Bovine Herpesvirus Research Articles

Modulation of granzymes mRNA expression in neural tissue of BoAHV-1 and BoAHV-5-infected cattle

Bovine alphaherpesviruses (BoAHV)-1 and 5 are neurotropic viruses of cattle which differ in their neuropathogenic potential. BoAHV-5 is responsible for non-suppurative meningoencephalitis in calves while BoAHV-1 can occasionally cause encephalitis. Granzymes (GZMs) are serine-proteases that participate in CD8+ T cells-mediated killing of virally-infected cells upon release through perforin (PFN)-formed pores in the cell membrane. Recently, six GZMs have been identified in cattle (A, B, K, H, M and O). However, their expression in bovine tissues has not been evaluated. In this study, the mRNA expression levels of PFN and GZMs A, B, K, H and M in the nervous system of calves experimentally-inoculated with BoAHV-1 or BoAHV-5 were analyzed at the three distinct stages of the infectious cycle of alphaherpesviruses: acute infection, latency and reactivation. This is the first report describing the expression of GZMs in bovine neural tissue and the first analysis of GZM expression in the context of bovine alphaherpesviruses neuropathogenesis. The findings revealed that PFN and GZM K are upregulated during BoAHV-1 or BoAHV-5 acute infection. In contrast to BoAHV-1, during BoAHV-5 latency a significant up-regulation of PFN, GZM K and GZM H was detected. PFN and GZM A, K and H expression was also up-regulated during BoAHV-5 reactivation. Therefore, a distinct pattern of PFN and GZM expression is evident along the infectious cycle of each alphaherpesvirus and this might contribute to the differences in BoAHV-1 and BoAHV-5 neuropathogenesis.

Development of Matrix-Embedded Bovine Tracheal Organoids to Study the Innate Immune Response against Bovine Respiratory Disease

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle. Bovine herpesvirus-1 (BHV-1) is one of the main culprits of BRD; however, research on BHV-1 is hampered by the lack of suitable models for infection and drug testing. In this study, we established a novel bovine tracheal organoid culture grown in a basement membrane extract type 2 (BME2) matrix and compared it with the air–liquid interface (ALI) culture system. After differentiation, the matrix-embedded organoids developed beating cilia and demonstrated a transcriptomic profile similar to the ALI culture system. The matrix-embedded organoids were also highly susceptible to BHV-1 infection and immune stimulation by Pam2Cys, an immunomodulator, which resulted in robust cytokine production and tracheal antimicrobial peptide mRNA upregulation. However, treatment of bovine tracheal organoid cultures with Pam2Cys was not sufficient to inhibit viral infection or replication, suggesting a role of the non-epithelial cellular microenvironment in vivo.

Characterization of BoHV-4 ORF45.

Bovine herpesvirus 4 (BoHV-4) is a Gammaherpesvirus belonging to the Rhadinovirus genus. The bovine is BoHV-4's natural host, and the African buffalo is BoHV-4's natural reservoir. In any case, BoHV-4 infection is not associated with a specific disease. Genome structure and genes are well-conserved in Gammaherpesvirus, and the orf 45 gene and its product, ORF45, are one of those. BoHV-4 ORF45 has been suggested to be a tegument protein; however, its structure and function have not yet been experimentally characterized. The present study shows that BoHV-4 ORF45, despite its poor homology with other characterized Rhadinovirus ORF45s, is structurally related to Kaposi's sarcoma-associated herpesvirus (KSHV), is a phosphoprotein, and localizes in the host cell nuclei. Through the generation of an ORF45-null mutant BoHV-4 and its pararevertant, it was possible to demonstrate that ORF45 is essential for BoHV-4 lytic replication and is associated with the viral particles, as for the other characterized Rhadinovirus ORF45s. Finally, the impact of BoHV-4 ORF45 on cellular transcriptome was investigated, an aspect poorly explored or not at all for other Gammaherpesvirus. Many cellular transcriptional pathways were found to be altered, mainly those involving p90 ribosomal S6 kinase (RSK) and signal-regulated kinase (ERK) complex (RSK/ERK). It was concluded that BoHV-4 ORF45 has similar characteristics to those of KSHV ORF45, and its unique and incisive impact on the cell transcriptome paves the way for further investigations.

