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Background CD4+ recent thymic emigrants (CD4+ RTEs) constitute a subset of T cells recently generated in the thymus and exported into peripheral blood. CD4+ RTEs have increased copy numbers of T-cell receptor excision circles (TREC). They are characterized by the expression of CD31 on naive CD4 T-cells. We aimed to validate a flow-cytometry assay to enumerate CD4+ RTEs and assess its performance in relation to TREC measurement. Methods CD4+ RTEs cell count in peripheral blood was measured to determine sample stability, precision, linearity, and to establish reference ranges. TRECs were measured using qPCR assay performed with DNA isolated from peripheral blood. CD4+ RTEs, TRECs, and flow cytometry results for major T-cell markers were assessed in 50 infants less than 2 years of age. Results Inter-and intra-assay precisions (% CV) were 1.5–12.2 and 1.5–7.0, respectively. Linearity studies showed that the results are linear over a range of 0.7 to 403.0 CD4+ RTEs/μL of blood. There was 84% agreement (42 of 50) between CD4+ RTEs and TRECs qualitative results for the infant samples. CD4+ RTEs reference ranges in 17 healthy children was in agreement with published data, while that of the healthy adults were 51–609 cells/μL of blood. Conclusion The validation results provide acceptable measures of the CD4+ RTEs test performance within CAP/CLIA frameworks. CD4+ RTEs and TRECs assays show high agreement in the infant population. The CD4+ RTEs test can be used as a confirmation for the TREC results along with or as an alternative to T-cell phenotyping in infants with repeatedly low TRECs concentrations. © 2016 International Clinical Cytometry Society

Background Mass cytometry can differentiate more channels than conventional flow cytometry. However, for clinical use, standardization and agreement with well-established methods is paramount. We compared mass cytometry to standard clinical flow cytometry. Methods Mass and flow cytometry were performed in parallel on peripheral blood samples from 25 healthy individuals. Antibody staining was performed on the same samples at the same time, and analyzed for granulocyte, monocyte, lymphocyte, T, B, NK, CD4 and CD8 percentages. Validation parameters included comparison to flow cytometry, inter- and intra-assay precision and establishment of reference intervals. Results There was a positive correlation between mass and flow cytometry for the eight populations studied (R2 between 0.26 and 0.97). Slopes of the best-fit lines varied from 0.50 to 1.21 (fluorescence/mass). No significant differences in variance were found (F-test, P > 0.05). However, paired t-tests were significantly different for four of the eight markers (granulocytes, NK cells, T cells and CD4 cells), resulting in different reference intervals. Signal intensities were correlated for monocytes, lymphocytes, T, CD4 and CD8 cells (R2 = 0.41-0.57). The mass cytometry intra-assay precisions were 0.7-8.5% and inter-assay precisions 1.5-13.8%. Conclusion Mass and flow cytometry evaluations of whole blood for major cell populations correlate with similar precision and signal intensity. However, for clinical use, separate reference interval studies are required. Cell population identification should rely on gating strategies that take advantage of the characteristics offered by each method. © 2019 International Clinical Cytometry Society.

A peripheral membrane protein that is interactive with lymphocytic choriomeningitis virus (LCMV) was purified from cells permissive to infection. Tryptic peptides from this protein were determined to be alpha-dystroglycan (alpha-DG). Several strains of LCMV and other arenaviruses, including Lassa fever virus (LFV), Oliveros, and Mobala, bound to purified alpha-DG protein. Soluble alpha-DG blocked both LCMV and LFV infection. Cells bearing a null mutation of the gene encoding DG were resistant to LCMV infection, and reconstitution of DG expression in null mutant cells restored susceptibility to LCMV infection. Thus, alpha-DG is a cellular receptor for both LCMV and LFV.

Shellfish allergy affects approximately 2% of the population and can cause immediate hypersensitivity reactions such as urticaria, swelling, difficulty breathing, and, in some cases, anaphylaxis. Tropomyosin is the major shrimp allergen and binds IgE in two-thirds of patients. A total of 38 shrimp-allergic patients and 20 negative control subjects were recruited and evaluated on the basis of history, skin prick testing, specific immunoglobulin E (IgE) levels, and peripheral blood mononuclear cell proliferation in response to shrimp tropomyosin or shrimp tropomyosin-derived peptides. Of the classically allergic patients by history, 59% tested positive for serum shrimp IgE antibodies. Of patients with shrimp-specific IgE in sera, 70% also had significant IgE levels specific for shrimp tropomyosin. Peripheral blood mononuclear cells from classically shrimp-allergic patients proliferated in a dose-dependent manner in response to to tropomyosin. In addition, a T-cell line derived from a shrimp-allergic patient proliferated specifically in response to tropomyosin-derived peptides. These studies suggest a strategy for immunotherapy using a tropomyosin-derived T-cell epitope vaccination.

