A glycoprotein E gene-deleted bovine herpesvirus 1 as a candidate vaccine strain

A bovine herpesvirus 1 (BoHV-1) defective in glycoprotein E (gE) was constructed from a Brazilian genital BoHV-1 isolate, by replacing the full gE coding region with the green fluorescent protein (GFP) gene for selection. Upon co-transfection of MDBK cells with genomic viral DNA plus the GFP-bearing gE-deletion plasmid, three fluorescent recombinant clones were obtained out of approximately 5000 viral plaques. Deletion of the gE gene and the presence of the GFP marker in the genome of recombinant viruses were confirmed by PCR. Despite forming smaller plaques, the BoHV-1△gE recombinants replicated in MDBK cells with similar kinetics and to similar titers to that of the parental virus (SV56/90), demonstrating that the gE deletion had no deleterious effects on replication efficacy in vitro. Thirteen calves inoculated intramuscularly with BoHV-1△gE developed virus neutralizing antibodies at day 42 post-infection (titers from 2 to 16), demonstrating the ability of the recombinant to replicate and to induce a serological response in vivo. Furthermore, the serological response induced by recombinant BoHV-1△gE could be differentiated from that induced by wild-type BoHV-1 by the use of an anti-gE antibody ELISA kit. Taken together, these results indicated the potential application of recombinant BoHV-1 △gE in vaccine formulations to prevent the losses caused by BoHV-1 infections while allowing for differentiation of vaccinated from naturally infected animals.

A glycoprotein E gene-deleted bovine herpesvirus 1 as a candidate vaccine strain.

A bovine herpesvirus 1 (BoHV-1) defective in glycoprotein E (gE) was constructed from a Brazilian genital BoHV-1 isolate, by replacing the full gE coding region with the green fluorescent protein (GFP) gene for selection. Upon co-transfection of MDBK cells with genomic viral DNA plus the GFP-bearing gE-deletion plasmid, three fluorescent recombinant clones were obtained out of approximately 5000 viral plaques. Deletion of the gE gene and the presence of the GFP marker in the genome of recombinant viruses were confirmed by PCR. Despite forming smaller plaques, the BoHV-1△gE recombinants replicated in MDBK cells with similar kinetics and to similar titers to that of the parental virus (SV56/90), demonstrating that the gE deletion had no deleterious effects on replication efficacy in vitro. Thirteen calves inoculated intramuscularly with BoHV-1△gE developed virus neutralizing antibodies at day 42 post-infection (titers from 2 to 16), demonstrating the ability of the recombinant to replicate and to induce a serological response in vivo. Furthermore, the serological response induced by recombinant BoHV-1△gE could be differentiated from that induced by wild-type BoHV-1 by the use of an anti-gE antibody ELISA kit. Taken together, these results indicated the potential application of recombinant BoHV-1 △gE in vaccine formulations to prevent the losses caused by BoHV-1 infections while allowing for differentiation of vaccinated from naturally infected animals.

Effects of inactivated parapoxvirus ovis on cellular and humoral events of the innate immune response in mice.

The immunostimulating properties of inactivated parapoxvirus ovis (iPPVO) have long been demonstrated in vivo and in vitro, yet the biological and molecular mechanisms involved remain largely unknown. We herein report that intraperitoneal inoculation of iPPVO in mice results in stimulation of several events of the innate immune response. Increased interferon I (IFN-I) activity was demonstrated in sera of mice treated with iPPVO at 6 and 12 h post-inoculation (hpi), and enhanced expression of IFN-γ (15-fold increase) and IL-12 (6-fold) mRNA was detected in the spleen of treated mice at 24 and 48 hpi, respectively. A significant increase in neutrophil activity (p<0.01) was observed at 6 hpi in the blood of iPPVO treated mice. In addition, increased phagocytic activity by peritoneal macrophages of iPPVO-treated mice (p<0.01) was detected in vivo (from 24 to 72 hpi) and in vitro (12 to 96 hpi). Bactericidal activity of sera mice treated with iPPVO against Escherichia coli was also increased (p<0.05) at 24 and 72 hpi. Taken together, these results demonstrate that iPPVO administration leads to a transient stimulation of selected innate immune mechanisms, likely contributing to the immunostimulant effects observed against viral and bacterial infections in vivo.

Inactivated Parapoxvirus ovis induces a transient increase in the expression of proinflammatory, Th1-related, and autoregulatory cytokines in mice.