Determination of protection provided by two modified live bovine herpesvirus-1 vaccines against bovine herpesvirus-1 conjunctivitis in calves

A study was performed in 2 phases to determine the ability of 2 different modified live (MLV) bovine herpesvirus-1 (BoHV-1)vaccines to protect against conjunctivitis caused by a recent field isolate of BoHV-1. In phase I, BoHV-1 isolates were used todetermine the ability of the isolates to cause conjunctivitis and to establish a challenge model for phase II. Fifteen calves wererandomly assigned to 1 of 3 groups: 5 calves received 5 mL containing 107 tissue culture infectious dose (TCID)50/mL of BoHV-1adminisered intraocularly (topically) for 2 days, 5 calves received 5 mL of inoculum consisting of 107TCID50/mL of BoHV-1 administered intranasally for 2 days, and 5 calves served as unchallenged sentinels and received phosphatebuffered saline, 5 mL administered by the intranasal and 5 mL by the intraocular route for 2 days. All 15 calves developedpyrexia (rectal temperature greater than 103.5°F (39.7°C)), were BoHV-1 virus isolation positive from ocular swabsand 10/15 calves had increased ocular discharge and conjunctivitis. In phase II, 10 calves were intranasally vaccinated witha MLV BoHV-1, 10 calves were parenterally vaccinated with a MLV BoHV-1, and 5 calves were unvaccinated. All 25 calveswere challenged by receiving 5 mL ntraocularly of 107 TCID50/mL of BoHV-1 for 2 consecutive days. Following challenge, calves were examined daily, and blood (serum), nasal swabs, and conjunctival swab samples were obtained on study days 0, 3-7, 10, 14 and 28. Both vaccines significantly decreased post challenge ocular lesions, clinical scores, and quantity of virus isolated.

Seroprevalence of Infectious Bovine Rhinotracheitis (IBR) in Indian Buffaloes

Background: Infectious bovine rhinotracheitis (IBR) is an infectious disease of bovines causing respiratory problem, abortions and reduced milk yield leading to huge economic losses. IBR is endemic in India. India ranks first in buffalo population. Reports on seroprevalence of IBR in cattle in India are available but not in buffaloes. The study was aimed at conducting a seroprevalence of IBR in Indian buffaloes. Methods: A total of 1000 buffalo serum samples collected randomly from top 10 milk producing states of India and were tested for IBR antibodies using Avidin-Biotin (AB) ELISA. Result: Cumulative seropositivity was found to be 43.30%. Punjab and Andhra Pradesh states showed highest (58%) and lowest (27%) seropositivity respectively and were part of north and south zone of the country. India has highest number of buffalo population. Presently, India do not vaccinate bovines against IBR and should take call on vaccination of dairy buffalo after having witnessed a high seroprevalence of IBR. This is the first report of such kind of number of buffalo samples tested for IBR antibodies.

The effect of neonatal vaccination for bovine respiratory disease in the face of a dual challenge with bovine viral diarrhea virus and Mannheimia hemolytica.

