The capacity of UL49.5 proteins to inhibit TAP is widely distributed among members of the genus Varicellovirus.

The lifelong infection by varicelloviruses is characterized by a fine balance between the host immune response and immune evasion strategies used by these viruses. Virus-derived peptides are presented to cytotoxic T lymphocytes by major histocompatibility complex (MHC) class I molecules. The transporter associated with antigen processing (TAP) transports the peptides from the cytosol into the endoplasmic reticulum, where the loading of MHC-I molecules occurs. The varicelloviruses bovine herpesvirus 1 (BoHV-1), pseudorabies virus, and equid herpesviruses 1 and 4 have been found to encode a UL49.5 protein that inhibits TAP-mediated peptide transport. To investigate to what extent UL49.5-mediated TAP inhibition is conserved within the family of Alphaherpesvirinae, the homologs of another five varicelloviruses, one mardivirus, and one iltovirus were studied. The UL49.5 proteins of BoHV-5, bubaline herpesvirus 1, cervid herpesvirus 1, and felid herpesvirus 1 were identified as potent TAP inhibitors. The varicella-zoster virus and simian varicellovirus UL49.5 proteins fail to block TAP; this is not due to the absence of viral cofactors that might assist in this process, since cells infected with these viruses did not show reduced TAP function either. The UL49.5 homologs of the mardivirus Marek's disease virus 1 and the iltovirus infectious laryngotracheitis virus did not block TAP, suggesting that the capacity to inhibit TAP via UL49.5 has been acquired by varicelloviruses only. A phylogenetic analysis of viruses that inhibit TAP through their UL49.5 proteins reveals an interesting hereditary pattern, pointing toward the presence of this capacity in defined clades within the genus Varicellovirus.

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP.

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I-restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL 49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL 49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL 49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL 49.5 proteins block TAP as well, these data indicate that UL 49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL 49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL 49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL 49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL 49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL 49.5. Taken together, these results classify the UL 49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.

Development of a polymerase chain reaction for the detection of Anguillid herpesvirus DNA in eels based on the herpesvirus DNA polymerase gene.

Anguillid herpesvirus (AnHV, also known as Herpesvirus anguillae or HVA) is found in both Japanese and European eels. Based on restriction enzyme analysis a small number of differences were found between AnHV isolated from Japanese eels and from European eels. The total genome size of both is about 245 kb, which is confirmed by alternating-field electrophoresis. Using a set of degenerate primers based on conserved regions within DNA-directed DNA polymerase coding regions, a 463 base pair fragment was isolated from both Japanese and European AnHV. Nucleotide sequence analysis showed that the cloned regions of both viruses have identical sequences. Based on this part of the DNA-polymerase sequence, primers were selected and used to develop a sensitive PCR to detect AnHV DNA in eel tissue samples. To avoid false negative results and to estimate the number of AnHV genome copies found in tissues, 100 copies of an internal control plasmid were added to the tissue samples. This semi-quantitative AnHV PCR can be used for both the European and Japanese isolates of AnHV, detects as few as 10 genome copies and is 100 times more sensitive than standard virus isolation.

A monoclonal antibody-based ELISA allows discrimination between responses induced by bovine herpesvirus subtypes 1 (BoHV-1.1) and 2 (BoHV-1.2).

Bovine herpesvirus type 1 (BoHV-1) has distinct subtypes according to genomic characterization. Immune responses induced by BoHV-1 subtype 1 (BoHV-1.1) are not distinguishable from those induced by BoHV-1 subtype 2 (BoHV-1.2) through conventional serological methods. In the present report, an enzyme linked immunosorbent assay is described that allows discrimination between immune responses in cattle immunized with either subtype, based on a monoclonal antibody that recognizes specifically the amino-terminal region of glycoprotein C (gC) on BoHV-1.1 strains, thus not reacting with BoHV-1.2a. The test displayed a sensitivity of 92%, specificity of 90% and a good correlation with serum neutralization tests on samples from BoHV-1.1-immunized calves (kappa = 0.799). The test may be useful to provide new insights into the roles played by each of these two subtypes in the epidemiology of BoHV-1 infections.