The immunostimulatory properties of inactivated Parapoxvirus ovis (iPPVO) have long been investigated in different animal species and experimental settings. In this study, we investigated the effects of iPPVO on cytokine expression in mice after intraperitoneal inoculation. Spleen and sera collected from iPPVO-treated mice at intervals after inoculation were submitted to cytokine mRNA determination by real-time PCR (qPCR), serum protein concentration by ELISA, and interferon (IFN)-α/ß activity by bioassay. The spleen of iPPVO-treated animals showed a significant increase in mRNA expression of all cytokines assayed, with different kinetics and magnitude. Proinflammatory cytokines interleukin (IL)-1ß, tumor necrosis factor-alpha (TNF-α), and IL-8 mRNA peaked at 24 hours postinoculation (hpi; 5.4-fold increase) and 48 hpi (3- and 10-fold increases), respectively. A 15-fold increase in IFN-γ and 6-fold IL-12 mRNA increase were detected at 48 and 24 hpi, respectively. Increased expression of autoregulatory cytokines (Th2), mainly IL-10 and IL-4, could be detected at later times (72 and 96 hpi) with peaks of 4.7- and 4.9-fold increases, respectively. IFN-I antiviral activity against encephalomyocarditis virus was demonstrated in sera of treated animals between 6 and 12 hpi, with a >90% reduction in the number of plaques. Measurement of serum proteins by ELISA revealed increased levels of IL-1, TNF-α, IL-12, IFN-γ, and IL-10, with kinetics similar to those observed by qPCR, especially for IL-12 and IFN-γ. These data demonstrate that iPPVO induced a transient and complex cytokine response, initially represented by Th1-related cytokines followed by autoregulatory and Th2 cytokines.

Inactivated Parapoxvirus ovis induces a transient increase in the expression of proinflammatory, Th1-related, and autoregulatory cytokines in mice

The immunostimulatory properties of inactivated Parapoxvirus ovis (iPPVO) have long been investigated in different animal species and experimental settings. In this study, we investigated the effects of iPPVO on cytokine expression in mice after intraperitoneal inoculation. Spleen and sera collected from iPPVO-treated mice at intervals after inoculation were submitted to cytokine mRNA determination by real-time PCR (qPCR), serum protein concentration by ELISA, and interferon (IFN)-α/β activity by bioassay. The spleen of iPPVO-treated animals showed a significant increase in mRNA expression of all cytokines assayed, with different kinetics and magnitude. Proinflammatory cytokines interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), and IL-8 mRNA peaked at 24 hours postinoculation (hpi; 5.4-fold increase) and 48 hpi (3- and 10-fold increases), respectively. A 15-fold increase in IFN-γ and 6-fold IL-12 mRNA increase were detected at 48 and 24 hpi, respectively. Increased expression of autoregulatory cytokines (Th2), mainly IL-10 and IL-4, could be detected at later times (72 and 96 hpi) with peaks of 4.7- and 4.9-fold increases, respectively. IFN-I antiviral activity against encephalomyocarditis virus was demonstrated in sera of treated animals between 6 and 12 hpi, with a >90% reduction in the number of plaques. Measurement of serum proteins by ELISA revealed increased levels of IL-1, TNF-α, IL-12, IFN-γ, and IL-10, with kinetics similar to those observed by qPCR, especially for IL-12 and IFN-γ. These data demonstrate that iPPVO induced a transient and complex cytokine response, initially represented by Th1-related cytokines followed by autoregulatory and Th2 cytokines.

A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is immunogenic for calves and confers protection upon homologous challenge and BoHV-1 challenge.

A recombinant bovine herpesvirus 5 lacking thymidine kinase and glycoprotein E genes (BoHV-5gEΔTKΔ) was evaluated as a live experimental vaccine. In a first experiment, ten-months-old calves were vaccinated intramuscularly (n=9) or remained as controls (n=8) and 42 days later were challenged with BoHV-5 or BoHV-1 intranasally. The four control calves challenged with BoHV-5 developed severe depression and neurological signs and were euthanized in extremis at days 13 and 14 pos-infection (pi); the five vaccinated animals challenged with BoHV-5 remained healthy. The titers of virus shedding were reduced (p<0.01) from days 3 to 7 post-infection (pi) in vaccinated animals. Control and vaccinated calves challenged with BoHV-1 presented mild transient respiratory signs; yet the magnitude of virus shedding was reduced (p<0.05) in vaccinated animals (days 5, 9 and 11pi). In a second experiment, young calves (100-120 days-old) were vaccinated (n=15) or kept as controls (n=5) and subsequently challenged with a BoHV-1 isolate. Control calves developed moderate to severe rhinitis and respiratory distress; two were euthanized in extremis at days 5 and 9 pi, respectively. In contrast, vaccinated animals were protected from challenge and only a few developed mild and transient nasal signs. The duration and titers of virus shedding after challenge were reduced (p<0.05) in vaccinated animals comparing to controls. In both experiments, vaccinated animals developed antibodies to gE only after challenge. These results demonstrate homologous and heterologous protection and are promising towards the use of the recombinant BoHV-5gEΔTKΔ in vaccine formulations to control BoHV-5 and BoHV-1 infections.