Bovine respiratory disease is the greatest threat to calf health. In this study, colostrum-fed dairyXbeef calves were vaccinated at ∼30days of age with an adjuvanted parenteral vaccine containing modified live bovine viral diarrhea virus (BVDV) type 1 and type 2, bovine herpesvirus 1 (BHV-1), bovine parainfluenza type 3 virus (PI3V) and bovine respiratory syncytial virus (BRSV) andM. haemolyticatoxoid (Group 1), or intranasal temperature-sensitive BHV-1, BRSV and PI3V concurrently witha parenteral vaccine containing modified live BVDV type 1 and type 2 andM. haemolyticatoxoid (Group 2) or a placebo (Group 3). The calves were challenged ∼150days post vaccination intranasally with BVDV 1b and then 7days later intratracheally withM. haemolytica. The calves wereeuthanized 6days after theM. haemolyticachallenge. Clinical signs following BVDV infection were similar in all groups. There was increased rectal temperatures in the Groups 2 and 3 on day 3 and in Group 3 on days 8-13. Group 1 animals had a slight leukopenia following BVDV infection while Groups 2 and 3 had greater leukopenia. BVDV type 1 and 2 serum titers increased in Group 1 following vaccination while these titers waned in Groups 2 and 3. There were higher levels of BVDV in the buffy coats and nasal samples in Group 2 and Group 3 versus Group 1 (p<0.01). Interferon-gamma response was higher (p<0.01) in Group 1 animals than Groups 2 and 3. Group 1 had the lowest percent pneumonic tissue (1.6%) while Group 2 vaccinates had 3.7% and the control Group 3 was 5.3%. Vaccination in the face of maternal antibody with a parenteral adjuvanted vaccine resulted in better protection than the regimen of an intranasal vaccine anda parenteral adjuvanted BVDV andM haemolyticacombination vaccine in a BVDV-M. haemolyticadual challenge.

A retrospective evaluation of pooled serum ELISA testing in the frame of the French eradication program for infectious bovine rhinotracheitis.

Pooled serum testing using whole-virus indirect ELISA has been recently recognized as an official method for surveillance of bovine herpesvirus 1 (BoHV1) in cattle herds in Europe. In this study, a retrospective analysis of data from the French BoHV1 surveillance campaign 2018-2019, including 7434 BoHV1-free certified herds and 157 infected herds, was performed in order to evaluate the diagnostic specificity and sensitivity of two pooled serum indirect ELISAs (from IDEXX and IDVet), in comparison with individual testing by blocking ELISAs targeting the gB and gE proteins. Pooled serum testing showed a relative specificity higher than 97.5% and a detection rate of 100% since all gB+/gE+ samples were found in positive pools. At the herd level, no more than one false positive pool was observed in most of BoHV1-free certified herds, leading to a herd relative specificity of 85.1% and 86.0% for the IDEXX and IDVet pooled serum ELISAs, respectively. Among infected herds tested by pool sizes up to 10 sera (n=122), 46% of herds were detected through pools of size 10 containing a single positive sample, 23% through pools of size 10 containing at least two positive samples, and 31% through pools of smaller sizes. A complementary study based on manually constituted pools revealed that at least one positive sample in 100% and 93.4% of herds could be detected individually by pools of size 10 with the IDEXX and IDVet ELISAs, respectively. However, pooled serum ELISAs were influenced by the level of individual reactivity, since pools composed of either one weak-positive sample or one gB+/gE- sample could yield negative results. Altogether, these results provided the first evidence that pooled serum testing (pool size up to 10) is a suitable strategy for surveillance of BoHV1-free cattle farms.

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.

A REVIEW ON ANTIVIRAL PLANTS EFFECTIVE AGAINST DIFFERENT VIRUS TYPES

The treatment of microbial infections has proven challenging for humans in recent years. Synthetic medications, such as antimicrobial agents, are used for treating these infections. Antimicrobials derived from natural sources have gained popularity as an alternative to manufactured medications due to their lack of adverse effects. Plants, which play a significant role in this setting, have historically served as a reliable natural defence against several pathogens. In this study, studies on plants used against viral diseases are mentioned. Studies on viruses that cause disease have been compiled in the literature. According to the findings, it has been reported in the literature that many different plant species are effective against herpes simplex virus (HSV-1, HSV-2), human immunodeficiency virus (HIV), influenza virus (A, B) and parainfluenza, Poliovirus, Astrovirus, Parvovirus, Sindbis virus (SINV), Feline calicivirus (FCV), Rhinovirus, Echovirus, Rotavirus, Bovine alphaherpesvirus 1 (BoHV-1), Reovirus, Vaccinia virus (VACV), Cardiovirus A (Encephalomyocarditis virus; EMCV), Coxsackie virus, Semliki forest, Measles virus, Newcastle disease virus (NDV), Coronavirus, Adenovirus (ADV-3, ADV-5, ADV-8, ADV -11), Canine distemper virus (CDV), Lumpy skin disease virus (LSDV), Hepatitis A, B, C virus and Enterovirus. To combat viruses, plants can be considered a potentially invaluable natural resource.