Cytomegalovirus (CMV) infection is one of the most important infectious complications of transplantation. Monitoring CMV-specific CD8 T cell immunity is useful for predicting active CMV infection and for directing targeted antiviral therapy. In this study, we examined four basic parameters for validation of CMV-specific tetramer staining and peptide stimulation assays that cover five most frequent HLA class I alleles. We also examined the potential use of CMV-specific CD8+T cell numbers and functional and cytolytic responses in two autologous HSCT recipients treated for multiple myeloma. The coefficient of variation (CV %) of the precision within assays was 3.1−24% for HLA-tetramer staining, 2.5−47% for IFN-γ, and 3.4−59.7% for CD107a/b production upon peptide stimulation. The precision between assays was 5−26% for tetramer staining, 4−24% for IFN-γ, and 5−48% for CD107a/b. The limit of detection was 0.1−0.23 cells/μL of blood for tetramer staining, 0−0.23 cell/μL for IFN-γ, and 0.11−0.98 cells/μL for CD107a/b. The assays were linear and specific. The reference interval with 95% confidence level was 0−18 cells/μL for tetramer staining, 0−2 cells/μL for IFN-γ, and 0–3 cells/μL for CD107a/b. Our results provide acceptable measures of test performance for CMV immune competence assays for the characterization of CD8+T cell responses posttransplant measured in the absolute cell count perμL of blood.

IL-2 provides a memory differentiation signal to CD8+ T cells during the primary response that impacts the ability of the subsequent memory pool to mount a successful recall response. In this study, we find that although primary effector CTL development is modestly decreased in the absence of IL-2, the persistence of short-term and long-term effector memory CD8+ T cells on pathogen clearance is greatly diminished. Furthermore, secondary challenge of CD8+ memory T cells lacking the high-avidity IL-2R results in a failure to repopulate the effector pool. The role of IL-2 in promoting effector differentiation is not shared with the highly related cytokine, IL-15. Although IL-15 supports the survival of effector CD8+ T cells after pathogen clearance, its absence does not impair either primary or secondary effector CTL differentiation, nor does it impact the differentiation of long-term effector memory CD8+ T cells. These findings indicate a unique role for IL-2, but not IL-15, in promoting the differentiation not only of primary effector CD8+ T cells, but also of CD8+ memory T cells capable of secondary effector differentiation.

The parameters controlling the generation of robust CD4+ T cell recall responses remain poorly defined. In this study, we compare recall responses by CD4+ and CD8+ memory T cells following rechallenge. Homologous rechallenge of mice immune to either lymphocytic choriomeningitis virus or Listeria monocytogenes results in robust CD8+ T cell recall responses but poor boosting of CD4+ T cell recall responses in the same host. In contrast, heterologous rechallenge with a pathogen sharing only a CD4+ T cell epitope results in robust boosting of CD4+ T cell recall responses. The disparity in CD4+ and CD8+ T cell recall responses cannot be attributed to competition for growth factors or APCs, as robust CD4+ and CD8+ T cell recall responses can be simultaneously induced following rechallenge with peptide-pulsed dendritic cells. Instead, CD4+ T cell recall responses are dependent on the duration of the secondary challenge. Increasing the rechallenge dose results in more potent boosting of CD4+ T cell recall responses and artificially limiting the duration of secondary infection following heterologous rechallenge adversely impacts the magnitude of CD4+ T cell, but not CD8+ T cell, recall responses. These findings suggest that rapid pathogen clearance by secondary CTL following homologous rechallenge prevents optimal boosting of CD4+ T cell responses and therefore have important practical implications in the design of vaccination and boosting strategies aimed at promoting CD4+ T cell-mediated protection.