Experimental infection of calves with recombinants of bovine herpesvirus 5 defective in glycoprotein E (gE), thymidine kinase (TK) and both, gE/TK / Infecção experimental de bezerros com recombinantes do herpesvírus bovino tipo 5 defectivos na glicoproteína E (gE), timidina quinase (TK) e ambos, gE/TK

This article describes an investigation on the virulence/attenuation of bovine herpesvirus type 5 (BoHV-5) recombinants deleted in the genes encoding glycoprotein E (BoHV-5gEΔ), thymidine kinase (BoHV-5TKΔ), and both gE and TK (BoHV-5gEΔTKΔ). Seronegative calves (80 to 90 days-old) inoculated with the parental strain (SV-507/99, n=5) shed virus in nasal secretions for up to 15 days (average 10.8 days). Duration of virus shedding was 11 days for BoHV-5gΔ, 9.6 days for BoHV-5TKΔ and 6.2 days for BoHV-5gEΔTKΔ groups. The highest titers were observed between days 1 and 6 post-infection (pi) for SV-507/99 (10(6.8)TCID50/mL), 10(5.1)TCID50/mL (BoHV-5gEΔ), 10(5.9)TCID50/mL (BoHV-5TKΔ) and 10(4.7)TCID50/mL (BoHV-5gEΔTKΔ). Calves inoculated with the parental virus presented anorexia, profound apathy and loss of body condition. Two calves were euthanized in extremis on days 10 and 11 pi; infectious virus was recovered from several areas of the brain. In contrast, calves inoculated with the recombinants remained healthy and a few presented a mild and transient nasal secretion. Dexamethasone (Dx) administration at day 42 pi resulted in virus shedding by all controls calves (mean duration 3.7 days), by 2/5 of BoHV-5TKΔ calves (two days) and 2/5 of BoHV-5gEΔ (one day). No virus shedding was detected in BoHV-5gEΔTKΔ calves upon Dx treatment. PCR examination of brain sections of calves euthanized at day 30 post Dx treatment revealed the presence of latent viral DNA widely distributed in the brain of SV-507/99 calves. Latent viral DNA was detected in a few sections (3/30) of the brains of BoHV-5gEΔ calves and was not detected in the brains of calves inoculated with BoHV-5TKΔ and BoHV-5gEΔTKΔ. These results show that the single BoHV-5 mutants (gE and tk-deleted) are attenuated for calves and establish and/or reactivate latent infection inefficiently. The double mutant BoHV-5gEΔTKΔ is fully attenuated and appears not to establish or not reactivate efficiently from latent infection. Thus, these recombinants, especially the double mutant BoHV-5gEΔTKΔ, display an adequate phenotype for use in modified-live vaccine formulations.

Este artigo descreve uma investigação da virulência/atenuação de recombinantes do herpesvírus bovino tipo 5 (BoHV-5) com deleções nos genes da glicoproteína E (BoHV-5gEΔ), timidina quinase (BoHV-5TKΔ), e ambos gE e TK (BoHV-5gEΔTKΔ). Bezerros soronegativos (80-90 dias de idade) inoculados com o vírus parental SV-507/99 (n=5) excretaram o vírus em secreções nasais por até 15 dias (média de 10,8 dias). Nos animais inoculados com os recombinantes, a duração da excreção viral foi de 11 dias (BoHV-5gEΔ), 9,6 dias (BoHV-5TKΔ) e 6,2 dias (BoHV-5gEΔTKΔ). Os maiores títulos foram observados entre os dias 1 e 6 pós-inoculação (pi), sendo de 10(6,8)TCID50/mL para o SV-507/99, 10(5,1)TCID50/mL (BoHV-5gEΔ), 10(5,9)TCID50/mL (BoHV-5TKΔ) e 10(4,7)TCIΔ50/mL (BoHV-5gEΔTKΔ). Os bezerros inoculados com o vírus parental apresentaram anorexia e apatia; três deles mostraram apatia profunda e perda da condição corporal. Dois bezerros foram eutanasiados in extremis nos dias 10 e 11 pi, respectivamente e o vírus foi isolado de várias regiões do encéfalo. Já os bezerros inoculados com os recombinantes permaneceram saudáveis; alguns apresentaram uma secreção nasal serosa transitória. Administração de dexametasona (Dx) no dia 42 pi resultou em excreção viral por todos os bezerros inoculados com o vírus parental (duração média de 3,7 dias), por 2 de 5 bezerros dos grupos BoHV-5TKΔ (dois dias) e BoHV-5gEΔ (um dia). Os bezerros inoculados com o duplo mutante BoHV-5gEΔTKΔ não excretaram o vírus após o tratamento com Dx. Pesquisa de DNA viral por PCR no dia 30 pós-Dx revelou uma ampla distribuição do DNA do vírus parental no encéfalo; poucas seções (3/30) foram positivas no encéfalo dos animais do grupo BoHV-5gEΔ, e não detectou-se DNA latente no encéfalo dos animais dos grupos BoHV-5TKΔ e BoHV-5gEΔTKΔ. Esses resultados demonstram que os mutantes simples (gE and tk-deletados) são atenuados para bezerros e estabelecem e/ou reativam infecção latente ineficientemente. Já o duplo mutante BoHV-5gEΔTKΔ é atenuado e parece não estabelecer e/ou não reativar eficientemente a infecção latente. Portanto, os vírus recombinantes, e em especial o duplo mutante BoHV-5gEΔTKΔ apresentam um fenótipo compatível com a sua inclusão em vacinas vivas modificadas.