A molecular survey using a validated real-time PCR assay finds no evidence of bovine alphaherpesvirus 2 in samples from animals with suspected vesicular disease in Brazil between 2014 and 2017.

Bovine alphaherpesvirus 2 (BoHV-2) is the etiologic agent of bovine mammillitis (BM) and pseudo-lumpy skin disease. BM is also important because its clinical presentation can be confused with foot-and-mouth disease (FMD), making it necessary to establish differential diagnoses and perform additional laboratory tests. The objective of this work was to use a validated real-time PCR assay to test for the presence of BoHV-2 in samples from cattle and buffalo with suspected vesicular disease in Brazil. The method could detect the virus at a concentration of 0.5 fg/µL and had 99.4% amplification efficiency, a repeatability error of only 4.1%, and good reproducibility with other reagents. No evidence of BoHV-2 causing vesicular disease in cattle and buffalo was found in this work. This study was able to validate a new methodology for detection of BoHV-2 and evaluate its usefulness for investigating outbreaks of vesicular disease Brazil. The importance of BoHV-2 in cases involving other clinical signs should still be studied using the qPCR developed in this work.

Herpesvírus bovino tipo 1 (BoHV-1): método de diagnóstico e sua influência na qualidade espermática em touros infectados experimentalmente / Bovine Herpes virus type 1 (BoHV-1): Diagnosis method and its influence on spermatic quality in experimentally infected bulls

O herpesvírus bovino tipo-1 (BoHV-1) é um vírus amplamente distribuído no Brasil e no mundo, havendo um crescente número de estudos envolvendo métodos de diagnóstico e o seu impacto na reprodução animal. O objetivo deste trabalho foi identificar o material genético do BoHV-1 no sêmen de touros infectados experimentalmente por meio da técnica de PCR e avaliar a influência do vírus sobre a qualidade espermática desses animais. A técnica de PCR foi satisfatória, permitindo identificar a presença do material genético do vírus no sêmen de todos os animais a partir de sete dias pós-infecção, com persistência de 21 até 28 dias. Apesar da presença do vírus BoHV-1 por um longo período no sêmen dos animais experimentais, não foram observados efeitos deletérios na qualidade do sêmen fresco e nem após a criopreservação.(AU)

Bovine Herpesvirus type-1 (BoHV-1) is a virus widely distributed in Brazil and worldwide, with a growing number of studies involving diagnostic methods and their impact on animal reproduction. The objective of this work was to identify the genetic material of BoHV-1 in the semen of experimentally infected bulls through the PCR technique, and to evaluate the influence of the virus on the sperm quality of these animals. The PCR technique was satisfactory, allowing for the identification of the presence of the genetic material of the virus in the semen of all the animals from 7 days post infection, with persistence of 21 to 28 days. Despite the presence of the BoHV-1 virus over a long period in the semen of the experimental animals, no deleterious effects were observed on the quality of either fresh semen or semen after the cryopreservation.(AU)

Herpesvírus bovino tipo 1 (BoHV-1): método de diagnóstico e sua influência na qualidade espermática em touros infectados experimentalmente / Bovine Herpes virus type 1 (BoHV-1): Diagnosis method and its influence on spermatic quality in experimentally infected bulls

O herpesvírus bovino tipo-1 (BoHV-1) é um vírus amplamente distribuído no Brasil e no mundo, havendo um crescente número de estudos envolvendo métodos de diagnóstico e o seu impacto na reprodução animal. O objetivo deste trabalho foi identificar o material genético do BoHV-1 no sêmen de touros infectados experimentalmente por meio da técnica de PCR e avaliar a influência do vírus sobre a qualidade espermática desses animais. A técnica de PCR foi satisfatória, permitindo identificar a presença do material genético do vírus no sêmen de todos os animais a partir de sete dias pós-infecção, com persistência de 21 até 28 dias. Apesar da presença do vírus BoHV-1 por um longo período no sêmen dos animais experimentais, não foram observados efeitos deletérios na qualidade do sêmen fresco e nem após a criopreservação.(AU)

Bovine Herpesvirus type-1 (BoHV-1) is a virus widely distributed in Brazil and worldwide, with a growing number of studies involving diagnostic methods and their impact on animal reproduction. The objective of this work was to identify the genetic material of BoHV-1 in the semen of experimentally infected bulls through the PCR technique, and to evaluate the influence of the virus on the sperm quality of these animals. The PCR technique was satisfactory, allowing for the identification of the presence of the genetic material of the virus in the semen of all the animals from 7 days post infection, with persistence of 21 to 28 days. Despite the presence of the BoHV-1 virus over a long period in the semen of the experimental animals, no deleterious effects were observed on the quality of either fresh semen or semen after the cryopreservation.(AU)

Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology / Caracterização de Anticorpos Monoclonais reativos ao Herpesvírus bovino tipo 1 selecionados por Phage Display

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.

