RAMPVIS: Answering the challenges of building visualisation capabilities for large-scale emergency responses.

The effort for combating the COVID-19 pandemic around the world has resulted in a huge amount of data, e.g., from testing, contact tracing, modelling, treatment, vaccine trials, and more. In addition to numerous challenges in epidemiology, healthcare, biosciences, and social sciences, there has been an urgent need to develop and provide visualisation and visual analytics (VIS) capacities to support emergency responses under difficult operational conditions. In this paper, we report the experience of a group of VIS volunteers who have been working in a large research and development consortium and providing VIS support to various observational, analytical, model-developmental, and disseminative tasks. In particular, we describe our approaches to the challenges that we have encountered in requirements analysis, data acquisition, visual design, software design, system development, team organisation, and resource planning. By reflecting on our experience, we propose a set of recommendations as the first step towards a methodology for developing and providing rapid VIS capacities to support emergency responses.

In Search of Patient Zero: Visual Analytics of Pathogen Transmission Pathways in Hospitals.

Pathogen outbreaks (i.e., outbreaks of bacteria and viruses) in hospitals can cause high mortality rates and increase costs for hospitals significantly. An outbreak is generally noticed when the number of infected patients rises above an endemic level or the usual prevalence of a pathogen in a defined population. Reconstructing transmission pathways back to the source of an outbreak - the patient zero or index patient - requires the analysis of microbiological data and patient contacts. This is often manually completed by infection control experts. We present a novel visual analytics approach to support the analysis of transmission pathways, patient contacts, the progression of the outbreak, and patient timelines during hospitalization. Infection control experts applied our solution to a real outbreak of Klebsiella pneumoniae in a large German hospital. Using our system, our experts were able to scale the analysis of transmission pathways to longer time intervals (i.e., several years of data instead of days) and across a larger number of wards. Also, the system is able to reduce the analysis time from days to hours. In our final study, feedback from twenty-five experts from seven German hospitals provides evidence that our solution brings significant benefits for analyzing outbreaks.

Animation, Small Multiples, and the Effect of Mental Map Preservation in Dynamic Graphs.

In this paper, we present the results of a human-computer interaction experiment that compared the performance of the animation of dynamic graphs to the presentation of small multiples and the effect that mental map preservation had on the two conditions. Questions used in the experiment were selected to test both local and global properties of graph evolution over time. The data sets used in this experiment were derived from standard benchmark data sets of the information visualization community. We found that small multiples gave significantly faster performance than animation overall and for each of our five graph comprehension tasks. In addition, small multiples had significantly more errors than animation for the tasks of determining sets of nodes or edges added to the graph during the same timeslice, although a positive time-error correlation coefficient suggests that, in this case, faster responses did not lead to more errors. This result suggests that, for these two tasks, animation is preferable if accuracy is more important than speed. Preserving the mental map under either the animation or the small multiples condition had little influence in terms of error rate and response time.

[Molecular and biological aspects of Arterivirus]. / Aspects moléculaires et biologiques des artérivirus.

Arteriviruses are a group of small enveloped viruses with a genome composed of a positive, single-stranded, 5'-capped and 3'-polyadenylated RNA molecule. These viruses belong to the Arteriviridae family which together with the Coronaviridae and Roniviridae families constitutes the order Nidovirales. Arteriviruses have a marked tropism for macrophages and induce persistent infections. Several studies have reported the unique genomic organization of these viruses together with replication and transcription mechanisms for the synthesis of genomic and sub-genomic viral RNAs which, for the latter, encode viral structural proteins. Several sequences are now available for these viruses. Recent studies about the modulation of innate immune response by arteriviruses also demonstrated a key-role of particular non structural proteins in that process. Finally, certain pro- and anti-apoptotic mechanisms associated to Arteriviruses have been elucidated; however the exact significance of theses apoptotic processes in arterivirus pathogenesis has yet to be determined.

Bovine immunodeficiency virus: identification of a long terminal repeat sequence with enhanced promoter activity.

