Bovine tongue epithelium-derived cells: A new source of bovine mesenchymal stem cells.

Mesenchymal stem cells (MSCs) possess the ability to differentiate into multiple cell lineages, and thus, confer great potential for use in regenerative medicine and biotechnology. In the present study, we attempted to isolate and characterize bovine tongue tissue epithelium-derived MSCs (boT-MSCs) and investigate the culture conditions required for long-term culturing of boT-MSCs. boT-MSCs were successfully isolated by the collagenase digestion method and their proliferative capacity was maintained for up to 20 or more passages. We observed a significant increase in the proliferation of boT-MSCs during the 20 consecutive passages under low-glucose Dulbecco's modified Eagle's medium culture condition among the three culture conditions. These boT-MSCs presented pluripotency markers (octamer-binding transcription factor 3/4 (Oct3/4) and sex determining region Y-box2 (Sox2)) and cell surface markers, which included CD13, CD29, CD44, CD73, CD90, CD105, CD166, and major histocompatibility complex (MHC) class I (MHC-I) but not CD11b, CD14, CD31, CD34, CD45, CD80, CD86, CD106, CD117, and MHC-II at third passage. Moreover, these boT-MSCs could differentiate into mesodermal (adipocyte, osteocyte, and chondrocyte) cell lineages. Thus, the present study suggests that the tongue of bovines could be used as a source of bovine MSCs.

Adverse Effects of Classical Swine Fever Virus LOM Vaccine and Jeju LOM Strains in Pregnant Sows and Specific Pathogen-Free Pigs.

In Jeju island of South Korea, a classical swine fever (CSF) non-vaccinated region, many pig farmers insisted on abortion and stillbirth in pregnant sows and high mortality of suckling/weaning piglets by circulating CSF virus from 2014 to 2018. We investigated whether CSF viruses isolated from pigs in Jeju Island (Jeju LOM) have recovered their pathogenicity by conducting experiments using pregnant sows and specific pathogen-free (SPF) pigs. The CSF modified live LOM vaccine (MLV-LOM) and Jeju LOM strains induced abortion and stillbirth in pregnant sows. Viral antigens were detected in the organs of fetuses and stillborn piglets in the absence of specific pathological lesions associated with the virulent CSF virus in both groups (MLV-LOM and Jeju LOM strain). However, antigen was detected in one newborn piglet from a sow inoculated with a Jeju LOM strain, suggesting that it may cause persistent infections in pigs. SPF pigs inoculated with the MLV-LOM or Jeju LOM strains were asymptomatic, but virus antigen was detected in several organ and blood samples. Virus shedding in both groups of animals was not detected in the feces or saliva until 21 days post inoculation. The serum concentration of the three major cytokines, IFN-α, TNF-α, and IL-10, known to be related to lymphocytopenia, were similar in both groups when the MLV-LOM or Jeju LOM strains were inoculated into SPF pigs. In conclusion, Jeju LOM strains exhibited most of the characteristics of the MLV-LOM in pigs and resulted in the same adverse effects as the MLV-LOM strain.

Impact of a Live Attenuated Classical Swine Fever Virus Introduced to Jeju Island, a CSF-Free Area.

Here, we examine the effects of LOM(Low virulence of Miyagi) strains isolated from pigs (Jeju LOM strains) of Jeju Island, where vaccination with a live attenuated classical swine fever (CSF) LOM vaccine strain was stopped. The circulation of the Jeju LOM strains was mainly caused by a commercial swine erysipelas (Erysipelothrix rhusiopathiae) vaccine mixed with a LOM vaccine strain, which was inoculated into pregnant sows of 20 pig farms in 2014. The Jeju LOM strain was transmitted to 91 pig farms from 2015 to 2018. A histopathogenic investigation was performed for 25 farms among 111 farms affected by the Jeju LOM strain and revealed pigs infected with the Jeju LOM strain in combination with other pathogens, which resulted in the abortion of fetuses and mortality in suckling piglets. Histopathologic examination and immunohistochemical staining identified CSF-like lesions. Our results also confirm that the main transmission factor for the Jeju LOM strain circulation is the vehicles entering/exiting farms and slaughterhouses. Probability estimates of transmission between cohabiting pigs and pigs harboring the Jeju LOM strain JJ16LOM-YJK08 revealed that immunocompromised pigs showed horizontal transmission (r = 1.22). In a full genome analysis, we did not find genetic mutation on the site that is known to relate to pathogenicity between Jeju LOM strains (2014-2018) and the commercial LOM vaccine strain. However, we were not able to determine whether the Jeju LOM strain (2014-2018) is genetically the same virus as those of the commercial LOM vaccine due to several genetic variations in structure and non-structure proteins. Therefore, further studies are needed to evaluate the pathogenicity of the Jeju LOM strain in pregnant sow and SPF pigs and to clarify the characteristics of Jeju LOM and commercial LOM vaccine strains.