A especificidade dos anticorpos monoclonais (mAb) aos alvos desejados torna estas moléculas ade-quadas para uso em diagnóstico ou terapia de uma vasta gama de agentes patogênicos. Biblioteca de anticorpos apresentados em fagos filamentosos é uma metodologia para a produção de mAbs, poden-do ser utilizada como alternativa à tecnologia de hibridoma convencional, tradicionalmente empregada para este fim. Neste trabalho, a tecnologia de Phage display foi usada para construir, selecionar e ca-racterizar uma biblioteca combinatorial de fragmentos de anticorpos de cadeia única (scFv) contra o Herpesvírus bovino tipo 1 (BoHV-1) a partir do repertório imune de galinhas imunizadas com o vírus. A análise in silico dos domínios hipervariáveis das cadeias pesadas dos anticorpos revelou uma alta frequência de fragmentos scFv com baixa variabilidade, sugerindo que a seleção foi provavelmente conduzida e favorecida por poucos antígenos virais mais imunogênicos. A reatividade dos fragmentos scFv selecionados contra BoHV-1 foi demonstrada por Fago-ELISA. Observou-se um aumento signi-ficativo da reatividade dos anticorpos após seis ciclos de seleção, evidenciando sua utilização como molécula para o diagnóstico de BoHV-1. A estratégia aqui descrita abre a possibilidade do uso da tec-nologia de Phage display como ferramenta na seleção de anticorpos monoclonais com potencial uso tanto para o diagnóstico quanto para terapia de doenças infecciosas e/ou parasitárias de interesse veterinário.

Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology / Caracterização de Anticorpos Monoclonais reativos ao Herpesvírus bovino tipo 1 selecionados por Phage Display

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.(AU)

A especificidade dos anticorpos monoclonais (mAb) aos alvos desejados torna estas moléculas ade-quadas para uso em diagnóstico ou terapia de uma vasta gama de agentes patogênicos. Biblioteca de anticorpos apresentados em fagos filamentosos é uma metodologia para a produção de mAbs, poden-do ser utilizada como alternativa à tecnologia de hibridoma convencional, tradicionalmente empregada para este fim. Neste trabalho, a tecnologia de Phage display foi usada para construir, selecionar e ca-racterizar uma biblioteca combinatorial de fragmentos de anticorpos de cadeia única (scFv) contra o Herpesvírus bovino tipo 1 (BoHV-1) a partir do repertório imune de galinhas imunizadas com o vírus. A análise in silico dos domínios hipervariáveis das cadeias pesadas dos anticorpos revelou uma alta frequência de fragmentos scFv com baixa variabilidade, sugerindo que a seleção foi provavelmente conduzida e favorecida por poucos antígenos virais mais imunogênicos. A reatividade dos fragmentos scFv selecionados contra BoHV-1 foi demonstrada por Fago-ELISA. Observou-se um aumento signi-ficativo da reatividade dos anticorpos após seis ciclos de seleção, evidenciando sua utilização como molécula para o diagnóstico de BoHV-1. A estratégia aqui descrita abre a possibilidade do uso da tec-nologia de Phage display como ferramenta na seleção de anticorpos monoclonais com potencial uso tanto para o diagnóstico quanto para terapia de doenças infecciosas e/ou parasitárias de interesse veterinário.(AU)

Delivery of recombinant vaccines against bovine herpesvirus type 1 gD and Babesia bovis MSA-2c to mice using liposomes derived from egg yolk lipids.

Liposomes prepared from total egg yolk lipid extracts were used to deliver experimental DNA vaccines to mice consisting of pCI-neo plasmids encoding bovine herpesvirus type 1 (BoHV-1) gD or Babesia bovis MSA-2c. A significantly higher proportion of mice in the B. bovis MSA-2c group, but not those in the BoHV-1 gD group, developed detectable immunoglobulin G responses when vaccinated with liposome encapsulated DNA in comparison with mice vaccinated with naked DNA. In both groups, antibody titres were similar between mice vaccinated with liposome encapsulated DNA and naked DNA.

Molecular and in vitro characterization of field isolates of bovine herpesvirus-1.