Variants of the recently discovered avian gyrovirus 2 are detected in Southern Brazil and The Netherlands.

A genome of a virus preliminarily named avian gyrovirus 2 (AGV2), a close relative to chicken anemia virus, was recently discovered in a chicken in the state of Rio Grande do Sul, Southern Brazil. To study the occurrence of AGV2 in Rio Grande do Sul and the neighboring state Santa Catarina, a number of adult chickens (n=108 and n=48, respectively) were tested for the presence of AGV2 DNA. An AGV2-specific PCR was developed, optimized and used to analyze DNA extracted from clinical samples. AGV2 DNA was detected in 98/108 (90.7%) of samples collected in the state of Rio Grande do Sul and 29/48 (60.4%) of the samples collected in the state of Santa Catarina. In order to check whether AGV2 DNA would be detected in samples from a geographically distant region, DNA from brain samples of 21 diseased chickens from the Netherlands were tested independently, by the same method. In such specimens, 9/21 (42.9%) brain tissue samples were found to contain AVG2 DNA. Sequence analysis of some of the PCR products demonstrated that the amplified AGV2 sequences could vary up to 15.8% and could preliminarily be divided in three groups. This indicated the occurrence of variants of AGV2, which may reflect differences in geographical origin and/or in biological properties. The data presented here provides evidence that AGV2 seems fairly distributed in chickens in Southern Brazil and that AGV2 also circulates in the Netherlands. Besides, circulating viruses display genetic variants whose significance should be further examined, particularly to determine whether AGV2 would play any role in chicken diseases.

Haemonchus contortus: characterization of the baculovirus expressed form of aminopeptidase H11.

Recombinant form of Haemonchus contortus aminopeptidase H11, an intestinal membrane glycoprotein considered to be in its native form the most promising vaccine candidate, was produced in insect cells, characterised and tested in pilot vaccination-challenge trial on sheep. The sequence of the cloned gene, obtained by RT PCR isolated from adult worms, showed 97% identity to the highly immunogenic H11 clone, described by Graham et al., (database accession number AJ249941.1). A 1305 bp fragment of H11 was expressed in E. coli and used to raise a specific antiserum, which recognized recombinant forms of H11 and 110 kDa protein from H. contortus extract. H11 was expressed by baculovirus recombinants in insect cells in full length and as a fusion protein with H. contortus glutathione S-transferase (GST). The baculovirus produced recombinant antigens were used without adjuvants to immunize sheep, which resulted in 30% (full length H11) and 20% (GST-H11) reduction of worm burden. These animal experiments indicated that, although the protection induced by in vitro produced protein is lower than in case of H11 isolated from worms, recombinant forms of aminopeptidase may be considered as antigens for the control of haemonchosis.

Vaccination with a gE-negative bovine herpesvirus type 1 vaccine confers insufficient protection to a bovine herpesvirus type 5 challenge.

In the present study, cross-protection to bovine herpesvirus type 5 (BHV-5) induced by bovine herpesvirus type 1 (BHV-1) vaccination was examined following inoculation of rabbits and calves with a glycoprotein E (gE)-negative BHV-1 vaccine and subsequent challenge with BHV-5. Rabbits (n=5) and calves (n=8) were vaccinated [five rabbits intranasally (IN), four calves IN and four intramuscularly (IM)] with 7.1 log(10)median tissue culture infective dose (TCID(50)) of the BHV-1 vaccine. Rabbits and calves were challenged IN [rabbits 2 weeks post-vaccination (pv); calves 5 weeks pv] with 9.1log(10)TCID(50) of BHV-5. Two out of five vaccinated rabbits died after challenge with typical BHV-5 disease, as did 3/5 non-vaccinated controls. In calves, 4/8 vaccinated animals displayed mild signs of disease, whereas 6/6 non-vaccinated controls developed signs of disease, so severe that 2/6 had to be killed. Besides, nasal virus shedding post-challenge was not reduced by vaccination. At necropsy, on day 21 post-challenge, typical BHV-5 lesions were evident in brain tissues of both vaccinated and non-vaccinated calves. Dexametasone administration at 180 days post-infection did not reactivate clinical signs despite BHV-5 shedding in nasal secretions of both vaccinated and non-vaccinated calves. These results show that the BHV-1 vaccine evaluated here did not confer protection to BHV-5 in rabbits. In calves, BHV-1 vaccination did confer some protection to BHV-5 induced clinical disease, but it did not prevent infection and had no effect on nasal virus shedding or on the development of encephalitic lesions.