Infectious bovine rhinotracheitis – A review

In many parts of the world, domestic as well as wild cattle are affected by infectious bovine rhinotracheitis. The causative organism is Varicellovirus in the subfamily Alphaherpesvirinae of the family Herpesviridae. This review is mainly concerned with the etiology, epidemiology, economic importance, clinical presentation of infectious bovine rhinotracheitis, diagnosis, treatment, prevention and control apart from its status in Ethiopia.

The Cell-Mediated Immune Response against Bovine alphaherpesvirus 1 (BoHV-1) Infection and Vaccination.

Bovine Alphaherpesvirus 1 (BoHV-1) is one of the major respiratory pathogens in cattle worldwide. Infection often leads to a compromised host immune response that contributes to the development of the polymicrobial disease known as "bovine respiratory disease". After an initial transient phase of immunosuppression, cattle recover from the disease. This is due to the development of both innate and adaptive immune responses. With respect to adaptive immunity, both humoral and cell-mediated immunity are required to control infection. Thus, several BoHV-1 vaccines are designed to trigger both branches of the adaptive immune system. In this review, we summarize the current knowledge on cell-mediated immune responses directed against BoHV-1 infection and vaccination.

Serological Detection of SRMV, Bovine Viral Diarrhea Virus, BVDV and BEFV in Camels (Camelus dromedarius) in Southwest Iran

Background: Small ruminant morbillivirus (SRMV), bovine viral diarrhea virus (BVDV), bovine herpes virus-1 (BHV-1), and bovine ephemeral fever virus (BEFV) are among the most important viruses of farm animals. Objectives: This study aimed at the serological detection of SRMV, BVDV, BHV-1, and BEFV in the camel population of Khuzestan Province, located in southwest Iran. Methods: A total of 150 camel blood samples were randomly collected from free-ranging, seemingly healthy camels of both sexes and various ages in 8 different regions of Khuzestan Province, Iran. The sera were tested for SRMV, BVDV, BHV-1, and BEFV with serum neutralization test. Results: The seropositive samples for SRMV, BVDV, and BHV-1 were 1(0.67%), 7(4.67%), and 11(7.33%), respectively. There was no seroconversion against BEFV in the serum samples. Conclusion: The camel population of Khuzestan Province is subclinically infected with SRMV, BVDV, and BHV-1 and could play a significant role in the epizootiology of these viral diseases in this region, which is very important for the control and eradication programs.

The immunogenicity and efficacy of acommercially available Infectious Bovine Rhinotracheitis (IBR) virus vaccine against a Pakistani field IBR strain.

Infectious bovine rhinotracheitis (IBR) is a highly communicable disease of cattle and wild ruminants that is caused by Bovine alphaherpesvirus 1 (BoHV‑1). For IBR control, several developed countries have adopted the immunization and eradication programs focusing on IBR‑positive animals. In Pakistan, livestock producers are importing commercially available vaccine of BoHV‑1, but no studies on the efficacy of these commercial vaccines against local isolates are available. Therefore, the present study was aimed to evaluate the efficacy of a commercially available vaccine of BoHV‑1 against local field isolates of virus. The rabbit model was used and the vaccine was evaluated for immunogenicity and protection after challenge with a highly virulent strain of a field virus. The immune response was measured by virus neutralization titers (VNT). This vaccine induced a humoral response in rabbits but that was not sufficient to completely protect the vaccinated animals against the wild‑type BoHV‑1 strain challenge. While a low virus titer compared to control rabbits was observed in the vaccinated rabbits (p<0.05), there was no sterilizing immunity or freedom from infection. However, complete freedom from disease, for example, the absence of pyrexia was noticed in the vaccinated group. In conclusion, the present study demonstrated that imported vaccine stock provoked only a partial protection against indigenous isolated of BoHV‑1. However, tests performed on rabbits are preliminary, as only those performed on the source species can determine more reliable results.

