Marker Vaccine Research Articles

Stability of closed and needle-punctured vials of Porvac® subunit vaccine against classical swine fever subjected to thermal stress.

Classical Swine Fever (CSF) is still one of the most economically important viral diseases of pigs. In endemic countries, the disease is controlled mostly through vaccination; hence, the availability of safe and effective vaccines is of utmost importance. Vaccines intended for application in developing countries must also be thermally stable, since the infrastructure needed to maintain a cold chain in those countries is usually lacking. Porvac® is a second-generation subunit marker vaccine against CSF that has demonstrates to be safe and protective. Previous studies have also shown that the vaccine is stable for 1 week at 37 oC and have a shelf life of at least 36 months at 2-8 oC. The aim of this work was to further explore the accelerated stability of Porvac® by assessing the physicochemical properties of the emulsion, and the safety and immunogenicity of the vaccine subjected to more drastic conditions of thermal stress: (1) 25 oC for 12 months; (2) 30oC and 37 oC for one month and (3) 15 days at 37°C after the cap of the vials had been needle-punctured. The vaccine subjected to all these conditions did not show significant changes in the physicochemical properties of the emulsion; did not produce local or systemic adverse reactions in pigs, and the chromatographic profile of the recovered antigen was preserved. All vaccinated swine developed neutralizing antibody titers ≥ 1:1000 at 28 days post vaccination. Porvac® is stable in all the experimental conditions tested, even after cap puncture, and retains the capacity to induce high titers of neutralizing antibodies, well above the threshold of protection. These results reinforce the robustness of the vaccine, and support its use as a very attractive alternative to modified live vaccines in developing countries endemic for CSF.

Assessment of the Safety Profile of Chimeric Marker Vaccine against Classical Swine Fever: Reversion to Virulence Study.

Chimeric marker vaccine candidates, vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, have been generated and their efficacy and capability to differentiate infected from vaccinated animals were confirmed in previous studies. The safety profile of the two chimeric marker vaccine candidates, particularly in the potential reversion to virulence, was evaluated. Each virus was administered to pigs with a dose equivalent to the vaccination dose, and pooled tonsil homogenates were subsequently inoculated into further pigs. Chimeric virus vGPE-/PAPeV Erns displayed the most substantial attenuation, achieving this within only two passages, whereas vGPE-/PhoPeV Erns was detectable until the third passage and disappeared entirely by the fourth passage. The vGPE- strain, assessed alongside, consistently exhibited stable virus recovery across each passage without any signs of increased virulence in pigs. In vitro assays revealed that the type I interferon-inducing capacity of vGPE-/PAPeV Erns was significantly higher than that of vGPE-/PhoPeV Erns and vGPE-. In conclusion, the safety profile of the two chimeric marker vaccine candidates was affirmed. Further research is essential to ensure the stability of their attenuation and safety in diverse pig populations.

Development of a live attenuated vaccine candidate for equid alphaherpesvirus 1 control: a step towards efficient protection.

Equid alphaherpesvirus 1 (EqAHV1) is a viral pathogen known to cause respiratory disease, neurologic syndromes, and abortion storms in horses. Currently, there are no vaccines that provide complete protection against EqAHV1. Marker vaccines and the differentiation of infected and vaccinated animals (DIVA) strategy are effective for preventing and controlling outbreaks but have not been used for the prevention of EqAHV1 infection. Glycoprotein 2 (gp2), located on the envelope of viruses (EqAHV1), exhibits high antigenicity and functions as a molecular marker for DIVA. In this study, a series of EqAHV1 mutants with deletion of gp2 along with other virulence genes (TK, UL24/TK, gI/gE) were engineered. The mutant viruses were studied in vitro and then in an in vivo experiment using Golden Syrian hamsters to assess the extent of viral attenuation and the immune response elicited by the mutant viruses in comparison to the wild-type (WT) virus. Compared with the WT strain, the YM2019 Δgp2, ΔTK/gp2, and ΔUL24/TK/gp2 strains exhibited reduced growth in RK-13 cells, while the ΔgI/gE/gp2 strain exhibited significantly impaired proliferation. The YM2019 Δgp2 strain induced clinical signs and mortality in hamsters. In contrast, the YM2019 ΔTK/gp2 and ΔUL24/TK/gp2 variants displayed diminished pathogenicity, causing no observable clinical signs or fatalities. Immunization with nasal vaccines containing YM2019 ΔTK/gp2 and ΔUL24/TK/gp2 elicited a robust immune response in hamsters. In particular, compared with the vaccine containing the ΔTK/gp2 strain, the vaccine containing the ΔUL24/TK/gp2 strain demonstrated enhanced immune protection upon challenge with the WT virus. Furthermore, an ELISA for gp2 was established and refined to accurately differentiate between infected and vaccinated animals. These results confirm that the ΔUL24/TK/gp2 strain is a safe and effective live attenuated vaccine candidate for controlling EqAHV1 infection.