Bovine herpesvirus 1 UL49.5 protein inhibits the transporter associated with antigen processing despite complex formation with glycoprotein M.

Bovine herpesvirus 1 (BHV-1) interferes with peptide translocation by the transporter associated with antigen processing (TAP). Recently, the UL49.5 gene product of BHV-1 was identified as the protein responsible for the observed inhibition of TAP. In BHV-1-infected cells and virions, the UL49.5 protein forms a complex with glycoprotein M (gM). Hence, it was investigated whether UL49.5 can combine the interactions with gM and the TAP complex. In cell lines constitutively expressing both UL49.5 and gM, UL49.5 appears to be required for functional processing of gM. Immunofluorescence-confocal laser scanning microscopy demonstrated that both proteins are interdependent for their redistribution from the endoplasmic reticulum to the trans-Golgi network. Remarkably, expression of cloned gM results in the abrogation of the UL49.5-mediated inhibition of TAP and prevents the degradation of the transporter. However, in BHV-1-infected cells, differences in UL49.5 and gM expression kinetics were seen to create a window of opportunity at the early stages of infection, during which time the UL49.5 protein can act on TAP without gM interference. Moreover, in later periods, non-gM-associated UL49.5 can be detected in addition to the UL49.5/gM complex. Thus, it has been deduced that different functions of UL49.5, editing of gM processing and inhibition of TAP, can be combined during BHV-1 infection.

Fasciola hepatica procathepsin L3 protein expressed by a baculovirus recombinant can partly protect rats against fasciolosis.

Fasciola hepatica juveniles express immunodominant cathepsin L proteins, which are mainly found in their immature, procathepsin form. A gene encoding such a procathepsin L (FheCL3) was expressed by a baculovirus recombinant and by Saccharomyces cerevisiae. The glycosylated FheCL3 proteins obtained by both systems were used in a vaccination/challenge experiment in rats. Both antigens evoked similar antibody responses, but only the baculovirus expressed FheCL3 caused a significant protection against the number of liver flukes (52% protection, P=0.01), whereas the S. cerevisiae expressed FheCL3 did not. In a second experiment in rats, deglycosylated versions of both antigens were used, but this did not improve their efficacies.

Varicelloviruses avoid T cell recognition by UL49.5-mediated inactivation of the transporter associated with antigen processing.

Detection and elimination of virus-infected cells by cytotoxic T lymphocytes depends on recognition of virus-derived peptides presented by MHC class I molecules. A critical step in this process is the translocation of peptides from the cytoplasm into the endoplasmic reticulum by the transporter associated with antigen processing (TAP). Here, we identified the bovine herpesvirus 1-encoded UL49.5 protein as a potent inhibitor of TAP. The expression of UL49.5 results in down-regulation of MHC class I molecules at the cell surface and inhibits detection and lysis of the cells by cytotoxic T lymphocytes. UL49.5 homologs encoded by two other varicelloviruses, pseudorabies-virus and equine herpesvirus 1, also block TAP. Homologs of UL49.5 are widely present in herpesviruses, acting as interaction partners for glycoprotein M, but in several varicelloviruses UL49.5 has uniquely evolved additional functions that mediate its participation in TAP inhibition. Inactivation of TAP by UL49.5 involves two events: inhibition of peptide transport through a conformational arrest of the transporter and degradation of TAP by proteasomes. UL49.5 is degraded along with TAP via a reaction that requires the cytoplasmic tail of UL49.5. Thus, UL49.5 represents a unique immune evasion protein that inactivates TAP through a unique two-tiered process.