Memory T cells exhibit a high degree of heterogeneity in terms of their phenotype and functional characteristics. It has been proposed that the CCR7 chemokine receptor divides memory T cell populations into central memory T cells and effector memory T cells with distinct functions in secondary immune responses. We were interested whether this hypothesis holds true in experiments performed on Ag-specific CD8+ T cells. To identify CCR7+ cells, we engineered a fluorescent ligand for CCR7; results with the new CC chemokine ligand 19 chemotetramer were verified by staining with a CCR7 mAb. Staining with the CC chemokine ligand 19 chemotetramer reveals two subsets within CCR7+ cells: a CCR7int population containing memory cells and a CCR7high population containing naive T cells. Phenotypic analysis of MHC class I/peptide tetramer-positive cells revealed that HLA-A2-restricted CMV-specific CD8 T cells exhibit the lowest percentage of CCR7+ cells (0.5–5%), while HLA-A2-restricted flu- and HLA-B8-restricted EBV-specific CD8 T cells showed the highest (45–70%). Intracellular staining of unstimulated cells revealed that both CCR7int- and CCR7−-specific CD8 T cells exhibit a detectable level of perforin. Both CCR7int and CCR7− Ag-specific CD8+ T cells produced IFN-γ and TNF-α following short-term peptide stimulation. Therefore, our finding that CCR7+CD8+ T cells are able to exert immediate effector functions requires a substantial revision to the central and effector memory hypothesis.

We have investigated the involvement of cytoskeletal proteins in the morphogenesis of Black Creek Canal virus (BCCV), a New World hantavirus. Immunofluorescent staining of BCCV-infected cells revealed a filamentous pattern of virus antigen, the appearance of which was sensitive to treatment with cytochalasin D, an actin microfilament-depolymerizing drug. Double immunofluorescence staining of BCCV-infected Vero cells with anti-BCCV nucleocapsid (N) monoclonal antibody and phalloidin revealed a colocalization of the BCCV N protein with actin microfilaments. A similar, though less prominent, filamentous pattern was observed in BHK21 cells transiently expressing the BCCV N protein alone but not in cells expressing the BCCV G1 and G2 glycoproteins. Moreover, the association of the N protein with actin microfilaments was confirmed by coimmunoprecipitation with β-actin-specific antibody. Treatment of the BCCV-infected Vero cells at 3 days postinfection with cytochalasin D decreased the yield of released BCCV by 94% relative to the yield from untreated cells. Pretreatment of Vero cells with cytochalasin D prior to and during BCCV adsorption and entry had no effect on the outcome of virus production. These results indicate that actin filaments may play an important role in hantavirus assembly and/or release.

Shellfish allergy is an immune-mediated adverse reaction to allergenic shellfish and is responsible for significant morbidity and mortality. CD4 T cell responses play an important role in the pathophysiological mechanisms of sensitization and in production of IgE.We sought to identify and validate CD4 T cell shrimp tropomyosin-derived epitopes and characterize CD4 T cell responses in subjects with a clinical history of shellfish allergy.Using an in vitro MHC-peptide binding assay, we screened 91 overlapping peptides and identified 28 epitopes with moderate and strong binding capacities; 3 additional peptides were included based on MHC binding prediction score. These peptides were then examined in proliferation and cytokine release assays with T cells from allergic subjects.17 epitopes restricted to DRB(∗)01:01, DRB1(∗)03:01, DRB1(∗)04:01, DRB1(∗)09:01, DQB1(∗)02:01, DQB1(∗)03:02 and DQB1(∗)05:01 alleles were identified and validated by both the MHC binding and the functional assays. Two peptides showed specificities to more than one MHC class II allele. We demonstrated that these peptides exert functional responses in an epitope specific manner, eliciting predominantly IL-6 and IL-13.The identified epitopes are specific to common MHC class II alleles in the general population. Our study provides important data for the design of peptide-based immunotherapy of shrimp-allergic patients.