Safety and DIVA Capability of Novel Live Attenuated Classical Swine Fever Marker Vaccine Candidates in Pregnant Sows.

Classical Swine Fever (CSF), a highly contagious viral disease affecting pigs and wild boar, results in significant economic losses in the swine industry. In endemic regions, prophylactic vaccination and stamping-out strategies are used to control CSF outbreaks. However, sporadic outbreaks and persistent infections continue to be reported. Although the conventional attenuated CSF vaccines protect pigs against the disease, they do not allow for the differentiation of infected from vaccinated animals (DIVA), limiting their use as an eradication tool. In this study, three targeted attenuation strategies were employed to generate vaccine candidates based on the current prevalent CSFV group 2 strains GD18 and QZ07: a single deletion of H79 in Erns (QZ07-sdErnsH-KARD), double deletion of H79 and C171 in Erns (GD18-ddErnsHC-KARD and QZ07-ddErnsHC-KARD), and deletion of H79 in Erns combined with a 5-168 amino acids deletion of Npro (GD18-ddNpro-ErnsH-KARD). Additionally, a negative serological marker with four substitutions in a highly conserved epitope in E2 recognized by the monoclonal antibody 6B8 was introduced in each candidate for DIVA purposes. The safety of these four resulting vaccine candidates was evaluated in pregnant sows. Two candidates, GD18-ddErnsHC-KARD and QZ07-sdErnsH-KARD were found to be safe for pregnant sows and unlikely to cause vertical transmission. Both candidates also demonstrated potential to be used as DIVA vaccines, as was shown using a proprietary blocking ELISA based on the 6B8 monoclonal antibody. These results, together with our previous work, constitute a proof-of-concept for the rational design of CSF antigenically marked modified live virus vaccine candidates.

Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.

Identification of a linear B-cell epitope on the "puff" loop of the Senecavirus A VP2 protein involved in receptor binding.

Senecavirus A (SVA) is an important emerging swine pathogen that causes vesicular lesions in swine and acute death in newborn piglets. VP2 plays a significant role in the production of antibodies, which can be used in development of diagnostic tools and vaccines. Herein, the aim of the current study was to identify B-cell epitopes (BCEs) of SVA for generation of epitope-based SVA marker vaccine. Three monoclonal antibodies (mAbs), named 2E4, 1B8, and 2C7, against the SVA VP2 protein were obtained, and two novel linear BCEs, 177SLGTYYR183 and 266SPYFNGL272, were identified by peptide scanning. The epitope 177SLGTYYR183 was recognized by the mAb 1B8 and was fully exposed on the VP2 surface, and alanine scanning analysis revealed that it contained a high continuity of key amino acids. Importantly, we confirmed that 177SLGTYYR183 locates on "the puff" region within the VP2 EF loop, and contains three key amino acid residues involved in receptor binding. Moreover, a single mutation, Y182A, blocked the interaction of the mutant virus with the mAb 1B8, indicating that this mutation is the pivotal point for antibody recognition. In summary, the BCEs that identified in this study could be used to develop diagnostic tools and an epitope-based SVA marker vaccine.

Development of a dual immunochromatographic test strip to detect E2 and Erns antibodies against classical swine fever.