Evaluation of practices used to reduce the incidence of bovine respiratory disease in Australian feedlots (to November 2021).

Bovine respiratory disease (BRD) has been identified as the most significant infectious disease of feedlot cattle in eastern Australia.1 Bovine respiratory disease causes economic loss due to medication costs, mortalities, excessive feed inputs associated with increased time on feed, reduced sale prices and associated labour costs. Bovine respiratory disease is a complex multifactorial condition with multiple animal, environmental and management risk factors predisposing cattle to illness. A range of microorganisms are implicated in BRD with at least four viral and five bacterial species commonly involved individually or in combination. The viruses most commonly associated with BRD in Australia are bovine herpesvirus 1 (BHV1), bovine viral diarrhoea virus (BVDV or bovine pestivirus), bovine parainfluenza 3 virus (PI3) and bovine respiratory syncytial virus (BRSV). More recently, bovine coronavirus has been identified as a potential viral contributor to BRD in Australia.2 A number of bacterial species have also been recognised as important to the BRD complex; these include Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes and Mycoplasma bovis. Although one or more of the pathogens listed above can be isolated from clinical cases of BRD, there is no evidence that infection alone causes serious illness. This indicates that, in addition to specific infectious agents, other factors are crucial for the development of BRD under field conditions. These can be categorised as environmental, animal and management risk factors. These risk factors are likely to exert their effects through multiple pathways including reductions in systemic and possibly local immunity. For example, stressors such as weaning, handling at saleyards, transport, dehydration, weather conditions, dietary changes, comingling and pen competition might reduce the effectiveness of the immune system. Reduced immunocompetence can allow opportunistic infection of the lower airways with potential pathogens leading to the development of BRD. The objective of this paper is to critically review the evidence for management practices aimed at reducing the incidence of BRD in Australian feedlot cattle. Predisposing factors (Table 1) largely beyond the control of most feedlots, such as weather and exposure to respiratory viruses, are discussed separately, but these factors can generate indirect prevention responses that are discussed under the preventative practices categories. The current practices are classified as either animal preparation practices (Table 2) or feedlot management practices (Table 3).

Confirmation of the passive transfer of maternal antibodies to calves following vaccination of pregnant cows with an inactivated Mannheimia haemolytica and Bovine herpes virus type 1 vaccine.

The objective of this study was to evaluate the passive transfer of maternal antibodies to calves following vaccination of pregnant cows with an inactivated Mannheimia haemolytica (MH) and Bovine herpes virus type 1 (IBR) vaccine (Bovilis® MH + IBR). Sixty-two pregnant cows were allocated at random to two groups; one group was retained as a negative control group (T01), while the other group (T02) was vaccinated with Bovilis® MH + IBR on two occasions during their third trimester of pregnancy. Following calving, blood samples were collected from calves for the measurement of serum antibody titres to IBR and MH, with samples collected prior to suckling (Day 0) and on days 5 (±2), 14 (±3), 28, 56, 84, 112, 140, 168, 196, 224, 252 and 280. The group mean IBR blocking percentage remained low for T01 calves (calves born to T01 cows) between days 0 and 224 (range 4.5%-15.4%), while the group mean IBR blocking percentage increased for T02 calves (calves born to T02 cows) from 14.3% on Day 0 to 94.9% on Day 5 and remained significantly higher than T01 calves up until Day 252. The group mean MH titre (Log2) for T01 calves increased after suckling to 8.9 on Day 5, before declining and remaining stable (range 5.0-6.5). The group mean MH titre for T02 calves increased after suckling to 13.6 on Day 5 and then gradually declined; however, it remained significantly higher than T01 calves between days 5 and 140. Outcomes from this study have confirmed that colostral transfer of IBR and MH antibodies to newborn calves was successful and a high level of passive immunity was acquired by calves.