Bovine Herpesvirus-1 (BoHV-1) is distributed worldwide and is a major pathogen in cattle, being the causal agent of a variety of clinical syndromes. The aim of this study was to isolate and to characterize (molecular and biological characterization) BoHV-1 from 29 immunosuppressed animals. It was possible to obtain 18 isolates, each from a different animal, such as from the respiratory and reproductive tracts. In some cases the cytopathic effect was visible 12 hours post-inoculation, and became characteristic after 36-48 hours. Biological characteristics were evaluated and compared with Iowa and Colorado-1 reference strains, and differences were found in plaque size, virus titer measured by TCID50 and PFU/mL, and one step virus curves. These results showed that some isolates had a highly virulent-like behavior in vitro, compared to the reference strains, with shorter eclipse periods, faster release of virus into the supernatants, and higher burst size and viral titer. There were no differences in glycoprotein expression of BoHV-1 isolates, measured by Western blot on monolayers. Moreover, using restriction endonucleases analysis, most of the viruses were confirmed as BoHV-1.1 and just one of them was confirmed as BoHV-1.2a subtype. These findings suggest that some wild-type BoHV-1 isolates could be useful as seeds to develop new monovalent vaccines.

Characterization of interspecific recombinants generated from closely related bovine herpesviruses 1 and 5 through multiple PCR sequencing assays.

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses infecting cattle. In countries where both viruses circulate, co-infection of cattle is likely. It was shown that recombination occurs at a high frequency in cattle infected dually with two BoHV-1 mutants. In addition, interspecific recombinants are generated in cell culture co-infected with BoHV-1 and BoHV-5. Even if the process of interspecific recombination appears inefficient relative to intraspecific recombination, BoHV-1 and BoHV-5 may give rise to interspecific recombinants in co-infected cattle. Since molecular tools for differentiating BoHV-1 from BoHV-5 are limited and do not allow to localize recombination events between these closely related virus species, 13 PCR sequencing assays were developed to discriminate between BoHV-1 and BoHV-5 at regular intervals throughout the entire respective viral DNA genomes. These assays were used to determine the genetic background of two interspecific BoHV-1/-5 recombinants generated previously. The two crossover points where recombination events occurred between the parental strains were determined. This study provides a detailed analysis of two interspecific recombinant viruses generated in vitro from closely related alphaherpesviruses infecting the same natural host. It demonstrates that recombination can occur within very short fragments of sequence homology. This finding raises questions about the mechanisms involved in the strands exchange and resolution step of the homologous recombination used by herpesviruses. This method will allow monitoring generation of recombinants between closely related herpesvirus species both in vitro and in vivo.

Phylogenetic comparison of the carboxy-terminal region of glycoprotein C (gC) of bovine herpesviruses (BoHV) 1.1, 1.2 and 5 from South America (SA).

Different types and subtypes of bovine herpesvirus 1 and 5 (BoHV-1 and BoHV-5) have been associated to different clinical conditions of cattle, in such a way that type/subtype differentiation has become an essential tool for understanding the pathogenesis and epidemiology of BoHV infections. In search for a genomic region that would allow a clear distinction between BoHV-1 and BoHV-5, the carboxy-terminal portion of glycoprotein C (gC), corresponding to residues 321-450 (BoHV-1) and 301-429 (BoHV-5) of 23 South American (SA) isolates (Brazil mostly) was amplified and sequenced. The nucleotide sequence alignments revealed levels of genomic similarity ranging from 98.7 to 99.8% among BoHV-1 isolates, 88.3 to 92% between BoHV-1/BoHV-5 and 96 to 99.7% among BoHV-5 isolates. At the amino acid level, sequence similarity varied ranging from 97.5 to 99.5% among BoHV-1, 77.5 to 84.4% between BoHV-1/BoHV-5 and 92.1 to 99.5% (BoHV-5/BoHV-5). The isolates could be clearly separated into BoHV-1.1, BoHV-1.2 and BoHV-5 after phylogenetic analysis. The results suggest that the phylogenetic analysis performed here can be used as a potential molecular epidemiological tool for herpesviruses.

Rapid detection and differentiation of bovine herpesvirus 1 and 5 glycoprotein C gene in clinical specimens by multiplex-PCR.