We previously identified a new bovine immunodeficiency virus (BIV) trans-activator factor of transcription (Tat236) that was derived from a variant of BIV. Here, we report a new BIV long terminal repeat (LTR) sequence (LTRn) that was obtained by PCR from the DNA of cells infected with the BIV variant mentioned above. Sequence analysis indicated that the LTRn U3 region harbors three nucleic acid mutations at residue positions -194, -135 and -114 when compared to the original (wild-type) LTR sequence. Reporter gene assays indicated that LTRn promotes basal and Tat-mediated transactivation activity to levels significantly higher than those obtained with the wild-type LTR. Restoration experiments to the wild-type genotype indicated that both the -135 and -114 nucleic acid substitutions were responsible for the enhanced promoter activity of BIV LTRn.

Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal strain.

AIMS: The aim of this study was to purify and identify the bacteriocin produced by Pediococcus acidilactici MM33, a strain previously isolated from human gut. METHODS AND RESULTS: Purification of the bacteriocin was performed by cationic exchange chromatography followed by a reverse phase step. Biochemical and mass spectrometry analysis showed homology with pediocin PA-1. To verify if P. acidilactici MM33 carried the pediocin PA-1 gene, total DNA was used to amplify the pediocin gene. The PCR product obtained was then sequenced and the nucleotide sequence revealed to be identical to that of pediocin PA-1. Treatment of P. acidilactici MM33 with novobiocin resulted in a plasmid-cured strain without bacteriocin-producing capacity. Antimicrobial assay and molecular analysis demonstrated that this strain was ped(-) suggesting that the ped cluster is plasmid encoded. Antimicrobial assay revealed that pediocin was bactericidal against Listeria monocytogenes, showing a minimal inhibitory concentration (MIC) of 200 AU ml(-1). CONCLUSIONS: A two-step purification procedure was elaborated in this study. The bacteriocin secreted by the human strain P. acidilactici MM33 is carried on a plasmid and the amino acid sequence is identical to pediocin PA-1. SIGNIFICANCE AND IMPACT OF THE STUDY: Pediococcus acidilactici MM33 is the first human pediocin-producing strain reported and could be used as probiotic to prevent enteric pathogen colonization.

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates.

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.

The bovine immunodeficiency virus: cloning of a tat/rev cDNA encoding a novel Tat protein with enhanced transactivation activity.

Previous studies have shown that BIV may encode two types of Tat proteins of 103 and 108 amino acids, respectively. Here, we report the characterization of a new BIV Tat protein (Tat236) derived from a tat/rev cDNA. The tat/rev cDNA was obtained by reverse transcription-PCR from RNA extracted from cells infected with BIV. BIV was rescued by cell co-cultivation from the spleen of rabbits exposed for 3 years to the R29 isolate of BIV. Sequence analysis indicated that BIV Tat236 contains the first 98 amino acids of Tat103 and the 3' end 138 amino acids of Rev. Reporter gene assays indicated that transactivation of BIV long terminal repeat (LTR) by Tat236 is higher than by the original BIV Tat proteins in several cell types. By using overlapping deletion mutants, evidence was given that the predicted basic domain of Rev within Tat236 plays a major role in the observed enhanced transactivation activity of the protein. However, the intact functional domain of the original BIV Tat is required for efficient transactivation. This is the first report of a hybrid Tat protein from BIV or any lentiviruses that shows higher transactivation than the original transactivator Tat proteins.

Molecular cloning and characterization of beluga whale (Delphinapterus leucas) interleukin-1beta and tumor necrosis factor-alpha.