Cloning of BHV-UL25 Conserved Fragment in a Lentiviral Transfer Plasmid for the Preparation of a Monitoring Cell Line

: Bovine herpesvirus-1 (BHV-1) is the causative agent of a domestic and wild cow disease namely infectious bovine rhinotracheitis (IBR). Severe economic damage due to IBR is possible to occur and vaccines are not completely effective against the disease. Therefore, over the recent years, the development of effective and genetic-based therapies to control viral infections, such as IBR has been remarkable. A common way to evaluate such therapeutic strategies is cloning of the viral target sequence into appropriate vectors for the preparation of cell lines expressing viral subgenomic replicons. Due to the required duties of UL25 gene, serine protease substrate domain of this gene was cloned in pCDH-CMV-MCS-EF1-cGFP-T2A-Puro lentiviral vector at the upstream of GFP gene. The cloning accuracy was verified by restriction of enzyme digestion and sequencing. Thus, this recombinant plasmid will be available to produce lentiviral vectors with the desired gene; after infection of eukaryotic cells with such lentiviral vectors the target gene will be expressed.

Serum and Milk Levels of Antibodies to Bovine Viral Diarrhoea Virus, Bovine Herpesvirus-1 and -4, and Circulation of Different Bovine Herpesvirus-4 Genotypes in Dairy Cattle with Clinical Mastitis.

Bovine viral diarrhoea virus (BVDV) and bovine herpesvirus (BoHV)-1 and -4 are important causes of respiratory diseases and reproductive disorders of dairy cattle worldwide. Investigation of BVDV and BoHV-1 and -4 antibody levels in the serum and milk of dairy cattle in a group with clinical mastitis and a healthy group was undertaken using an indirect ELISA, and identification of the BoHV-4 genotypes in clinical mastitis cases was attempted by PCR and sequencing. Antibodies specific to BVDV, BoHV-1 and BoHV-4 were detected in the serum and milk of all dairy cattle with clinical mastitis. The cut-off values for BVDV and BoHV-1 in the sera and milk were extremely high in both healthy and mastitic animals. However, BoHV-4 antibodies were detected only in the clinically mastitic cattle, and BoHV-4 levels were higher in milk than in sera among these animals. Genotypes I and II of BoHV-4 were detected in the milk samples of four seropositive cows with clinical mastitis from the same herd. The results of this investigation demonstrate that clinical mastitis cases in the same herd may have aetiology in different BoHV-4 genotypes.

Neutralizing antibodies to bovine and bubaline alphaherpesviruses in water buffaloes (Bubalus bubalis).

Water buffaloes (Bubalus bubalis) have been introduced in many regions of the world as a source of animal protein. In many instances, bubaline cattle are reared close to or mixed with bovine or zebuine cattle. However, little is known about infectious diseases of bubaline and the interactions that may arise involving the microbiota of those species. Alphaherpesviruses of ruminants (bovine alphaherpesviruses types 1 and 5, BoHV-1, BoHV-5; bubaline alphaherpesvirus 1, BuHV-1) are highly cross-reactive in serological assays performed with bovine or zebuine sera. However, the profile of reactivity of bubaline cattle sera to alphaherpesviruses remains unknown. As such, it is not known which virus strain (or strains) would be most appropriate to be used as the challenge virus in the laboratory in search for alphaherpesvirus-neutralizing antibodies. In this study, the profile of neutralizing antibodies to alphaherpesviruses in bubaline sera was determined against different types/subtypes of bovine and bubaline alphaherpesviruses. Sera (n=339) were screened in a 24-h serum neutralization test (SN) against 100 TCID50 of each of the challenge viruses. From those, 159 (46.9 %) neutralized at least one of the viruses assayed; 131 (38.6%) sera neutralized the three viral strains used for screening. The viral strain that was neutralized by the largest number of sera was BoHV-5b A663 (149/159; 93.7%). A few sera neutralized only one of the challenge viruses: four sera neutralized BoHV-1 LA only; another neutralized BoHV-5 A663 only and four others neutralized BuHV-1 b6 only. SN testing with two additional strains gave rise to similar results, where maximum sensitivity (defined here as the largest number of sera that neutralized the challenge viruses) was obtained by adding positive results attained with three of the challenge strains. Differences in neutralizing antibody titers were not significant to allow inferences on which would be the most likely virus that induced the antibody responses detected here.

