact test (x2 GraphPad). All P-values were two-tailed and considered statistically significant when the associated probability was less than 0.05.

Table 1 Scoring system for the clinical signs of distemper disease in dogs infected with virulent strain ZJ 7. The score was given. Normal, (0) loss of appetite or depressed, (1) diarrhea and bloody stool or eye nose secretions increase or epidermal keratinization, (3) neural symptoms, (4) and dead, (5). Clinical observation

Score

Clinical observation

Score

Temperature performance

0 1 2 0 1 2 0

Loss of appetite or depressed Diarrhea and bloody stool or eye nose secretions increase or epidermal keratinization Neural symptoms

1 3 4

Dead

5

Body weight change

Normal

<39 8C 39.1–39.9 8C >40.0 8C Loss of weght 0–5% Loss of weight 5–10% Loss of weight >10%

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3. Results 3.1. Construction of chimeric clone and recovery and detection of chimeric viruses expressing CDV H gene To develop recombinant rabies virus (RV) expressing hemagglutin of canine distemper virus (CDV) (LBNSE-CDVH), a recombinant virus was produced using the RV infectious cDNA clone pLBNSE as the backbone as described under material and methods. Chimeric clone was shown in Fig. 1a, namely the hemagglutinin of CDV was inserted ahead of L gene in pLBNSE by BsiwI and NheI. The resulting chimeric clone was designated pLBNSE-CDVH and confirmed by sequencing. Several scattered nucleotide changes and resulting amino acid alterations including introduced restriction endonuclease (RE) sites were observed throughout the genome. The culture supernatant was collected at 5 days posttransfection. The cloned virus was further amplified by subsequent passages in mNA cells. The titer of passage-3 rescued virus was determined by direct immunofluorescence assay. It was indicated that the transfected cells possessed high levels of RV N protein and CDV H protein expression by FA or IFA and qRT-PCR (Fig. 3a and b). Fullgenome sequencing results showed that recovered virus LBNSE-CDV-H maintained the nucleotide sequence of the original plasmids pLBNSE-CDV-H.

the dogs vaccinated with LBNSE-CDV-H had high levels of VN antibodies of CDV and RV in their serum (Fig. 4a and b). To evaluate the protection conferred by vaccination with LBNSE-CDV-H, the dogs at 28 day post-immunization were challenged with virulent CDV by the intranasal route. The result showed vaccinated dogs with LBNSE-CDV-H and CDV3 were not observed any clinical signs of CDV infection after challenge. While the dogs in mock group developed systemic distemper. The 6 dogs vaccinated with the LBNSE developed an increase in body temperature, viremia and respiratory tract problems during 6 days after challenge inoculation with CDV-ZJ7. Temperature curves indicated the dogs in mock group exhibited fever with biphasic thermal response, which was character of canine distemper (Fig. 5a). Temperature of the dogs in mock group were significantly higher (p < 0.01) than those of negative group and vaccinated group at 5–6 dpi and 11–12 dpi. The clinical scores of the dogs in mock group were higher than control (Fig. 5b). Exclusively, RT-PCR test found that temporary viremia was presented in the dogs in vaccine group during 6 days after challenged (Table 2), at the same time persistent viremia in mock group. No abnormal clinical signs were observed in immunized dogs challenged with medium control during the course of the study, which suggests that the recombinant virus LBNSE-CDV H is safe and effective for dogs against CDV challenge. 3.4. Gross pathological and histopathological changes

3.2. Growth kinetics curve of recombinant RVs To study the replication kinetics of LBNSE-CDV-H and in greater detail, a multicycle growth curve was performed by infecting BSR cells at an MOI of 5. One concern about a recombinant RV expressing a very large gene such as CDV H gene was that this will affect the viral replication cycle, resulting in a recombinant RV that grows slowly or to low titers. From Fig. 1, we can get an information of growth kinetics of the reconstituted virus and the parental virus. The skeleton virus rLBNSE and the chimeric virus LBNSECDV-H showed semblable growth kinetics in BSR cells (Fig. 1b) and mNA cells (Fig. 1c). The recombinant RV grew very similarly and reached approximately the same titer at similar time points, indicating there were no differences in viral spread. We also performed a one-step growth curve to analyze a single cell cycle of viral replication. The highest titers reached to 108.5 FFU/ml within 4 days postinfection. Both two viruses showed approximately the same viral titer at all five time points. Therefore, the insertion of CDV H did not have any major impact on the viral life cycle, unlike the results for rhabdoviruses expressing HIV-1 genes (Schnell et al., 2000) or a foreign gene from a different location within the viral genome (McGettigan et al., 2003). 3.3. Immune response and protection in dogs after vaccination with chimeric virus against CDV We immunized dogs with the chimeric virus LBNSECDV-H. The MEM, CDV3 vaccine and rLBNSE were as mock, positive and negative respectively. At 21 days postinoculation, sera were collected for CDV and RV NA assays,