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are cytokines produced primarily by monocytes and macrophages with regulatory effects in inflammation and multiple aspects of the immune response. As yet, no molecular data have been reported for IL-1beta and TNF-alpha of the beluga whale. In this study, we cloned and determined the entire cDNA sequence encoding beluga whale IL-1beta and TNF-alpha. The genetic relationship of the cytokine sequences was then analyzed with those from several mammalian species, including the human and the pig. The homology of beluga whale IL-1beta nucleic acid and deduced amino acid sequences with those from these mammalian species ranged from 74.6 to 86.0% and 62.7 to 77.1%, respectively, whereas that of TNF-alpha varied from 79.3 to 90.8% and 75.3 to 87.7%, respectively. Phylogenetic analyses based on deduced amino acid sequences showed that the beluga whale IL-1beta and TNF-alpha were most closely related to those of the ruminant species (cattle, sheep, and deer). The beluga whale IL-1beta- and TNF-alpha-encoding sequences were thereafter successfully expressed in Escherichia coli as fusion proteins by using procaryotic expression vectors. The fusion proteins were used to produce beluga whale IL-1beta- and TNF-alpha-specific rabbit antisera.

Induction of apoptosis by equine arteritis virus infection.

Equine arteritis virus (EAV) is the etiological agent of equine viral arteritis, a contagious viral disease of equids. EAV is the prototype virus of the arteriviruses, a group of small enveloped viruses with positive single-stranded RNA genomes. Because apoptosis or programmed cell death is believed to play an important role in the biogenesis of several cytopathogenic viruses, we examined whether EAV was able to induce cell apoptosis in vitro. To do this, Vero cells were infected with EAV at a multiplicity of infection of 0.1 tissue culture infectious dose (TCID50) per cell, and analyzed at various time intervals for the appearance of apoptotic signs. Fragmentation of chromosomal DNA into nucleosomal oligomers and caspase activation were observed in the infected cells at the time (e.g. 24h postinfection) where a noticeable cytopathic effect was observed. The kinetics of the DNA fragmentation correlated with that of the production of progeny virus, so that viral multiplication was not interrupted by the apoptotic cell damage. All these data provide evidence that EAV is able to induce apoptotic cell death in vitro.

Clinical response and immunomodulation following experimental challenge of calves with type 2 noncytopathogenic bovine viral diarrhea virus.

Eight calves between 16 and 18 weeks of age that were seronegative to bovine viral diarrhea virus (BVDV), bovine leucosis virus and bovine immunodeficiency-like virus were infected (day 0) intranasally with the type 2 noncytopathogenic Canadian 24515 field isolate of BVDV in order to evaluate the effect of BVDV infection on certain clinical, hematological and immunological parameters. All virus-exposed animals developed fever and showed a significant (P < 0.05, 0.01 or 0.001) drop in the number of circulating leucocytes (neutrophils, lymphocytes and monocytes) by day 3 or 5 post-exposure (PE), which continued to the end of the experiment at day 12 PE. BVDV was consistently isolated from the peripheral blood buffy coat cells from day 5 PE, and also from selected tissues (spleen, thymus, mesenteric and submaxillary lymph nodes, small intestine, lungs and thyroid gland) that were collected at the time of euthanasia of the animals at day 12 PE. Diminished significant (P < 0.05) percentages of peripheral blood mononuclear cells (PBMCs) expressing at their surface either B7 and MHC II molecules were observed in virus-exposed calves at days 7, 10 and/or 12 PE, when compared to virus-nonexposed control calves (n = 5). However, no changes in the percentages of PBMCs expressing either B4 or MHC I molecules were observed throughout the experiment. Finally, a significant (P < 0.05 or 0.01) enhanced phagocytic capability of the PBMCs, as analyzed by flow cytometry, was observed in virus-exposed animals at days 3, 5, 7, 10 and 12 PE, when compared to control calves. These results demonstrated the virulence of the 24515 isolate of BVDV in 4 to 4.5 month-old calves, and suggest that type 2 BVDV infection in calves is associated with dysregulation of certain immunological functions.

Molecular cloning, phylogenetic analysis and expression of beluga whale (Delphinapterus leucas) interleukin 6.