Vaccination of pigs with a DNA construct expressing an influenza virus M2-nucleoprotein fusion protein exacerbates disease after challenge with influenza A virus.

In mice, vaccines inducing antibodies to the extracellular domain of the M2 protein (M2e) can confer protection to influenza A virus infection. Unlike the surface glycoproteins, haemagglutinin and neuraminidase, this domain of M2 is highly conserved and is therefore a potential broad-spectrum immunogen. In this study, the protection conferred by vaccines inducing antibodies to M2e was evaluated in a challenge model for swine influenza in pigs. A protein resulting from the fusion between M2e and the hepatitis B virus core protein (M2eHBc), with or without adjuvant, was evaluated. In addition, a DNA construct expressing a fusion protein between M2e and influenza virus nucleoprotein (M2eNP) was evaluated to see if the broad-spectrum protection conferred by antibodies could be further enhanced by T helper cells and cytotoxic T cells. All vaccines induced an antibody response against M2e, and the M2eNP DNA vaccine additionally induced an influenza virus-specific lymphoproliferation response. However, after challenge with a swine influenza virus (H1N1), no protection was observed in the vaccinated groups compared with the non-vaccinated control group. On the contrary, vaccinated pigs showed more severe clinical signs than the control pigs. The M2eNP DNA-vaccinated pigs showed the most severe clinical signs and three out of six pigs died on days 1 and 2 post-challenge. These results indicate that antibodies to M2e, especially in combination with cell-mediated immune responses, exacerbate disease. Thus, clinical signs after infection should be observed closely in further studies using M2e as an immunogen and caution should be exercised in using M2e in humans.

Binding of hematin by a new class of glutathione transferase from the blood-feeding parasitic nematode Haemonchus contortus.

The phase II detoxification system glutathione transferase (GST) is associated with the establishment of parasitic nematode infections within the gastrointestinal environment of the mammalian host. We report the functional analysis of a GST from an important worldwide parasitic nematode of small ruminants, Haemonchus contortus. This GST shows limited activity with a range of classical GST substrates but effectively binds hematin. The high-affinity binding site for hematin was not present in the GST showing the most identity, CE07055 from the free-living nematode Caenorhabditis elegans. This finding suggests that the high-affinity binding of hematin may represent a parasite adaptation to blood or tissue feeding from the host.

The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1.

The virion host shutoff (vhs) protein of alphaherpesviruses causes a rapid shutoff of host cell protein synthesis. We constructed a bovine herpesvirus 1 (BHV1) deletion mutant in which the putative vhs gene, UL41, has been disrupted. Whereas protein synthesis is inhibited within 3 h after infection with wild-type BHV1, no inhibition was observed after infection with the BHV1(vhs-) deletion mutant. These results indicate that the BHV1 UL41 gene product is both necessary and sufficient for shutoff of host cell protein synthesis at early times post-infection. Using the vhs deletion mutant, we investigated the mechanism of BHV1-induced down-regulation of MHC class I cell surface expression. In contrast to BHV1 wild-type infection, the BHV1(vhs-) mutant allows detection of MHC class I molecules at much later time-points after infection. This illustrates the role the vhs protein plays in MHC class I down-regulation. However, even after infection with BHV1(vhs-), MHC class I cell surface expression is impaired. In BHV1(vhs-)-infected cells, MHC class I molecules are retained within the endoplasmic reticulum (ER). Moreover, the transporter associated with antigen presentation (TAP) is still blocked. Temporal control of viral protein expression using chemical inhibitors shows that viral protein(s) expressed within the early phase of BHV1 infection are responsible for ER retention of MHC class I molecules. These results indicate that multiple mechanisms are responsible for down-regulation of MHC class I molecules in BHV1-infected cells.