Assessment of the efficacy of an attenuated live marker classical swine fever vaccine (Flc-LOM-BErns) in pregnant sows.

Here, we constructed an attenuated live marker classical swine fever (CSF) vaccine (Flc-LOM-BErns) to eradicate CSF. This was done by taking infectious clone Flc-LOM, which is based on an attenuated live CSF vaccine virus (LOM strain), and removing the full-length classical swine fever virus (CSFV) Erns sequences and the 3' end (52 base pairs) of the CSFV capsid. These regions were substituted with the full-length bovine viral diarrhoea virus (BVDV) Erns gene sequence and the 3' end (52 base pairs) of the BVDV capsid gene. Sows were vaccinated with the Flc-LOM-BErns vaccine 3 weeks before insemination and then challenged with virulent CSFV at the early, mid- or late stages of pregnancy. We then examined transplacental transmission to the foetuses. Piglets born to sows vaccinated with Flc-LOM-BErns did not show vertical infection, regardless of challenge time. In addition, CSFV challenge did not affect the delivery date, weight or length of the foetus. Pregnant sows inoculated with the Flc-LOM-BErns vaccine were anti-CSF Erns antibody-negative and anti-BVDV Erns antibody-positive. Challenge of pregnant sows with virulent CSFV resulted in anti-CSF Erns antibody positivity. These results strongly indicate that differential diagnosis can be conducted between the Flc-LOM-BErns vaccinated animal and virulent CSFV affected animal by detecting antibody against BVDV Erns or CSF Erns gene. Therefore, the Flc-LOM-BErns vaccine may fulfil the function of differential diagnosis which required for DIVA vaccine.

Complete Genome Sequences of Classical Swine Fever Virus Subgenotype 2.1 and 2.2 Strains Isolated from Vietnamese Pigs.

Classical swine fever virus (CSFV) strains ND20 and HY78 were detected from infected pigs in the Xuan Truong-Nam Dinh and Hung Yen provinces in North Vietnam in 2014 and 2015, respectively. The most prevalent CSFV subgenotypes in Vietnam are 2.1 and 2.2, and these two complete genome sequences will help the CSFV prevention policy in Vietnam. In particular, subgenotype 2.2 (ND20 strain) has been reported less worldwide, so it is worth sharing information about this subgenotype.

Genetic characterization of bovine coronavirus in Vietnam.

A maximum clade credibility tree constructed using the full-length spike (S) and hemagglutinin-esterase genes revealed that Vietnamese Bovine coronavirus (BCoV) strains belong to a single cluster (C1); therefore, they might share a common origin with Cuban and Chinese BCoV strains. The omega values of cluster 1 (C1) and cluster 2 (C2) were 0.15734 and 0.11613, respectively, and naive empirical bayes analysis identified two amino acid positions (179 and 501) in the S protein in C1 and three amino acid positions (113, 501, and 525) in that of C2 that underwent positive selection (p > 99%). The evolutionary rate of C1 was estimated to be 7.6206 × 10-4 substitutions/site/year, and the most recent common ancestor (tMRCA) of Vietnamese BCoVs was estimated to date back to 1962 (95% HPD 1950-1973). The effective population sizes of C1 and C2 underwent a rapid reduction after 2000 and 2004, respectively.

Complete Genome Sequence of H1N1 Swine Influenza Virus from Pigs in the Republic of Korea in 2016.

We report here the genome sequence of the influenza A virus strain A/swine/Korea/61/2016, isolated from swine in the Republic of Korea. On the basis of sequence analysis, A/swine/Korea/61/2016 is marked from swine H1N1 influenza virus.

Evolutionary dynamics of classical swine fever virus in South Korea: 1987-2017.