It is essential to consider a practical antibody test to successfully implement marker vaccines and validate vaccination efficacy against classical swine fever virus (CSFV). The test should include a serological antibody assay, combined with a tool for differentiating infected from vaccinated animals (DIVA). The immunochromatographic test strip (ICS) has been exclusively designed for detecting CSFV E2 antibodies while lacking in detecting Erns antibodies, which can be employed and satisfy DIVA strategy. This study developed a novel ICS for detecting CSFV E2/Erns dual-antibody. The effectiveness of ICS in evaluating the DIVA capability of two novel chimeric pestivirus vaccine candidates was assessed. Recombinant E2 or Erns protein was transiently expressed in the plant benthamiana using Agrobacterium tumefaciens. ICS was subsequently assembled, and goat anti-rabbit IgG and recombinant CSFV E2 or Erns protein were plated onto the nitrocellulose membrane as control and test lines, respectively. The sensitivity and specificity of ICS were evaluated using sera with different neutralizing antibody titers or positive for antibodies against CSFV and other pestiviruses. The coincidence rates for detecting E2 and Erns antibodies between ICS and commercial enzyme-linked immunosorbent assay (ELISA) kits were also computed. ICS performance for DIVA capability was evaluated using sera from pigs vaccinated with conventional vaccine or chimeric vaccine candidates. E2 and Erns proteins were successfully expressed in N. benthamiana-produced recombinant proteins. ICS demonstrated high sensitivity in identifying CSFV E2 and Erns antibodies, even at the low neutralizing antibody titers. No cross-reactivity with antibodies from other pestiviruses was confirmed using ICS. There were high agreement rates of 93.0 and 96.5% between ICS and two commercial ELISA kits for E2 antibody testing. ICS also achieved strong coincidence rates of 92.9 and 89.3% with two ELISA kits for Erns antibody detection. ICS confirmed the absence of CSFV Erns-specific antibodies in sera from pigs vaccinated with chimeric vaccine candidates. E2 and Erns proteins derived from the plant showed great potential and can be used to engineer a CSFV E2/Erns dual-antibody ICS. The ICS was also highly sensitive and specific for detecting CSFV E2 and Erns antibodies. Significantly, ICS can fulfill the DIVA concept by incorporating chimeric vaccine candidates.

Investigation of the safety and protective efficacy of an attenuated and marker M. bovis-BoHV-1 combined vaccine in bovines.

Bovine respiratory disease (BRD) is one of the most common diseases in the cattle industry worldwide; it is caused by multiple bacterial or viral coinfections, of which Mycoplasma bovis (M. bovis) and bovine herpesvirus type 1 (BoHV-1) are the most notable pathogens. Although live vaccines have demonstrated better efficacy against BRD induced by both pathogens, there are no combined live and marker vaccines. Therefore, we developed an attenuated and marker M. bovis-BoHV-1 combined vaccine based on the M. bovis HB150 and BoHV-1 gG-/tk- strain previously constructed in our lab and evaluated in rabbits. This study aimed to further evaluate its safety and protective efficacy in cattle using different antigen ratios. After immunization, all vaccinated cattle had a normal rectal temperature and mental status without respiratory symptoms. CD4+, CD8+, and CD19+ cells significantly increased in immunized cattle and induced higher humoral and cellular immune responses, and the expression of key cytokines such as IL-4, IL-12, TNF-α, and IFN-γ can be promoted after vaccination. The 1.0× 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- combined strain elicited the most antibodies while significantly increasing IgG and cellular immunity after challenge. In conclusion, the M. bovis HB150 and BoHV-1 gG-/tk- combined strain was clinically safe and protective in calves; the mix of 1.0 × 108 CFU of M. bovis HB150 and 1.0 × 106 TCID50 BoHV-1 gG-/tk- strain was most promising due to its low amount of shedding and highest humoral and cellular immune responses compared with others. This study introduces an M. bovis-BoHV-1 combined vaccine for application in the cattle industry.

Assessment of BoAHV-1 Seronegative Latent Carrier by the Administration of Two Infectious Bovine Rhinotracheitis Live Marker Vaccines in Calves.

Seronegative latent carriers (SNLCs) are animals that carry the virus without detectable antibodies and pose a risk for disease transmission and diagnostic challenges, suggesting the importance of consideration of marker vaccines in managing them. Therefore, in this study, we evaluated two modified live infectious bovine rhinotracheitis (IBR) marker vaccines (single and double deletions) for their ability to generate SNLC calves. These vaccines were administered to four groups (n = 3 in each group) of three-month-old calves in the presence or absence of passive immunity. Three hundred days after the first vaccination and after confirming the IBR seronegativity of all animals, dexamethasone was administered intravenously for five consecutive days. Only animals immunized with the modified live IBR marker vaccine (single deletion) in the absence of passive immunity exhibited a more enduring immune response than those vaccinated in the presence of passive immunity. Moreover, the administration of a modified live IBR marker vaccine (double deletion) to calves with passive immunity generated SNLC. These findings underscore the potential of live IBR marker vaccine (double-deletions) to aid serological diagnostic tools and develop vaccination protocols in achieving the desired immune response, particularly in the context of latent carrier status, offering valuable insights into optimizing vaccination strategies for effective IBR control.

