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Equine herpesvirus type 1 (EHV-1) UL11 is a 74-amino-acid tegument protein encoded by ORF51 of the EHV-1 genome. EHV-1 UL11 was previously reported by other researchers using the RacL22 and RacH strains to be nonessential for viral replication in cultured cells. Here, we constructed UL11 mutant viruses including a UL11 null mutant and three C-terminal truncated mutants, for further characterization of EHV-1 UL11 using bacterial artificial chromosome (BAC) technology based on the neuropathogenic strain Ab4p. EHV-1 Ab4p UL11 was localized to juxtanuclear and Golgi regions as reported by other researchers. We found that no progeny viruses were produced by transfection of fetal equine kidney cells and rabbit kidney (RK-13) cells with the UL11 null mutant and truncation mutant BAC DNAs. However, mutant viruses were generated after transfection of RK13-UL11 cells constitutively expressing EHV-1 UL11 with the mutant BAC DNAs. In conclusion, UL11 of EHV-1 Ab4p is essential for replication in cultured cells.

Viruses related to equine herpesvirus type 1 (EHV-1) were isolated from an aborted fetus of an onager (Equus hemionus) in 1984, an aborted fetus of Grevy's zebra (Equus grevyi) in 1984 and a Thomson's gazelle (Gazella thomsoni) with nonsuppurative encephalitis in 1996, all in the USA. The mother of the onager fetus and the gazelle were kept near plains zebras (Equus burchelli). In phylogenetic trees based on the nucleotide sequences of the genes for glycoproteins B (gB), I (gI), and E (gE), and teguments including ORF8 (UL51), ORF15 (UL45), and ORF68 (US2), the onager, Grevy's zebra and gazelle isolates formed a genetic group that was different from several horse EHV-1 isolates. Within this group, the onager and gazelle isolates were closely related, while the Grevy's zebra isolate was distantly related to these two isolates. The epizootiological origin of the viruses is discussed.

Infectious bursal disease virus (IBDV) capsid protein, VP2, contains a hypervariable domain that is recognized by virus-neutralizing antibodies. The virus-neutralizing epitope is highly conformation-dependent and the domain is speculated to be involved in the virus-target cell interaction. In this study, a polyclonal anti-idiotypic antibody (anti-id) was generated by the sequential immunization of a rabbit with a virus-neutralizing monoclonal antibody GI-11 which recognizes the VP2 hypervariable domain. Although the anti-id, which mimics the conformational epitope in the VP2 hypervariable domain, was expected to inhibit the virus infection, the anti-id did not interfere with either the virus binding or the infection to the target cell.

A flow cytometric virus binding assay that directly visualizes the binding of infectious bursal disease virus (IBDV) to its target cells was established. The chicken B lymphoblastoid cell line, LSCC-BK3, which is permissive for IBDV infection, bound high levels of the virus. Another B lymphoblastoid cell line, LSCC-1104-B1, bound low levels of the virus, although it was nonpermissive. No virus binding was detected in nonpermissive T lymphoblastoid cell lines. In the binding assay to heterogeneous cell populations of chicken lymphocytes, IBDV (a highly virulent OKYM strain) bound to 94% cells in the lymphocytes prepared from the bursa of Fabricius, 37% cells in those prepared from the spleen, 3% cells in those prepared from the thymus, and 21% cells in those prepared from the blood. Most of the cells, which bound the virus, were surface immunoglobulin M (SIgM)-positive, but a small number of them were SIgM-negative. Additionally, the binding of IBDV to the LSCC-BK3 cells was affected by treatment of the cells with proteases and N-glycosylation inhibitors. These findings may indicate that the IBDV host range is mainly controlled by the presence of a virus receptor composed of N-glycosylated protein associated with the subtle differentiation stage of B-lymphocytes represented mostly by SIgM-bearing cells.

Twenty hybridoma cell lines producing monoclonal antibodies (MAbs) against serotype 1 infectious bursal disease virus (IBDV) of GBF-1 and the attenuated GBF-1E strains were produced. The MAbs recognized major structural proteins VP2 and VP3. MAb recognition sites were mapped using recombinant Escherichia coli clones which expressed N-terminal and (or) C-terminal truncated virus antigens, and competitive-binding assays. At least 3 conformation-dependent serotype 1 specific virus neutralizing antigenic sites and a linear antigenic site were defined on VP2 and VP3, respectively. Two of the conformational virus neutralizing antigenic sites were localized in the central area of VP2 consisting of 156 amino acid residues, and the linear epitope was localized in C-terminal 105 amino acid residues of VP3. Another conformational virus neutralizing antigenic site recognized with the virus neutralizing MAb GK-5 was not defined because GK-5 did not react with virus antigen expressed in recombinant E. coli. The conformational antigenic site was supposed to be composed of tertiary or quaternary protein structure, which may not be constructed in recombinant E. coli.