The 5' UTR (n=102) and full-length E2 (n=37) genes of classical swine fever viruses (CSFVs) circulating in South Korea over the past 30 years (1987-2017) were examined to determine the evolutionary rate and estimated time of the most recent common ancestor (tMRCA). From 2000, the Korean classical swine fever (CSF) antigen changed from genotype 3 to 2, which comprises subgenotypes 2.1b (2002-2013) and 2.1d (2011-2017). There are genotypic variations in the full-length E2 gene of Korean CSFV genotypes 2.1b and 2.1d (seven separate amino acid substitutions); these are useful distinguishing markers. The mean substitution rate (×103 substitutions/site/year) for Korean CSFV was estimated to be 2.2088 (95% highest posterior density (HPD): lower, 1.7045; upper, 2.7574) and the mean tMRCA was estimated to be 1901 (95% HPD: lower, 1865; upper, 1933). The effective population size of Korean CSFV genotype 2 increased rapidly from 2002 to 2003, after which it remained constant. The occurrence of CSF in Korea is expected to decline in the future; however, it will likely be more prevalent in wild boar than in domestic pigs. Thus, there is a risk of transmission from wild boar to breeding pigs.

First detection and genetic characterization of porcine parvovirus 7 from Korean domestic pig farms.

Porcine parvovirus 7 (PPV7) was first detected in Korean pig farms in 2017. The detection rate of PPV7 DNA was 24.0% (30/125) in aborted pig fetuses and 74.9% (262/350) in finishing pigs, suggesting that PPV7 has circulated among Korean domestic pig farms. Phylogenetic analysis based on capsid protein amino acid sequences demonstrated that the nine isolated Korean strains (PPV-KA1-3 and PPV-KF1-6) were closely related to the previously reported USA and Chinese PPV7 strains. In addition, the Korean strains exhibit genetic diversity with both insertion and deletion mutations. This study contributes to the understanding of the molecular epidemiology of PPV7 in Korea.

Strategies to maintain Korea's animal rabies non-occurrence status.

Endemic animal rabies causes >99% of all human rabies cases; elimination of animal rabies reduces the rate of human infections. The most recent animal rabies cases in the Gangwon and Gyeonggi provinces of Korea occurred in November 2012 and February 2013, respectively. Here we explore ways to ensure that Korea remains animal rabies non-occurrence. The government must prevent rabies recurrence by vaccinating dogs, distributing bait vaccine in regions with a high rabies risk, performing laboratory-based surveillance, preventing the flow of rabies-suspect animals from neighboring countries, and enhancing border quarantine. As has already been shown in several developed countries, careful and ongoing rabies control will allow Korea to remain animal rabies-free.

Serosurvey of rabies virus, canine distemper virus, parvovirus, and influenza virus in military working dogs in Korea.

Rabies virus (RABV), canine distemper virus (CDV), canine parvovirus type-2 (CPV-2), and canine influenza A virus (CIV) are important contagious pathogens in canine populations. To assess post-vaccination immunity against RABV, CDV and CPV-2, and serological evidence of exposure to influenza A virus in military working dogs (MWDs) in Korea, we tested blood samples of 78 MWDs by fluorescent antibody virus neutralization (FAVN) for RABV, and by commercially available enzyme-linked immunosorbent assay (ELISA) for CDV, CPV-2, and CIV. Korean MWDs had high antibody-positive rates against RABV (97.4%, ≥0.5 IU/ml), CDV (94.8%), and CPV (100%). All dogs tested seronegative (0/78; 0%) for influenza A virus. Two 1-year-old dogs stationed in known rabies outbreak areas (Gangwon and Gyeonggi) exhibited VNA titers below the protective level (0.06 and 0.29 IU/ml, respectively). The breed and sex of MWDs were not significantly associated with antibody titers for RABV, CDV, or CPV; however, age was significantly associated with CPV antibody titers, while region of residence was associated with CDV antibody titer. Taken together, the data presented here provide important insights necessary for post-vaccination management and control of infectious diseases in MWDs.