More than a decade of research on Schmallenberg virus-Knowns and unknowns.

Schmallenberg virus, an arbovirus of the Orthobunyavirus genus that primarily infects ruminants, emerged in 2011 near the Dutch-German border region and subsequently caused a large number of abortions and the births of severely malformed newborns in the European livestock population. Immediate intensive research led to the development of reliable diagnostic tests, the identification of competent Culicoides vector species, and the elucidation of the pathogenesis in infected vertebrate hosts. In addition, the structure of the major antigenic domain has been elucidated in great detail, leading to the development of effective marker vaccine candidates. The knowledge gained over the last decade on the biology and pathogenesis of SBV and the experience acquired in its control will be of great value in the future for the control of any similar emerging pathogen of veterinary or public health importance such as Shuni or Oropouche virus. However, some important knowledge gaps remain, for example, the factors contributing to the highly variable transmission rate from dam to fetus or the viral factors responsible for the vector competence of Culicoides midges are largely unknown. Thus, questions still remain for the next decade of research on SBV and related viruses.

Progress in vaccines against peste des petits ruminants virus

Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of both domestic (goats and sheep) and wild small ruminants. Due to the devastating effect of this disease on livestock and livelihoods, the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (WOAH) endorsed the Global Strategy for the Control and Eradication of PPR (PPR GCES) and launched the PPR Global Eradication Programme (PPR GEP) to eradicate PPRV by 2030. In order to achieve this goal, a potent, safe and efficacious live-attenuated PPR vaccine with long-lasting immunity is available for immunoprophylaxis. However, the live-attenuated PPR vaccines are thermolabile and require maintenance of an effective cold chain to deliver to the field. In addition, infected animals cannot be differentiated from vaccinated ones (DIVA). To overcome these limitations, some new generation PPR vaccines have been developed: poxvirus vaccine, positive and negative marker vaccine through reverse genetic approach, chimeric vaccine, anti-idiotypic vaccine, subunit vaccine, virus-like particles vaccine, edible vaccine and combined vaccines. Novel recombinant PPR DIVA vaccines were evaluated in goats for safety and efficacy, and all vaccinated animals were clinically protected against an intranasal PPRV challenge. Furthermore, newly developed ELISAs were capable of differentiating between infected and vaccinated animals. Therefore, these DIVA vaccines and the associated tests can facilitate the serological monitoring process and speed up global PPR eradication through vaccination.

Performance of a Differentiation of Infected from Vaccinated Animals (DIVA) Classical Swine Fever Virus (CSFV) Serum and Oral Fluid Erns Antibody AlphaLISA Assay.

Classical swine fever virus (CSFV) is an OIE-listed disease that requires effective surveillance tools for its detection and control. The aim of this study was to develop and evaluate the diagnostic performance of a novel CSFV Erns IgG AlphaLISA for both serum and oral fluid specimens that would likewise be compatible with the use of CSFV E2 DIVA vaccines. Test performance was evaluated using a panel of well-characterized serum (n = 760) and individual (n = 528) or pen-based (n = 30) oral fluid samples from four groups of animals: (1) negative controls (n = 60 pigs); (2) inoculated with ALD strain wild-type CSFV (n = 30 pigs); (3) vaccinated with LOM strain live CSFV vaccine (n = 30 pigs); and (4) vaccinated with live CSFV marker vaccine on commercial farms (n = 120 pigs). At a cutoff of S/P ≥ 0.7, the aggregate estimated diagnostic sensitivities and specificities of the assay were, respectively, 97.4% (95% CI 95.9%, 98.3%) and 100% for serum and 95.4% (95% CI 92.9%, 97.0%) and 100% for oral fluid. The Erns IgG antibody AlphaLISA combined DIVA capability with solid diagnostic performance, rapid turnaround, ease of use, and compatibility with both serum and oral fluid specimens.