Black Creek Canal (BCC) virus is a newly identified virus associated with hantavirus pulmonary syndrome (HPS) in the southeastern United States. Nucleotide sequences were determined for the complete S and M and partial L genomic segments of a BCC virus isolate. Phylogenetic analysis indicates that each of the virus segments is unique, and there is no evidence of genetic reassortment having occurred between this and other previously characterized hantaviruses. All hantavirus-seropositive, wild-caught Sigmodon hispidus which were tested by polymerase chain reaction for the presence of BCC virus RNA were found to be positive, consistent with a chronic virus infection. This finding, together with serologic evidence, confirms S. hispidus as the primary rodent reservoir of this virus. Nucleotide sequence analysis of these PCR products indicates that BCC virus is genetically diverse and may pose a public health threat throughout much of the extensive range of this rodent species. Immunoprecipitation analysis demonstrated the presence of G1-, G2-, and N-specific antibodies in sera from BCC virus-infected patients with HPS and naturally infected S. hispidus. Comparison of the patterns of protein immunoprecipitation obtained with human acute- and convalescent-phase sera suggests that G1- and G2-specific antibodies increase relative to N-specific antibodies during the course of infection. The ratio of G1 to N protein immunoprecipitation by rodent or human acute-phase sera allowed differentiation of BCC and Sin Nombre virus infections.

Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While data obtained with MHC tetramers prepared with small molecule fluorophores has been reported, considerable lot-to-lot variation among conventional streptavidin conjugates to small molecules prevents routine preparation of such reagents. We now report robust preparation of MHC tetramers with small molecule fluorophores, using a recombinant mutant of streptavidin incorporating a carboxy-terminal cysteine in each of the four identical subunits that is conjugated to maleimide derivatives of any of several small molecule fluorophores. These reagents significantly expand the versatility of the MHC tetramer methodology.

CD1d presentation of alpha-galactosyl ceramides to natural killer T cells has been a focal point of the study of regulatory T cells. KRN7000, an alpha-galactosyl ceramide originally generated from structure activity studies of antitumor properties of marine sponge glycolipids, is currently the most commonly used agonist ligand and is used to stain NKT cells. However, this glycolipid suffers from poor solubility and availability. We have developed an alpha-galactosyl ceramide with improved solubility over KRN7000 that effectively stains NKT cells, both mouse and human, and stimulates cytokine release at low concentrations.

Hantavirus pulmonary syndrome (HPS) is a recently recognized viral zoonosis. The first recognized cases were caused by a newly described hantavirus. Sin Nombre virus (previously known as Muerto Canyon virus), isolated from Peromyscus maniculatus (deer mouse). We describe a 33-year-old Floridian man who resided outside the ecologic range of P maniculatus but was found to have serologic evidence of a hantavirus infection during evaluation of azotemia associated with adult respiratory distress syndrome. Small mammal trapping conducted around this patient's residence demonstrated the presence of antihantaviral antibodies in 13% of Sigmodon hispidus [cotton rat). Serologic testing using antigen derived from the Black Creek Canal hantavirus subsequently isolated from this rodent established that this patient was acutely infected with this new pathogenic American hantavirus. HPS is not confined to the geographical distribution of P maniculatus and should be suspected in individuals with febrile respiratory syndromes, perhaps associated with azotemia, throughout the continental United States.

Black Creek Canal virus (BCCV) is a New World hantavirus which is associated with hantavirus pulmonary syndrome. We have examined the site of expression of the BCCV nucleocapsid protein (NBCCV) in the absence of BCCV glycoproteins and found that the majority of the protein is localized to the Golgi region. Immunofluorescence analysis of BHK21 cells expressing the NBCCV and La Crosse virus nucleocapsid protein (NLACV) showed different intracellular localization patterns of these proteins within the same cell: NLACV is cytoplasmic, whereas NBCCV is perinuclear. NBCCV was found to be colocalized with α-mannosidase II, a marker for the Golgi complex. Also, NBCCV was found to be associated with microsomal membranes following cell fractionation. Sedimentation analysis in density gradients revealed that the membrane association of NBCCV is sensitive to treatments with high-salt and high-pH solutions, which indicates that NBCCV is a peripheral membrane protein. Analysis of NBCCV truncation mutants revealed that the 141-amino-acid C-terminal portion of this protein was capable of targeting green fluorescent protein to the perinuclear region. The difference in the intracellular localization between the NBCCV and NLACV proteins suggests that the mechanisms involved in the morphogenesis of New World hantaviruses are distinct from that documented for other members of the Bunyaviridae family.