Geographic distribution and molecular analysis of porcine reproductive and respiratory syndrome viruses circulating in swine farms in the Republic of Korea between 2013 and 2016.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes devastating disease characterized by reproductive failure and respiratory problems in the swine industry. To understand the recent prevalence and genetic diversity of field PRRSVs in the Republic of Korea, open reading frames (ORFs) 5 and 7 of PRRSV field isolates from 631 PRRS-affected swine farms nationwide in 2013-2016 were analyzed along with 200 Korean field viruses isolated in 2003-2010, and 113 foreign field and vaccine strains. RESULTS: Korean swine farms were widely infected with PRRSVs of a single type (38.4 and 37.4% for Type 1 and Type 2 PRRSV, respectively) or both types (24.2%) with up to approximately 83% nucleotide sequence similarity to prototype PRRSVs (Lelystad or VR2332). Phylogenetic analysis based on the ORF5 nucleotide sequence revealed that Korean Type 1 field isolates were classified as subgroups A, B, and C under subtype 1, while Korean Type 2 field isolates were classified as lineages 1 and 5 as well as three Korean lineages (kor A, B, and C) with the highest infection prevalence in subgroup A (50.5%) and lineage 5 (15.3%) for Type 1 and Type 2 PRRSV, respectively, among ORF5-positive farms. In particular, the lineages kor B and C were identified as novel lineages in this study, and lineage kor B comprised only the field viruses isolated from Gyeongnam Province in 2014-2015, establishing regionally unique genetic characteristics. It has also recently been confirmed that commercialized vaccine-like viruses (subgroup C) of Type 1 PRRSV and NADC30-like viruses of Type 2 PRRSV (lineage 1) are spreading rapidly in Korean swine farms. The Korean field viruses were also expected to be antigenically variable as shown in the high diversity of neutralizing epitopes and N-glycosylation sites. CONCLUSIONS: This up-to-date information regarding recent field PRRSVs should be taken into consideration when creating strategies for the application of PRRS control measures, including vaccination in the field.

Effects of three-dimensional spheroid culture on equine mesenchymal stem cell plasticity.

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy fields. We optimize culture conditions of equine adipose tissue-derived MSCs (eAD-MSCs) for treatment of horse fractures. To investigate enhancing properties of three-dimensional (3D) culture system in eAD-MSCs, we performed various sized spheroid formation and determined changes in gene expression levels to obtain different sized spheroid for cell therapy. eAD-MSCs were successfully isolated from horse tailhead. Using hanging drop method, spheroid formation was generated for three days. Quantitative real-time PCR was performed to analyze gene expression. As results, expression levels of pluripotent markers were increased depending on spheroid size and the production of PGE2 was increased in spheroid formation compared to that in monolayer. Ki-67 showed a remarkable increase in the spheroid formed with 2.0 × 105 cells/drop as compared to that in the monolayer. Expression levels of angiogenesis-inducing factors such as VEGF, IL-6, IL-8, and IL-18 were significantly increased in spheroid formation compared to those in the monolayer. Expression levels of bone morphogenesis-inducing factors such as Cox-2 and TGF-ß1 were also significantly increased in spheroid formation compared to those in the monolayer. Expression levels of osteocyte-specific markers such as RUNX2, osteocalcin, and differentiation potential were also significantly increased in spheroid formation compared to those in the monolayer. Therefore, spheroid formation of eAD-MSCs through the hanging drop method can increases the expression of angiogenesis-inducing and bone morphogenesis-inducing factors under optimal culture conditions.

An enhanced immunochromatographic strip test using colloidal gold nanoparticle-labeled dual-type N proteins for detection of antibodies to PRRS virus.

Porcine reproductive and respiratory syndrome (PRRS) is recognized as one of the most important infectious diseases causing serious economic loss in the swine industry worldwide. Due to its increasing genetic diversity, a rapid and accurate diagnosis is critical for PRRS control. The immunochromatographic strip test (ICST) is a rapid and convenient type of immunoassay. In this study, an on-site immunochromatographic assay-based diagnostic method was developed for detection of PRRS virus (PRRSV)-specific antibodies. The method utilized colloidal gold nanoparticle-labeled dual-type nucleocapsid proteins encoded by open reading frame 7. We evaluated 991 field samples from pig farms and 66 serum samples from experimentally PRRSV-inoculated pigs. Based on true PRRSV-specific antibody-positive or -negative sera determined by immunofluorescence assay and IgM enzyme-linked immunosorbent assay (ELISA), the specificity and sensitivity of the ICST were 97.5% and 91.1%, respectively, similar to those of a commercial ELISA (IDEXX PRRS X3 Ab). More importantly, the ICST was completed within 15 min and could detect the PRRSV-specific antibody at an earlier stage of infection (3-7 days) than that of ELISA (7+ days). The results demonstrate that the developed ICST has great potential as an on-farm diagnostic method, providing excellent diagnostic performance in a quick and convenient manner.

Efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs.

PURPOSE: The first aim of this study was to develop a novel inactivated porcine epidemic diarrhea virus (PEDV) vaccine using the recently isolated Korean PEDV QIAP1401 strain and to evaluate its protective efficacy in growing pigs. The second was to determine the optimum adjuvant formulation of the inactivated PEDV vaccine that induces protection against viral challenge. MATERIALS AND METHODS: To generate high titers of infectious PEDV, the QIAP1401 isolate was passaged in Vero cells. The experimental vaccines were prepared from a binary ethyleneimine-inactivated QIAP1401 strain passaged sequentially 70 times (QIAP1401-p70), formulated with four commercial adjuvants, and administered twice intramuscularly to growing pigs. Challenge studies using a virulent homologous strain of PEDV QIAP1401-p11, which was passaged 11 times after isolation, were performed to assess protection against disease progression and viral shedding during the 15-day observation period. The vaccine-induced antibody responses were measured in serum samples collected at predetermined time points by indirect enzyme-linked immunosorbent assay and virus neutralization test. RESULTS: The QIAP1401-p70 strain had 42 amino acid (aa) mutations, including a 25 aa deletion, and was selected as the inactivated PEDV vaccine candidate. Although none of the pigs that received the experimental vaccines were completely protected against subsequent viral challenge, they exhibited a significantly higher immune response than did non-vaccinated control pigs. Among the vaccine groups, the highest antibody responses were observed in the pigs that received an oil-based multiphasic water/oil/water (W/O/W) emulsion adjuvanted vaccine, which delayed the onset of clinical symptoms and viral shedding. CONCLUSION: A novel inactivated PEDV vaccine formulated with a W/O/W emulsion adjuvant was both immunogenic and protective against viral challenge.

Isolation and characterization of a new porcine epidemic diarrhea virus variant that occurred in Korea in 2014.

Outbreaks of porcine epidemic diarrhea (PED) have resulted in significant economic losses in the swine industry, and another PED outbreak occurred in 2014 in Korea. Isolating and culturing PED virus (PEDV) allow investigations into its pathogenesis and the development of vaccines and diagnostic assays. In this study, we successfully isolated two PEDV isolates (QIAP1401 and QIAP1402) from naturally infected piglets at Jeju-do, Korea. Viral propagation was confirmed in Vero cells based on cytopathic effect, immunofluorescence assay, reverse transcription-polymerase chain reaction, and electron microscopic analyses. The QIAP401 isolate propagated well in Vero cells for 70 passages, with titers of 106.5 to 107.0 50% tissue culture infectious dose/mL, which increased gradually with passaging. The nucleotide and amino acid sequences of the QIAP1401 isolate were determined and compared with those of other PEDV isolates. The QIAP1401 isolate was determined to be closely related to the USA/Minnesota271/2014 strain (> 99.9% nucleotide similarity) that was isolated in the USA in 2014. Phylogenetic analysis based on several PEDV genes suggested that a new PEDV variant is circulating in the Korean swine industry, with 93.08% similarity to the SM98 strain isolated in 1998. In addition, the QIAP1401 strain showed strong virulence in 3-day-old piglets and 11-week-old growing pigs.

Mass vaccination has led to the elimination of rabies since 2014 in South Korea.

PURPOSE: Rabies is one of the most fatal diseases, but it is 100% preventable in animals by vaccination. In this study, we present the epidemiological features of, and national preventive measures against, rabies in Korea. MATERIALS AND METHODS: Data related to rabies and the population density of raccoon dogs in Korea were collected from the Animal and Plant Quarantine Agency, the Korean Centers for Disease Control and Prevention, and the National Institute of Environmental Research. Rabies diagnosis was confirmed with a fluorescent antibody test using brain samples of animals in accordance with the procedures described by the World Organization for Animal Health. Serological assays for dogs and cattle were conducted using the fluorescent antibody virus neutralization test. RESULTS: From 1993 to 2016, a total of seven human rabies cases and 437 animal rabies cases in five different species were reported. An increase in the distribution of bait vaccine seemed to be related to a dramatic decrease in rabies prevalence in endemic rabies regions. Two Korean provinces and the capital city, Seoul, were involved in rabies outbreaks. Korean rabies strains are most closely related to the eastern Chinese strain belonging to the Arctic-like lineage. The yearly seropositive rates ranged from 50.4% to 81.2% in dogs and from 25% to 60.5% in cattle residing in endemic rabies regions. CONCLUSION: This study indicates that national preventive measures, including mass vaccination and distribution of bait vaccines, have contributed to a substantial decrease in the number of rabies cases in Korea.