Efficacy of an orally administered classical swine fever live marker vaccine (Flc-LOM-BErns strain) in pigs

In several countries, classical swine fever (CSF) has not been detected in domestic pigs, but has been detected in wild boars, making the disease difficult to control. To overcome this problem, we inoculated pigs with a CSF live marker vaccine (Flc-LOM-BErns strain), which has “distinguish infection from vaccinated animals (DIVA)” function, to determine whether it is suitable as an oral vaccine specifically for wild boars. Pigs inoculated intramuscularly or orally with the Flc-LOM-BErns vaccine were challenged 2 or 4 weeks later, respectively, with virulent CSFV. Pigs administered the oral Flc-LOM-BErns strain (105.0 and 6.0 TCID50/dose), and those vaccinated intramuscularly (103.0 TCID50/dose), had normal numbers of leukocytes and normal body temperature. Also, they generated protective neutralizing antibodies and anti-BVDV Erns antibodies. In addition, all pigs in these groups survived, with no CSFV RNA detected in feces, spleen, or other organs. Thus, the Flc-LOM-BErns vaccine shows excellent safety and efficacy, while having DIVA function and suitability for oral inoculation.

Safety and Immunogenicity of Chimeric Pestivirus KD26_E2LOM in Piglets and Calves.

A chimeric pestivirus (KD26_E2LOM) was prepared by inserting the E2 gene of the classical swine fever virus (CSFV) LOM strain into the backbone of the bovine viral diarrhea virus (BVDV) KD26 strain. KD26_E2LOM was obtained by transfecting the cDNA pACKD26_E2LOM into PK-15 cells. KD26_E2LOM chimeric pestivirus proliferated to titers of 106.5 TCID50/mL and 108.0 TCID50/mL at 96 h post-inoculation into PK-15 cells or MDBK cells, respectively. It also reacted with antibodies specific for CSFV E2 and BVDV Erns, but not with an anti-BVDV E2 antibody. Piglets (55-60 days old) inoculated with a high dose (107.0 TCID50/mL) of KD26_E2LOM produced high levels of CSFV E2 antibodies. In addition, no co-habiting pigs were infected with KD26_E2LOM; however, some inoculated pigs excreted the virus, and the virus was detected in some organs. When pregnant sows were inoculated during the first trimester (55-60 days) with a high dose (107.0 TCID50/mL) of KD26_E2LOM, anti-CSFV E2 antibodies were produced at high levels; chimeric pestivirus was detected in one fetus and in the ileum of one sow. When 5-day-old calves that did not consume colostrum received a high dose (107.0 TCID50/mL) of KD26_E2LOM, one calf secreted the virus in both feces and nasal fluid on Day 2. A high dose of KD26_E2LOM does not induce specific clinical signs in most animals, does not spread from animal to animal, and generates CSFV E2 antibodies with DVIA functions. Therefore, chimeric pestivirus KD26_E2LOM is a potential CSFV live marker vaccine.

Evaluation of Hematological Profiles and Monocyte Subpopulations in Water Buffalo Calves after Immunization with Two Different IBR Marker Vaccines and Subsequent Infection with Bubaline alphaherpesvirus-1.

Bubaline alphaherpesvirus-1 (BuAHV-1) and Bovine alphaherpesvirus-1 (BoAHV-1) are respiratory viruses that can cause an infection known as "Infectious Bovine Rhinotracheitis" (IBR) in both water buffalo and bovine species. As the main disease control strategy, vaccination can protect animals from clinical disease through the development of specific humoral and cell-mediated immune responses. In the present study, the time-related circulatory kinetics of hematological profile and bubaline monocyte subsets have been investigated in vaccinated buffalo calves after challenge infections with BuAHV-1. Thirteen buffalo calves were selected and grouped into the VAX-1 group, which received an IBR-live-attenuated gE-/tk-deleted marker vaccine; the VAX-2 group, which received an IBR-inactivated gE-deleted marker vaccine; the CNT group, which remained an unvaccinated control. Fifty-five days after the first vaccination, the animals were infected with 5 × 105.00 TCID50/mL of wild-type BuAHV-1 strain via the intranasal route. Whole blood samples were collected at 0, 2, 4, 7, 10, 15, 30, and 63 days post-challenge (PCDs) for the analysis of hematological profiles and the enumeration of monocyte subsets via flow cytometry. The analysis of leukocyte compositions revealed that neutrophils were the main leukocyte population, with a relative increase during the acute infection. On the other hand, a general decrease in the proportion of lymphocytes was observed early in the post-infection, both for the VAX-1 and VAX-2 groups, while in the CNT group, the decrease was observed later at +30 and +63 PCDs. An overall infection-induced increase in blood total monocytes was observed in all groups. The rise was especially marked in the animals vaccinated with an IBR-live-attenuated gE-/tK-deleted marker vaccine (VAX-1 group). A multicolor flow cytometry panel was used to identify the bubaline monocyte subpopulations (classical = cM; intermediate = intM; and non-classical = ncM) and to investigate their variations during BuAHV-1 infection. Our results showed an early increase in cMs followed by a second wave of intMs. This increase was observed mainly after stimulation with live-attenuated viruses in the VAX-1 group compared with the animals vaccinated with the inactivated vaccine or the non-vaccinated animal group. In summary, the present study characterized, for the first time, the hematological profile and distribution of blood monocyte subsets in vaccinated and non-vaccinated water buffalo in response to experimental infection with BuAHV-1. Although not experimentally proven, our results support the hypothesis of a linear developmental relationship between monocyte subsets.