A method for the infectivity discrimination of enveloped DNA viruses using palladium compounds pre-treatment followed by real-time PCR.

Cultivation-based assays represent the gold standard for the assessment of virus infectivity; however, they are time-consuming and not suitable for every virus type. Pre-treatment with platinum (Pt) compounds followed by real-time PCR has been shown to discriminate between infectious and non-infectious RNA viruses. This study examined the effect of Pt and palladium (Pd) compounds on enveloped DNA viruses, paying attention to two significant pathogens of livestock - bovine herpesvirus-1 (BoHV-1) and African swine fever virus (ASFV). Native or heat-treated BoHV-1 suspension was incubated with the spectrum of Pt/Pd compounds. Bis(benzonitrile)palladium(II) dichloride (BB-PdCl 2) and dichloro(1,5-cyclooctadiene) palladium(II) (PdCl 2-COD) produced the highest differences found between native and heat- -treated viruses. Optimized pre-treatment conditions (1 mM of Pd compound, 15 min, 4°C) were applied on both virus genera and the heat inactivation profiles were assessed. A significant decrease in the detected quantity of BoHV-1 DNA and ASFV DNA after heat-treatment (60°C and 95°C) and consequent incubation with Pd compounds was observed. BB-PdCl 2 and PdCl 2-COD could help to distinguish between infectious and non-infectious enveloped DNA viruses such as BoHV-1 or ASFV.

Immunogenicity of an inactivated vaccine for intravaginal application against bovine alphaherpesvirus type 5 (BoHV-5).

The present study was carried out to evaluate the intravaginal vaccine potential against bovine alphaherpesvirus type 5 (BoHV-5). Sixty three cows were divided into seven groups (n: 9) and inoculated intravaginally (VA) or intramuscularly (IM) with inactivated BoHV-5, associated with the recombinant B subunit of the heat-labile enterotoxin of E. coli (rLTB), 2-hydroxyethylcellulose (Drug Delivery System A - DDS-A) or Poloxamer 407 (Drug Delivery System B - DDS-B) as follows: G1 (DDS-A + BoHV-5+ rLTB), G2 (DDS-A + BoHV-5), G3 (DDS-B + BoHV-5+ rLTB), G4 (DDS-B + BoHV-5), G5 (BoHV-5+ rLTB), G6 (Negative control) e G7 (Positive control). The local and systemic humoral responses were measured by indirect ELISA (IgA and IgG) and serum neutralization tests, and the cellular response was measured by a quantitative direct ELISA (IL-2 and IFN-Gamma). The results showed the group inoculated by the IM route, G5, demonstrated the highest levels of IgG in the vaginal mucosa among the experimental groups (p<0.05). In the groups tested with polymers (G1 and G3) in the vaginal mucosa, even higher levels of IgG were seen in comparison to the positive control (G7; p<0.01). Higher levels of IgA were also noted in relation to the other groups (p<0.05) on days 30, 60 and 90 post-inoculations. The groups G1 and G3 also provided higher titers of neutralizing antibodies (Log2) in relation to other treatments (p<0.01) 90 days after inoculation. In the nasal mucosa, there was an increase in the levels of IgA and IgG with the use of vaccines from groups G1 and G3, in relation to the positive control, G7 (p<0.05) at 60 and 90 days after the first inoculation. Moreover, neutralizing antibodies titers were detected at 60 and 90 days by serum neutralization. The inclusion of the evaluated polymers resulted in a superior response (p<0.05) of immunoglobulins and IL-2 and IFN-Gamma in relation to the treatment using only rLTB (G5). This data demonstrates the capabilities of a vaccine with an intravaginal application in cattle to stimulate a local and systemic immune response.