Caracterização antigênica e molecular de oito amostras do vírus da doença de Aujeszky isoladas no estado do Rio Grande do Sul em 2003 / Antigenic and molecular characterization of eight samples of Aujeszky's disease virus isolated in the state of Rio Grande do Sul, Brazil, in 2003

A doença de Aujeszky ou pseudoraiva (DA), causada pelo vírus da pseudoraiva (PRV) é a maior preocupação na produção de suínos. No estado do Rio Grande do Sul, Brasil, a DA foi somente detectada em 1954, em bovino. Em 2003, ocorreram dois surtos de encefalite em granjas na região norte do estado, fronteira com o estado de Santa Catarina. O vírus da doença de Aujeszky (VDA) foi isolado a partir de animais coletados em oito granjas distintas da região e submetido a análises antigênicas e moleculares. As amostras de VDA isoladas foram comparadas com as amostras padrão NIA-3 e NP. A caracterização antigênica dos mesmos foi realizada com testes de imunoperoxidase frente a um painel de anticorpos mono-clonais (Mabs) preparado contra epitopos de glicoproteinas virais (gB, gC, gD e gE). A caracterização genômica foi realizada através da análise restrição enzimática (REA) sobre o genoma total das amostras, com a enzima de restrição (REA) Bam HI. O perfil antigênico das oito amostras isoladas no Rio Grande do Sul, bem como os apresentados pelas amostras padrão NIA-3 e NP, foram similares. A REA revelou que todos as oito amostras do Rio Grande do Sul apresentaram um arranjo genômico do tipo II, genótipo frequentemente encontrado em surtos prévios de DA em outros estados do Brasil. Os resultados aqui obtidos indicam que as oito amostras isoladas no Rio Grande do Sul são similares.

Construction and characterization of a glycoprotein E deletion mutant of bovine herpesvirus type 1.2 strain isolated in Brazil

This paper describes the construction and characterization of a Brazilian strain of bovine herpesvirus type 1.2a (BoHV-1.2a) with a deletion of the glycoprotein E (gE) gene. The deletion was introduced by co-transfection of a deletion fragment containing the 5 and 3 gE flanking regions and genomic DNA of wild type BoHV-1 into bovine cells. Isolation of gE deletion mutant was performed by immunoperoxidase staining with an anti-gE monoclonal antibody. Viral clones were plaque purified and further examined by restriction endonuclesase digestion and Southern blot hybridization. This gE deletion mutant will be evaluated as a vaccinal virus, in order to determine its potential use for a differential vaccine

In vitro characterization of gE negative bovine herpesvirus types 1.1 (BHV-1.1) and 1.2a (BHV-1.2a)

O presente estudo teve como objetivo a caracterização das propriedades de crescimento in vitro de uma amostra brasileira de herpesvírus bovino tipo 1.2a que apresenta uma deleção no gene que codifica a glicoproteína E (BHV-1.2a gE-). Os tamanhos de placa, cinética de penetração e cinética de multiplicação do vírus BHV-1.2a gE- foram estudados e comparados com o vírus parental, bem como com um vírus BHV-1.1 gE- recombinante, o qual é derivado de uma amostra européia de BHV-1.1. Em termos de cinética de penetração, não foram observadas diferenças significativas quando comparados os vírus gE- com os parentais. A determinação da cinética de multiplicação não demonstrou diferenças significativas entre os quatro vírus estudados. Foi entretanto observado que 11 horas pós infecção os dois vírus gE- foram excretados das células em títulos significativamente maiores do que os vírus parentais. Não foram observadas diferenças significativas quando comparados os diâmetros de placas formadas pelos dois vírus parentais. Da mesma forma, os diâmetros de placas dos vírus gE- foram semelhantes nos três tipos celulares estudados. Entretanto, a comparação dos diâmetros de placas entre os vírus gE- e os parentais mostrou uma redução significativa das placas dos vírus gE- em todos os tipos celulares. Esta característica indica que a falta da gE teve o mesmo efeito em ambos os subtipos de BHV-1, representado por uma disseminação viral célula-célula reduzida.