Interleukin 6 (IL-6) is a cytokine produced primarily by the monocytes/macrophages with regulatory effects in hematopoiesis, acute phase response, and multiple aspects of the immune response. IL-6 exerts its activity through its binding to specific high affinity receptors at the surface of target cells. As yet, no molecular data have been reported for the beluga whale IL-6. In this study, we cloned and determined the entire beluga whale IL-6-encoding cDNA sequence by reverse transcription-polymerase chain reaction (RT-PCR) sequencing, and analysed its genetic relationship with those from several mammalian species including human, rodent, ruminant, carnivore and other marine species. The identity levels of beluga whale IL-6 nucleic and deduced amino acid sequences with those from these mammalian species ranged from 62.3 to 97.3%, and 42.9 to 95.6%, respectively. Phylogenetic analysis based on amino acid sequences showed that the beluga whale IL-6 was most closely related to that of the killer whale. Thereafter, beluga whale IL-6-encoding sequence was successfully expressed in Escherichia coli by using the pTHIOHisA expression vector for the production of a recombinant fusion protein. The immunogenicity of the recombinant fusion protein was then confirmed as determined by the production of a beluga whale IL-6-specific rabbit antiserum.

A viral transmembrane recombinant protein-based enzyme-linked immunosorbent assay for the detection of bovine immunodeficiency virus infection.

The expression of bovine immunodeficiency virus (BIV) truncated transmembrane envelope protein (designated hereafter tTM) in insect cells has been described previously (Abed, Y., St-Laurent, G., Zhang, H., Jacobs, R.M., Archambault, D., 1999. Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane proteins expressed from recombinant baculovirus. Clin. Diagn. Lab. Immunol. 6, 168-172). In this study, a tTM-based enzyme-linked immunosorbent assay (ELISA) was developed for the serodetection of BIV infection. A total of 109 bovine sera including 86 BIV-negative and 23 BIV-positive serum samples were tested. The ELISA results were compared with those of three Western blot assays using, as test antigens, cell culture-derived whole virus proteins (WB1), and the tTM (WB2) and p26 (WB3) fusion proteins expressed from recombinant baculovirus in insect cells, respectively. The concordances of the ELISA results with those of the WB1, WB2, and WB3 were 97.2, 100 and 97.2%, respectively. The tTM protein-based ELISA and Western blot permitted the detection of BIV infection in cattle whose sera failed to react with the p26 fusion protein and the whole virus protein preparation. The tTM recombinant protein was also used to study the kinetics of appearance of antibodies against BIV transmembrane envelope protein in rabbits infected experimentally with BIV. Antibodies to tTM were detected at 28 days post-infection and persisted through the entire 36-39.5 months experimental time period. The results of this study showed that the tTM-ELISA might be useful for the serodetection of BIV-infected animals, and for basic studies on BIV replication life cycle.

Molecular cloning and phylogenetic analysis of beluga whale (Delphinapterus leucas) and grey seal (Halichoerus grypus) interleukin 2.

Interleukin 2 (IL-2) is a lymphokine produced by activated T helper lymphocytes which exerts immunoregulatory effects on a variety of immune cells, including T cells, activated B cells, natural killer cells, and lymphokine-activated killer cells. In this study, we cloned and determined the entire beluga whale (Delphinapterus leucas) and grey seal (Halichoerus grypus) IL-2-encoding cDNA sequences, and analysed their genetic relationships with those from several mammalian species obtained from the Genbank Database. The encoding nucleic acid sequences of beluga whale and grey seal IL-2 were 465 and 468 bp in length, respectively. The identity levels of IL-2 nucleic and deduced amino acid sequences from the beluga whale and grey seal with those from the other mammalian species, ranged from 59.9% to 89.5%, and 52.9% to 77.3%, and from 61.1% to 93.2%, and 58.7% to 88.4%, respectively. Phylogenetic analysis based on both nucleic and amino acid sequences showed that the beluga whale IL-2 was closely related to that of the ruminant species, which includes the bovine, while the grey seal IL-2 was closely related to that of the canine.

Detection of bovine retrospumavirus by the polymerase chain reaction.