The large (L) RNA segment of Lassa fever virus (LAS) encodes a putative RNA-dependent RNA polymerase (RdRp or L protein). Similar to other arenaviruses, the LAS L protein is encoded on the genome-complementary strand and is predicted to be 2218 amino acids in length (253 kDa). It has an unusually large non-coding region adjacent to its translation start site. The LAS L protein contains six motifs of conserved amino acids that have been found among arenavirus L proteins and core RdRp of other segmented negative-stranded (SNS) viruses (Arena-, Bunya- and Orthomyxoviridae). Phylogenetic analyses of the RdRp of 20 SNS viruses reveals that arenavirus L proteins represent a distinct cluster divided into LAS-lymphocytic choriomeningitis and Tacaribe-Pichinde virus lineages. Monospecific serum against a synthetic peptide corresponding to the most conserved central domain precipitates a 250 kDa product from LAS and lymphocytic choriomeningitis virus-infected cells.

Black Creek Canal (BCC) virus is a newly identified hantavirus from Florida which is carried by the cotton rat (Sigmodon hispidus) and is associated with hantavirus pulmonary syndrome (HPS). We have investigated the interaction of BCC virus with polarized epithelial cells to examine whether entry and release of this virus occur at specific plasma membrane domains. The polarized Vero C1008 monkey kidney cell line was grown on permeable filters and infected with BCC virus either through the apical or basolateral surface. As shown by indirect immunofluorescence and radioimmunoprecipitation analysis, cells infected through the apical surface demonstrated a high level of susceptibility to BCC virus infection. In contrast, Vero C1008 cells infected basolaterally exhibited a barely detectable level of BCC virus-synthesized proteins. Titration of virus from apical and basolateral media of infected cells has demonstrated that virus titers released from the apical surface are about 1,200-fold greater than the titer of virus released into the basolateral media. The site of BCC virus release from polarized cells is, therefore, different from that previously described for release of other members of the family Bunyaviridae and may reflect one of the determinants of hantavirus pathogenesis. In addition, we have shown that BCC viral glycoproteins are expressed at the plasma membrane on the apical surface of polarized cells. Electron microscopy studies of the infected cells revealed evidence of BCC virus budding at the plasma membrane. This strongly indicates that, in contrast to most other members of the Bunyaviridae, BCC virus is assembled at the plasma membrane. Since the same site of virus assembly was recently described for Sin Nombre virus, it is likely that all of the new American hantaviruses associated with HPS utilize this same type of virus maturation.

Numerous rodents were trapped for serologic and virologic studies following the identification of a hantavirus pulmonary syndrome (HPS) case in Dade County, Florida. Cotton rats (Sigmodon hispidus) were the most frequently capture rodent and displayed the highest seroprevalence to a variety of hantavirus antigens. Hantavirus genome RNA was detected in all the seropositive cotton rats tested, using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay. A virus was isolated from tissues of two seropositive cotton rats by cultivation of lung and spleen homogenates on Vero E6 cells. Nucleotide sequence information obtained by direct RT-PCR and the serologic relationships of this virus with the other hantaviruses indicate that this virus, Black Creek Canal virus, represents a new hantavirus distinct from the previously known serotypes.

The parameters controlling the generation of robust CD4+ T cell recall responses remain poorly defined. In this study we compared recall responses by CD4+ and CD8+ memory T cells following rechallenge. Homologous rechallenge of mice immune to either lymphocytic choriomeningitis virus (LCMV) or Listeria monocytogenes resulted in robust CD8+ T cell recall responses but poor boosting of CD4+ T cell recall responses in the same host. In contrast, heterologous rechallenge with a pathogen sharing only a CD4+ T cell epitope resulted in robust boosting of CD4+ T cell recall responses. Robust CD4+ and CD8+ T cell recall responses were simultaneously induced following rechallenge with peptide-pulsed dendritic cells, ruling out the possibility that the disparity in CD4+ and CD8+ T cell recall responses could be attributed to competition for growth factors or APCs. Instead, CD4+ T cell recall responses were dependant on the duration of the secondary challenge. Increasing the rechallenge dose resulted in more potent boosting of CD4+ T cell recall responses, and artificially limiting the duration of infection following heterologous rechallenge adversely impacted the magnitude of CD4+ T cell, but not CD8+ T cell, recall responses. These findings suggest that rapid pathogen clearance by secondary CTL following homologous rechallenge prevents optimal boosting of CD4+ T cell responses.