Generation and Efficacy of Two Chimeric Viruses Derived from GPE- Vaccine Strain as Classical Swine Fever Vaccine Candidates.

A previous study proved that vGPE- mainly maintains the properties of classical swine fever (CSF) virus, which is comparable to the GPE- vaccine seed and is a potentially valuable backbone for developing a CSF marker vaccine. Chimeric viruses were constructed based on an infectious cDNA clone derived from the live attenuated GPE- vaccine strain as novel CSF vaccine candidates that potentially meet the concept of differentiating infected from vaccinated animals (DIVA) by substituting the glycoprotein Erns of the GPE- vaccine strain with the corresponding region of non-CSF pestiviruses, either pronghorn antelope pestivirus (PAPeV) or Phocoena pestivirus (PhoPeV). High viral growth and genetic stability after serial passages of the chimeric viruses, namely vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns, were confirmed in vitro. In vivo investigation revealed that two chimeric viruses had comparable immunogenicity and safety profiles to the vGPE- vaccine strain. Vaccination at a dose of 104.0 TCID50 with either vGPE-/PAPeV Erns or vGPE-/PhoPeV Erns conferred complete protection for pigs against the CSF virus challenge in the early stage of immunization. In conclusion, the characteristics of vGPE-/PAPeV Erns and vGPE-/PhoPeV Erns affirmed their properties, as the vGPE- vaccine strain, positioning them as ideal candidates for future development of a CSF marker vaccine.

A Recombinant Duck Plague Virus Containing the ICP27 Deletion Marker Provides Robust Protection in Ducks.

Duck plague virus (DPV) is a member of Alphaherpesvirus genus and poses a major threat to waterfowl breeding. Genetic engineered vaccines that are capable of distinguishing naturally infected from vaccine-immunized animals are useful for eradicating duck plague. In this study, reverse genetics was used to develop an ICP27-deficient strain (CHv-ΔICP27), and its potential as a marker vaccination candidate was evaluated. The results showed that the CHv-ΔICP27 generated in this study exhibited good genetic stability in vitro and was highly attenuated both in vivo and in vitro. The level of neutralizing antibody generated by CHv-ΔICP27 was comparable to that induced by a commercial DPV vaccine, suggesting that it could protect ducks from virulent DPV attack. By using molecular identification techniques such as PCR, restriction fragment length polymorphism, immunofluorescence, Western blotting, and others, it is possible to differentiate the CHv-ΔICP27 from wild-type strains. Moreover, ICP27 can also be a potential target for the genetic engineering vaccine development of alphavirus or perhaps the entire herpesvirus family members due to the highly conservative of ICP27 protein in all herpesvirus family members. IMPORTANCE The development of distinguishable marker vaccines from natural infection is a key step toward eradicating duck plague. Here, we generated a recombinant DPV that carries an ICP27 deletion marker that could be easily distinguished from wild-type strain by molecular biological methods. It was highly attenuated in vitro and in vivo and could provide comparable protection to ducks after a single dose of immunizations, as commercial vaccines did. Our findings support the use of the ICP27-deficient virus as a marker vaccine for DPV control and future eradication.

Evaluation of an Immunization Protocol Using Bovine Alphaherpesvirus 1 gE-Deleted Marker Vaccines against Bubaline Alphaherpesvirus 1 in Water Buffaloes.