We determined the complete nucleotide sequence of an infectious bursal disease (IBD) virus (IBDV) isolate (designated KZC-104) from a confirmed IBD outbreak in Lusaka in 2004. The genome consisted of 3,074 and 2,651 nucleotides in the coding regions of segments A and B, respectively. Alignment of both nucleotide and deduced amino acid sequences and phylogenetic analysis revealed that the genome segment A of KZC-104 was derived from a very virulent (VV) strain, whereas its segment B was derived from a classical attenuated strain. On BLAST search, the full-length segment A and B sequences showed 98 % nucleotide sequence identity to the VV strain D6948 and 99.8 % nucleotide sequence identity to the classical attenuated strain D78. This is a unique IBDV reassortant strain that has emerged in nature, involving segment B of a cell-culture-adapted attenuated vaccine.

Nucleotide sequences of the VP2 hypervariable region (VP2-HVR) of 14 infectious bursal disease viruses (IBDVs) isolated in Tanzania from 2001 to 2004 were determined. Phylogenetic analysis showed that the isolates diverged into two genotypes and belonged to the very virulent (VV) type. In the phylogenetic tree, strains in one genotype clustered in a distinct group and were closely related to some strains isolated in western Africa, with nucleotide similarities of 96.1–96.8%, while strains in another genotype were clustered within the European/Asian VV type with nucleotide similarities ranging from 97.5 to 99.3%. Both genotypes were widely distributed throughout Tanzania, and had conserved putative virulence marker amino acids (aa) at positions 222(A), 242(I), 256(I), 294(I) and 299(S). Our findings demonstrate for the first time the existence of both African and European/Asian VV-IBDV variants in Tanzania.

Equine herpesvirus 1 was isolated from an onager in 1985, a zebra in 1986 and a Thomson's gazelle in 1996 in USA. The genetic relatedness and pathogenicity of these three viruses were investigated based on the nucleotide sequences of the glycoprotein G (gG) gene, experimental infection in hamsters, and comparison with horse isolates. The gG gene sequences of EHV-1 from onager and zebra were identical. The gG gene sequences of the gazelle isolate showed 99.5% identity to those of onager and zebra isolates. The gG gene sequences of EHV-1 isolated from horses were 99.9-100% identical and 98, 98 and 97.8% similar to gG from onager, zebra and gazelle isolates, respectively. Hamsters inoculated with onager, zebra and gazelle isolates had severe weight loss, compared with hamsters inoculated with horse isolates. The histopathological findings were related to the virulence of each isolate. The results indicated that EHV-1 isolates from onager, zebra and gazelle differ from horse EHV-1 and are much more virulent in hamsters.

The pathogenicity of a new neurotropic equine herpesvirus 9 (EHV-9) formerly designated gazelle herpesvirus 1 was evaluated using the goat as a representative of domesticated ruminants. Two goats inoculated intranasally with EHV-9 showed salivation, teeth grinding and other neurological disorders on day 8 post inoculation. One goat died 30 min after the onset of clinical signs and the other was sacrificed 3 h after the sudden onset of teeth grinding and foamy salivation. EHV-9 was recovered from peripheral white blood cells, the olfactory bulbs and brain, nasal swabs, concha, and lungs. Neuropathological lesions were located in the olfactory bulbs, cerebrum, midbrain and medulla oblongata with degeneration and necrosis of neurons, rarefaction, perivascular infiltration of mononuclear cells, and nodal glial reaction. EHV-9 antigen was detected in neurons in the lesions. These findings indicated that EHV-9 is highly pathogenic with high neurotropism for goats.

The nucleotide sequences of the genome segments A and B encoding the precursor polyprotein (NH2-VP2-VP4-VP3-COOH) and VP1 were determined for a highly virulent strain of infectious bursal disease virus (IBDV). The precursor polyprotein and VP1 coding regions of highly virulent OKYM strain consisted of 3039 nucleotides (1012 deduced amino acids) and 2640 nucleotides (879 deduced amino acids), respectively. Comparison of the deduced amino acid sequences of the highly virulent IBDV (HV-IBDV) with other serotype 1 and 2 sequences revealed 17 amino acid residues which were conserved only in the HV-IBDV. Among the 17 unique amino acid differences, 8 were in VP1, 4 were in VP2, 3 were in VP3 and 2 were in VP4. Although it is impossible to predict the effect of the unique amino acid residues without detailed knowledge of the three-dimensional structure and function of the proteins, they could affect the virulence of HV-IBDV. Alignment of the nucleic acid sequences of precursor polyprotein, VP1, VP2, VP3 and VP4 coding regions followed by distance analysis allowed the generation of phylogenetic trees. The same tree topology was obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. On the other hand, the tree topology of VP1 was quite different from that obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. These findings indicate that not a genetic recombination but a genetic reassortment may play an important role in the emergence of HV-IBDV.

Twenty five H4 avian influenza viruses of different origin were divided into 4 groups based on the reactivity patterns in hemagglutination-inhibition tests with 25 monoclonal antibodies prepared to two H4 influenza viruses of budgerigar and duck origin.