Interleukin-2 (IL-2) plays a central role in the differentiation of functionally competent CD8+ memory T cells. In this study, we found that in the absence of IL-2 signals, the differentiation of short-lived CD8+ effector T cells and tissue-residing effector memory CD8+ T cells in response to acute infection was greatly impaired. Due to their shared signaling apparatus, we explored the hypothesis that IL-15 signals might also contribute to the differentiation and survival of these populations. In line with this hypothesis, we observed an additional impairment in the differentiation and persistence of short-lived effector/effector memory cells in the spleen when both IL-2 and IL-15 signals were absent. Furthermore, in the absence of IL-2 and IL-15 signals this population disappeared completely by day 15. In contrast, joint inhibition of IL-2 and IL-15 signals had no further impact on the differentiation of tissue-residing CD8+ effector memory T cells. While IL-2 and IL-15 contributed to the differentiation of short-lived effector cells during the primary response, IL-15 continued to play a mandatory role in the survival of these cells during contraction and memory. These findings delineate a joint role for IL-15 and IL-2 in the differentiation of short-lived CD8+ effector cells but point to unique roles for IL-15 and IL-2 in promoting the survival of short-lived effector/effector memory cells in the spleen and the differentiation of tissue-residing effector memory cells, respectively.

SummaryRequirements for CD4+ T cell memory differentiation were analyzed with adoptively transferred SMARTA T cell receptor (TCR) transgenic cells specific for a lymphocytic choriomeningitis virus (LCMV) epitope. LCMV-induced effector and memory differentiation of SMARTA cells mimicked the endogenous CD4+ T cell response. In contrast, infection with a recombinant Listeria expressing the LCMV epitope, although resulting initially in massive SMARTA expansion, led to loss of effector function and rapid cell death characterized by high expression of the apoptosis regulator Bim. Defective memory differentiation was seen after stimulation of naive but not memory SMARTA cells, was independent of precursor frequency, and correlated with a lower TCR avidity compared to endogenous responders. In addition, long-lived endogenous CD4+ memory T cells skewed to a higher functional avidity over time. These results support a model in which CD4+ T cell memory differentiation and longevity depend on the strength of the TCR signal during the primary response.

Analysis of a limited number of laboratory strains of rabies virus had demonstrated the presence of a genome region bounded by two transcription termination and polyadenylation-like (TTP) signals (approximately 400 to 450 nucleotides apart) which was located between the end of the glycoprotein (G) coding sequence and the beginning of the L polymerase coding sequence. Although this region had been suggested to represent a remnant or pseudogene (~), no detailed analysis had been carried out to examine this possibility. Here we present the nucleotide sequence analysis of this genome region for several laboratory rabies virus strains and a large number of diverse rabies viruses detected directly in brain tissue of naturally infected animals, Only one distinct lineage of the laboratory strains and none of the wild-type rabies viruses contained the upstream -I-IP-like signal, indicating that only the downstream -I-IP motif is the authentic G mRNA transcription termination and polyadenylation and signal. Phylogenetic analysis of sequence differences provided no evidence of laboratory strains containing the two -I-IP-like signals being ancestral to any of the viruses possessing only the downstream -I-IP sequence motif. These data indicate that this region of the rabies virus genome encodes a G mRNA with a long 3 ' noncoding region with no evidence of a pseudogene. Rabies virus is a member of the Lyssavirus genus of the Rhabdoviridae family, a diverse group of animal, plant, and insect viruses, including vesicular stomatitis virus (VSV), rabies and related viruses, infectious hematopoietic necrosis virus (IHNV), and bovine ephemeral fever virus (BEFV) (1, 2). These viruses all possess nonsegmented, negative-sense, single-stranded RNA genomes of approximately 10 to 15 kb in length. The virus genomes commonly encode at least five mRNAs, which are located and also sequentially transcribed in the general pattern 3'-nucleocapsid protein (N), polymerase-associated phosphoprotein (P or M1), matrix protein (M or M2), glycoprotein(s) (G), and large polymerase protein (L)-5'. These genomes usually contain very little noncoding nucleotide sequence and gene junctions are small. The most common exception to this overall genetic structure is the genome region located between the G mRNA termination codon and the L mRNA initiation signal. In IHNV, an additional gene encoding a nonstructural protein (NV) is located in between the G and L genes (3). BEFV contains an additional nonstructural glycoprotein (GNs) in this genome area that has been proposed to have evolved by duplication of the original giycoprotein gene (4). Further, other small open reading frames