A multiplex polymerase chain reaction (multiplex-PCR) to detect and differentiate bovine herpesvirus 1 (BoHV-1) and 5 (BoHV-5) was developed using primers for the gene sequence that encodes the glycoprotein C. The technique was assessed against the BoHV-1 and BoHV-5 cell culture adapted strains, and clinical samples collected from animals with clinical signs of BoHV-1 (n = 10) or BoHV-5 (n = 7) infection and with diagnosis confirmed by virus isolation in cell culture and semi-nested PCR. Fifteen clinical samples from asymptomatic animals were included as control group. For the evaluation of the amplifiability of the extracted nucleic acid from clinical specimens was included a bovine internal control that amplified a 626 bp fragment of the ND5 gene present in the bovine mitochondrial DNA. For DNA extraction, a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate methods was used. The specificity of the BoHV-1 and BoHV-5 amplicons from standard strains were confirmed by sequence analysis. All the positive clinical samples for BoHV included in this study were characterized as BoHV-1 or BoHV-5 by the difference in length of the amplified product visualized in a agarose gel (354 bp size for BoHV-1, and 159 bp for BoHV-5). The internal control was amplified in all clinical specimens. Non-specific reactions were not observed when the multiplex-PCR was assessed with other viruses (bovine viral diarrhea virus and rabies virus) and BoHV-negative clinical samples from fetuses and adult cattle obtained from a slaughterhouse.

Production and characterization of monoclonal antibodies to a Brazilian bovine herpesvirus type 5.

Antigens of a bovine herpesvirus type 5 (BHV-5), isolated from a cow with a neurological infection in Rio Grande do Sul State, Brazil, were used to immunize BALB/c mice to produce monoclonal antibodies (mAbs). Eleven hybridomas secreting mAbs directed at BHV-5 antigens were obtained after two fusions and screening of 356 hypoxanthine-aminopterin-thymidine-resistant clones. The mAbs reacted at dilutions up to 1:500 (hybridoma culture supernatant) and up to >1:10,000 (ascitic fluid) in an indirect fluorescent antibody assay (IFA) and in immunoperoxidase staining of BHV-5-infected cells. Four mAbs (1D12, 2E2, 2G10 and 4E4) showed virus-neutralizing activity against the parental BHV-5 isolate. Five mAbs (1F3, 2A6, 2F9, 2G10 and HB24L) reacted in Western immunoblotting with a protein of approximately 90 kDa. Three other mAbs (2E2, 3D6 and 4E4) reacted in IFA with antigens of a BHV-1 mutant glycoprotein C- negative strain, demonstrating that they are directed at a viral antigen other than glycoprotein C. The eleven mAbs tested reacted with 20 BHV-5 field isolates and nine mAbs reacted with 10 BHV-1 isolates. Two mAbs (1F3 and 2F9) failed to react with BHV-1 field isolates, although they displayed a weak and nonreproducible reaction with the BHV-1 reference strain Los Angeles. These mAbs may be very useful in distinguishing between BHV-1 and BHV-5 infections since most of the traditional reagents and techniques are unable to do so. One mAb (2F9) was shown to bind to viral antigens by immunohistochemistry of histological sections of the brain of a BHV-5-infected calf. These results demonstrate that the mAbs produced here are suitable for use in a variety of immunological techniques and therefore may be useful for diagnostic and research purposes.

Restriction endonuclease and monoclonal antibody analysis of Brazilian isolates of bovine herpesviruses types 1 and 5.

Twelve Brazilian isolates and three reference strains of bovine herpesviruses (BHVs) were subjected to restriction endonuclease analysis (REA) and monoclonal antibody (MAb) analysis. Viral DNA was cleaved with BamHI, BstEII, EcoRI, HindIII and PstI. The monoclonal antibody panel allowed the differentiation between types 1 and 5 viruses, while REA with BstEII and HindIII showed the distinction between BHV-1 and -5 subtypes. Typical 1.1 and 1.2a patterns were observed with two isolates from respiratory disease. An isolate from semen of a clinically healthy bull displayed 1.2b profile, whereas another displayed a clear 5a pattern, which was never reported before. Seven out of nine Brazilian type 5 (BHV-5) isolates displayed REA patterns similar to the Australian BHV-5 strain N569 (BHV-5a), and differing from the Argentinean A663 strain (BHV-5b) virus. Another two BHV-5 isolates, which displayed an unusual MAb pattern of reactivity, showed a BstEII profile different from both reference strains of BHV-5. These two viruses were considered BHV-5 "non-a/non-b" subtype.

Development of a multiplex polymerase chain reaction for the differentiation of bovine herpesvirus-1 and -5.