A genetically modified rabies vaccine (ERAGS) induces protective immunity in dogs and cattle.

PURPOSE: The current live attenuated rabies vaccine must be replaced with a safer vaccine based on the ERAGS strain to prevent rabies in South Korea. We evaluated the safety and immunogenicity of a new strain in dogs and cattle. MATERIALS AND METHODS: The ERAGS strain, featuring two mutations altering two amino acids in a glycoprotein of rabies virus, was propagated in NG108-15 cells. We lyophilized the virus in the presence of two different stabilizers to evaluate the utilities of such preparations as novel rabies vaccines for animals. To explore safety and immunogenicity, dogs and cattle were inoculated with the vaccine at various doses via different routes and observed daily for 8 weeks post-inoculation (WPI). Immunogenicity was evaluated using a fluorescent antibody virus neutralization test or enzyme-linked immunosorbent assay. RESULTS: The two different stabilizers did not differ greatly in terms of maintenance of virus viability in accelerated stability testing. No clinical signs of rabies developed in dogs or cattle inoculated with the vaccines (107.0 FAID50/mL). Dogs and cattle inoculated intramuscularly with 105.0 FAID50/mL exhibited virus neutralization assay titers of 4.6 IU/mL and 1.5 to 0.87 IU/mL at 4 WPI, respectively. All control animals remained rabies virus-seronegative throughout, confirming that no contact transmission occurred between vaccinated and control animals. CONCLUSION: Our findings indicate that the new rabies vaccine is safe and immunogenic in dogs and cattle.

Traumatic esophageal perforation by a self bougienage.

The case report describes a 75-year-old female with esophageal perforation by a self bougienage. From a decade ago, she had performed an esophageal bougienage by herself with a 70 cm long self-made bougie whenever she suffered from food impaction. On the day of the accident, she went on an outing without carrying the bougie, she pushed a broken bough into her esophagus, eventually the bough perforated her thoracic esophagus. We found some food particles in a large mediastinal abscess cavity, and the perforated esophagus was repaired by interrupted sutures and reinforced with a pedicled intercostal musculopleural flap. We report an extremely rare case of esophageal perforation by a self bougienage.

Comparison of the protective efficacy between single and combination of recombinant adenoviruses expressing complete and truncated glycoprotein, and nucleoprotein of the pathogenic street rabies virus in mice.

BACKGROUND: Rabies is an important viral zoonosis that causes acute encephalitis and death in mammals. To date, several recombinant vaccines have been developed based on G protein, which is considered to be the main antigen, and these vaccines are used for rabies control in many countries. Most recombinant viruses expressing RABV G protein retain the G gene from attenuated RABV. Not enough is currently known about the protective effect against RABV of a combination of recombinant adenoviruses expressing the G and N proteins of pathogenic street RABV. METHODS: We constructed a recombinant adenovirus (Ad-0910Gsped) expressing the signal peptide and ectodomain (sped) of G protein of the Korean street strain, and evaluated the immunological protection conferred by a single and combination of three kinds of recombinant adenoviruses (Ad-0910Gsped and Ad-0910G with or without Ad-0910 N) in mice. RESULTS: A combination of Ad-0910G and Ad-0910 N conferred improved immunity against intracranial challenge compared to single administration of Ad-0910G. The Ad-0910G virus, expressing the complete G protein, was more immunogenic than Ad-0910Gsped, which expressed a truncated G protein with the transmembrane and cytoplasmic domains removed. Additionally, oral vaccination using a combination of viruses led to complete protection. CONCLUSIONS: Our results suggest that this combination of viruses is a viable new intramuscular and oral vaccine candidate.