Pathological lesions were found in all control dogs in mock group at necropsy, including mainly bronchitis, catarrhal pneumonia, gastroenteritis. Under microscopic examination, vascular gore, expansive glandular cavity around blood vessels (edema phenomenon), expansive glandular cavity surrounding microglia, and a small amount of inflammatory cells in blood vessels were shown in dog brains of mock group. In addition, denaturation of neurocyte nuclei and phenomenon of a little glial cell adhesion around nerve cells were manifested (Fig. 6). In immunized dogs, no pathological damage was found until 28 or 50 dpi. After the dogs were challenged at 28 dpi, none of immunized dogs had interstitial pneumonia. 4. Discussion As known, dogs are highly susceptible to CDV, which is a current problem for companion animals (Lan et al., 2005). Previous studies have shown that DNA plasmids encoding the H protein of CDV was used for immunization because the H protein appears essential for inducing neutralising antibodies against CDV in adult mice and mink (Dahl et al., 2004; Nielsen et al., 2012a). Cherpillod et al. ’s studies indicated that DNA vaccination of dogs with the H, F and N genes of CDV induced protection against severe distemper. Similarly, DNA plasmids encoding the H gene of MeV have been shown to induce VN antibodies for protecting macaques against measles (Pan et al., 2008; Polack et al., 2003). Additionally, the first canine vaccine developed and licensed using recombinant DNA technology was the canarypox-vectored recombinant CDV (rCDV) vaccine (Pastoret and Vanderplasschen, 2003). This kind of

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Fig. 3. Immunofluorescence analysis of CDV H protein expression. (a) Confluent BSR cells were infected with LBNSE and LBNSE-CDV-H at an MOI of 0.01. The infected cells were fixed and probed with serum against CDV and then incubated with a goat anti-mouse IgG H&L (DyLight 594) preadsorbed (ab96881), or derectly incubated with an FITC-conjugated to anti-dog antibody RV. Cells were analyzed by using a confocal laser microscope. (b) The BSR cells were infected with LBNSE (Mock) and LBNSE-CDV-H at an MOI of 0.1. RFU of amplified CDV HA by qRT-PCR at 48 h post infection was showed. * p < 0.05.

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Fig. 4. Immune response and protection of LBNSE-CDVH. (a) CDV NA titers and (b) RV NA titers of immunized animals. Groups of four dogs were inoculated with 106 TCID50 of LBNSE and LBNSE-CDV-H. Animals were observed daily for 3 weeks after inoculation. Blood samples were collected to detect NA to CDV (a) and RV (b) at 21 day after vaccination. NAs to RV were detected and normalized to international units (IU) by using the WHO standard.

recombinant viral vectored CDV vaccine, unlike the modified live CDV virus vaccines, cannot revert to a virulent form, because there is no CDV virus present in the canarypox vaccine. Previously our studies have shown that foreign proteins such as GM-CSF, MDC, and MIP-1a were stably expressed by RV-based vaccine vectors and could enhance the immunogenicity by recruiting and/or activating dendritic cells (DC) in vaccinated mice (Wen et al., 2011). In this study, CDV H gene was cloned into the RV genome and recombinant virus LBNSE-CDV-H was rescued. Generally, the incorporation of less than 4 kb into backbone did not interfere with the growth of the recombinant RV, as the

titers of an CDV H gene-expressing RV are not reduced significantly (Fig. 1b and c). After incorporation of CDV H gene, the recombinant virus still grew to a high final titer of about 108.5 FFU/ml. While the multicycle growth curve shows an additive effect of the expression of H gene in addition to slower growth due to the expression of a foreign gene between the G and L genes from the vector BNSP (Wen et al., 2011). Safety is a major concern for the use of every live viral vector, this RV based vectors expressing CDV H gene were completely safe in mice and apathogenic after intracranial infection (data not show). As mentioned above, RV-based vectors are very safe after peripheral inoculation, we exchanged the arginine at

Fig. 5. Clinical manifestation after challenge. The body temperature was measured after challenge with CDV (a). Data points represent the average of each group, and error bars indicate the standard deviation. Significant differences between groups were assessed by using the t test, ** p < 0.01. (b). the dogs were observed for clinical signs for three weeks after the challenge and clinical observation projects were scored.