European regulations on the control of infectious diseases provide measures to control Bovine alphaherpesvirus 1 (BoHV-1) infection in both cattle and buffalo. Owing to the reported serological cross-reactivity between BoHV-1 and Bubaline alphaherpesvirus 1 (BuHV-1), we hypothesized a new immunization protocol using BoHV-1 gE-deleted marker vaccines could protect water buffalo against BuHV-1. Five water buffaloes devoid of BoHV-1/BuHV-1-neutralizing antibodies were immunized with two commercial BoHV-1 gE-deleted marker vaccines at 0, 30, 210, and 240 post-vaccination days (PVDs). Five additional water buffaloes were used as controls. At 270 PVD (0 post-challenge days (PCDs), all animals were challenged intranasally with wild-type (wt) BuHV-1. The vaccinated animals produced humoral immunity (HI) as early as PVD 30 whereas, in control animals, antibodies were detected on PCD 10. After challenge infection, HI significantly increased in vaccinated animals compared to that in controls. Real-time PCR for gB revealed viral shedding in vaccinated animals from PCDs 2 to 10. In contrast, positive results were observed from PCDs 2 to 15 in the unvaccinated control group. Although the findings indicated the possible protection capabilities of the tested protocol, these findings did not support its protective roles in water buffaloes against wt-BuHV-1.

Construction and efficacy of a new live chimeric C-strain vaccine with DIVA characteristics against classical swine fever.

To develop the new classical swine fever (CSF) vaccine candidate with differentiating infected vaccinated animals (DIVA) characteristics, a chimeric CSF virus (CSFV) was constructed based on an infectious cDNA clone of the CSF vaccine C-strain. The 5'- and 3'-untranslated regions (UTRs) and partial E2 region (residues 690-860) of the C-strain were substituted with the corresponding regions of bovine viral diarrhoea virus (BVDV) to construct the chimeric cDNA clone pC/bUTRs-tE2. The chimeric virus rC/bUTRs-tE2 was generated by several passages of pC/bUTRs-tE2-transfected PK15 cells. Stable growth and genetic properties of rC/bUTRs-tE2 were obtained after 30 serial passages. Compared to parental rC/bUTRs-tE2 (1st passage), two residue mutations (M834K and M979K) located in E2 in rC/bUTRs-tE2 P30 were observed. Compared to the C-strain, rC/bUTRs-tE2 exhibited unchanged cell tropism and decreased plaque-forming ability. Substituting the C-strain UTRs with the BVDV UTRs resulted in significantly increased viral replication in PK15 cells. Compared to CSFV Erns-positive and BVDV tE2-negative antibody responses induced by the CSF vaccine C-strain, immunization of rabbits and piglets with rC/bUTRs-tE2 resulted in serological profiles of CSFV Erns- and BVDV tE2-positive antibodies, which are used to serologically discriminate pigs that are clinically infected and vaccinated. Vaccination of piglets with rC/bUTRs-tE2 conferred complete protection against lethal CSFV challenge. Our results suggest that rC/bUTRs-tE2 is a promising new CSF marker vaccine candidate.

Construction and Rescue of a Minigenome System for Lentogenic D58 Strain of Avian orthoavulavirus 1 (Newcastle Disease Virus)

Background: In reverse genetics studies, minigenomes of negative sense RNA viruses are used to model the process of virus replication and evaluate the virus rescue system in the course of construction of recombinant virus. In the current study, a minigenome (MG) rescue system for lentogenic D58 vaccine strain of Avian orthoavulavirus 1 (AOAV-1) (Newcastle disease virus) was developed to establish a reverse genetic system for this strain. Methods: The minigenome was constructed by assembling the trailer and leader regions of AOAV-1 D58 strain flanking the reporter gene, Aequorea coerulescens green fluorescence protein (AcGFP) in a modified transcription vector. Construction of this Minigenome was done using a simple restriction free technique-Splicing by Overlap Extension PCR. The constructed minigenome was evaluated for the reporter gene expression by transfection in T7 RNA polymerase expressing BSR/T7 cell line. Further, the replication and packaging ability of the constructed minigenome were also analyzed. Result: The reporter gene expression was confirmed in BSR/T7 cells which simulates the virus replication process and verifies the virus rescue system. In addition, this proves the applicability of restriction free cloning by Splicing by Overlap Extension (SOE) PCR for precise construction of the minigenome. The developed minigenome system can be used as a potential tool in the reverse genetic rescue of AOAV-1 D58 strain for the development of marker vaccine.