Bovine herpesvirus-1 (BHV-1) and bovine herpesvirus-5 (BHV-5) are closely related viruses which exhibit some important differences at the genetic and immunogenic levels which may explain the differences in their pathogenicity and epidemiological characteristics. A multiplex polymerase chain reaction (M-PCR) was developed to detect and differentiate between BHV-1 and BHV-5. In this M-PCR two pairs of primers (TK1, TK2 and GD1, GD2) were used in the same reaction mix to amplify a thymidine kinase genomic region (183 bp) of BHV-1 and one genomic region of the gLycoprotein D (564 bp) of BHV-5. The specificity of the M-PCR was demonstrated when using both primers pairs simultaneously with BHV-1 and BHV-5 templates. The two expected bands were amplified without the apparition of non-specific products. However, when other herpesvirus strains were used, there was no amplification. To evaluate the sensitivity of the assay, dilutions of purified viral DNA were made for M-PCR amplification. The detection limit was 7 pg for BHV-1 and 22 pg for BHV-5. It was also determined by comparing the M-PCR with viral isolation. M-PCR was able to detect one log10 more than viral isolation for BHV-1 and for BHV-5 was two logarithms lower. The applicability of M-PCR was demonstrated on different specimens. Twenty isolates from field samples (11 BHV-1 and nine BHV-5) were positive by M-PCR, and the results were completely coincident with previous characterization using the immunoperoxidase assay. M-PCR could detect viral DNA in organ samples from natural infections, such as semen and brain. In addition, M-PCR detected more positive samples than observation of the citophatic effect in cell culture of nasal swabs from experimentally infected animals in two different assays. Owing to the difference in size of the M-PCR products which allows easy identification in an electrophoretic run, it is not necessary to use extra blotting and hybridization steps or a second round of amplification to differentiate clearly between BHV-1 and BHV-5.

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in Argentina.

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.

Study of neutralizing monoclonal antibodies to bovine herpes virus type-1 (Cooper strain) by immunoperoxidase and immunoelectron microscopy.

Three neutralizing monoclonal antibodies (mAbs) that are specific against bovine herpes virus Type-1 (BHV-1) were studied as to their viral specificity by immunoperoxidase and immunoelectron microscopy. Microscopic examination of GBK BHV-1 infected cells revealed peroxidase activity represented by red-brown granular deposits in the nucleus and cytoplasm. No immunoperoxidase activity was observed in negative controls. For the ultrastructural observations, two approaches were used. Firstly we tested a pre-embedding technique using GBK infected cells, mAbs and gold conjugated-protein A. Gold particles were observed linked to the viral envelopes and to the host cell membrane. Alternatively, a second technique employed BHV-1 purified by potassium tartrate gradients, mAbs and gold conjugated-protein A. After performing the immune reaction, the samples were adsorbed to formvar-coated grids, stained with phosphotungstic acid and observed in a transmission electron microscope. Gold particles were mainly attached to the virion envelope.

A comparison of restrictive endonuclease sites of bovine herpesvirus type 1 isolates in Chile.

The Frutillar 82, F nasal 90, F vaginal 90, Puente Alto 77, VMLB 79, and Bio Bio 92 isolates of bovine herpesvirus 1 (BHV-1) from Chile were analysed by Eco RI, Bam HI and Hind III restriction enzymes (RE). The Los Angeles (LA) strain was used as reference. The results indicated that the fingerprints of F nasal 90 and F vaginal 90 were identical to the three RE used, but they were different from the other isolates. Major differences were found for Puente Alto 77 and VMLB 79 isolates with Eco RI, and for Puente Alto 77 with Hind III compared with the other Chilean isolates. It is concluded that Chilean BHV-1 isolates obtained from diverse lesions and geographic locations are genomically different from each other and from the other strains described in the literature.

Molecular characterization of South American bovine herpesvirus-1 isolates with monoclonal antibodies and SDS-PAGE.

Antigenic patterns of BHV-1 isolates from Argentina, Chile and Brazil were examined with a panel of monoclonal antibodies (MAbs) generated with BHV-1.1 and BHV-1.3 strains. Among the Argentinian isolates, 7/8 neurological cases showed BHV-1.3 characteristics and 10/12 non-neurological cases from all countries exhibited BHV-1.1/2 pattern. All viral isolates from genital infections showed BHV-1.1/2 characteristics. Chilean isolates exhibited BHV-1.1/2 pattern and one among 3 Brazilian viruses was defined as belonging to BHV-1.3. Results of the characterization using MAbs, were confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of representative isolates. A total correlation was observed between the reactivity with MAbs and the SDS-polyacrylamide analysis. Based on the data presented, it is concluded that the type 1.3, most frequently obtained from the brain of neurological cases, is present in Argentina and Brazil and coexists with BHV-1.1/2.