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Table 2 Detection of CDV-ZJ7 in blood and throat swabs of the experimental groups and control groups by RT-PCR on days 2, 4, 6, 8, 10, 12, 14 after challenge. Group

Days after challenge Samples

2

4

6

8

10

12

14

LBNSE

Throat swab Blood

+ +

+ +

+ +

+ +

+ +

+ +

+ +

LBNSE-CDV H

Throat swab Blood

+ 

 +

 

 

 

 

 

CDV3

Throat swab Blood

+ 

 

 

 

 

 

 

Medium

Throat swab Blood

+ +

+ +

+ +

+ +

+ +

+ +

+ +

position 333 (R333) with glutamic acid (E333) and asparagine (N194) with serine (S194) within the RV glycoprotein (G) (Wen et al., 2011). We have established an infection model in dog with a circulating wild type strain of CDV, Dog/ZJ7 (Tan et al., 2011), which is used to evaluate the protective capacity of the CDV vaccine strain. The present study demonstrated that one shot with LBNSE-CDV-H given to dogs induced detectable VN antibodies at 21 days. All dogs (n = 6) vaccinated with CDV recombinant virus had VN antibody titers more than 1:4 at the age of 6 weeks. The measurement of dogs vaccinated is standard methods used with attenuated live vaccines and is also believed to correlate with protection against distemper. Previous studies showed that DNA vaccination of adult dog with

both the H and N genes of CDV induced VN antibody titers of 1:4 which conferred with protective immunity (Dahl et al., 2004). In addition, the neutralizing antibody response to the recombinant vaccines was same as that seen in response to the attenuated CDV vaccine. These vaccines LBNSE-CDV-H and attenuated CDV3 had indistinguishable neutralizing antibody responses when administered parenterally to dogs. These results indicated that the rCDV vaccine was same as the attenuated CDV in inducing a neutralizing antibody response, and survival rates did not differ among the groups. The dogs immunized with LBNSE developed persistent lymphopenia and typical clinical symptoms for CDV infection. They excreted large amounts of infectious virus and had lower VN antibody titers compared to the other

Fig. 6. Photomicrographs of brains and lungs from inoculated and control dogs. Vascular gore, expansive glandular cavity around blood vessels (edema phenomenon), expansive glandular cavity surrounding microglia, and a small amount of inflammatory cells in blood vessels were shown in dog brains inoculated with LBNSE, then challenged with virulence CDV. The brains were normal in immune groups, LBNSE-CDVH and CDV3, either before or after infection. There were necrosis of alveolar septa, artery congestion and a large number of mucus in lungs in control group inoculated with LBNSE. But the lungs were normal in dogs of immune groups. The pathological changes were indicated with red arrows.

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12 dogs given CDV3 and recombinant virus LBNSE-CDV-H group. The differences in the outcome of the CDV infection between the dogs might be attributed to individual differences in immune capability. In this study, 6 dogs vaccinated with recombinant virus LBNSE-CDV-H were protected against disease and no signs of viremia or systemic infection were found, and the results of gross pathological and histopathological indicated that the LBNSE-CDV-H vaccinated dogs were protected against CDV infection as well (Fig. 6). There was no difference in the gross pathological and histopathological changes of the dogs inoculated with LBNSE-CDV-H and CDV3, but the LBNSE group suffered severe pathological damages. Previous study indicated an adenovirus-vectored footand-mouth disease capsid subunit vaccine could improve the efficacy of Ad5 vaccines against FMDV serotype O and induces cell immune response (Moraes et al., 2011). Though the humoral immune responses was mainly examined in our study, which was not mean without cellular immune responses. Since LBNSE-CDV-H, as a virus vectored vaccine, immunization leads to in vivo synthesis of CDV-H, one would expect a strong cell immune response. In conclusion, the results show that the chimeric virus can effectively protect the animals from challenge of the wild type CDV-ZJ7. The large foreign genes can be functionally expressed by RV-based vectors from a transcription unit, which further emphasizes their potential utility as efficacious antiviral vaccines. Our findings of solid immune response following recombinant virus which contain the H gene from CDV vaccine strain vaccinations encourage further studies on subunit protein immunizations in dogs, especially on aim at reducing the risks of attenuated strain got virulence in the field prevalence. Competing interests The authors declare that they have no competing interests. Authors’ contributions Conceived and designed the experiments: WFX, WYJ, FZF. Performed the experiments: WFX, ZSQ, ZHW. Analysed the data: WFX, LZG, TB, CSP. Wrote the paper: WFX, WYJ. All authors read and approved the final manuscript. Acknowledgements This work was supported by the Special Fund for Agroscientific Research in the Public Interest (201303042) and Innovation Team for Special Economic Animal Diseases Prevention and Control Research in Jilin Province (No. 20121823). References Appel, M., Robson, D.S., 1973. A microneutralization test for canine distemper virus. Am. J. Vet. Res. 34, 1459–1463. Appel, M.J., Harris, W.V., 1988. Antibody titers in domestic ferret Jills and their kits to canine distemper virus vaccine. J. Am. Vet. Med. Assoc. 193, 332–333.

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