A polymerase chain reaction (PCR) assay was developed for detection of bovine retrospumavirus (bovine syncytial virus; BSV) provirus DNA. Two different sets of oligonucleotide primers complementary to sequences located in the gag and the pol/env gene regions were used and compared for their ability to amplify the targeted BSV sequences by PCR. The results obtained from this study have shown that it is possible to amplify the BSV provirus DNA sequences not only from total DNA of BSV-infected cell cultures, but also from total DNA of various tissues and peripheral blood mononuclear cells (PBMCs) that were collected from two rabbits experimentally infected with BSV. Sensitivities of the PCR for amplification of BSV gag and pol/env nucleic acid sequences from cell culture total DNA were 10 ng and 10 pg of DNA, respectively, as determined by the analysis of the amplified PCR products on ethidium bromide-stained agarose gels. The specificity of the PCR for both primer sets tested was confirmed when the amplified cDNA products of the expected size reacted positively with the corresponding virus-specific digoxigenin-labeled cDNA probes in Southern blot chemiluminescent hybridization assays. No amplification was obtained when the BSV-specific primers were used in the PCR with DNA material specific to either bovine leukemia virus (BLV) or bovine immunodeficiency virus (BIV) provirus genomic DNA. No cross-hybridization was obtained when the BSV-specific cDNA probes were allowed to react with BLV or BIV provirus DNA. The PCR targeting the gag and pol/env gene regions of the BSV provirus genome may be an alternative to conventional methods for the confirmation of the presence of BSV in cell cultures used for virus isolation, and for the diagnosis of BSV infection from bovine peripheral blood leukocytes.

Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane envelope proteins expressed from recombinant baculovirus.

A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.

Genetic variation and phylogenetic analysis of open reading frames 3 and 4 of various equine arteritis virus isolates.

The genetic variation in equine arteritis virus (EAV) nonstructural (NS) protein-encoding open reading frames (ORF) 3 and 4 genes was investigated. Nucleotide and deduced amino acid sequences from seven different EAV isolates (one European, one American and five Canadian isolates) and the Arvac vaccine strain were compared with those of the Bucyrus reference strain. ORF 3 nucleotide and amino acid sequence identities amongst these isolates (including the Arvac vaccine strain) and the Bucyrus reference strain ranged from 85.6 to 98.8%, and 85.3 to 98.2%, respectively, whereas ORF 4 nucleotide and amino acid sequence identities ranged from 90.4 to 98.3%, and 90.8 to 97.4%, respectively. Phylogenetic tree analysis based on the ORF 3 nucleotide sequences showed that the European Vienna isolate could be classified into a genetically divergent group from all other isolates and the Arvac vaccine strain. In contrast, a phylogenetic relationship among all EAV isolates and the Arvac vaccine strain based on the ORF 4 nucleotide sequences was observed.

Sequence determination and genetic analysis of the leader region of various equine arteritis virus isolates.

The entire leader sequence of ten equine arteritis virus (EAV) isolates including the Bucyrus reference strain was determined and analyzed at the primary nucleotide and secondary structure levels. The leader sequence of eight EAV isolates was determined to be 206 nucleotides (nt) in length, whereas those of the 86AB-A1 and 86NY-A1 isolates were found to be 205 and 207 nt in length, respectively. The sequence identity of the leader sequences between the different isolates and the Bucyrus reference strain ranged from 94.2 to 98.5%. An AUG start codon found at position 14 in all EAV isolates could initiate an open reading frame (ORF) that could produce a polypeptide of 37 amino acids, except for the 86NY-A1 isolate where the intraleader polypeptide would contain 54 amino acids. Five patterns of computer-predicted RNA secondary structures were identified in the ten EAV leader regions analyzed. All EAV isolates showed three conserved stem-loops (designated A, B and C). An additional conserved stem-loop (D) was observed in six EAV isolates, including the Bucyrus reference strain. Based on the presence or absence of stem-loop D, all EAV isolates analyzed in this study could be tentatively classified into two genogroups (I and II). The significance of the intraleader ORF and the predicted secondary structures has yet to be determined.

Stabilization of human immunodeficiency virus type 1 envelope glycoprotein trimers by disulfide bonds introduced into the gp41 glycoprotein ectodomain.

Biochemical and structural studies of fragments of the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein have demonstrated that the molecular contacts between alpha helices allow the formation of a trimeric coiled coil. By introducing cysteine residues into specific locations along these alpha helices, the normally labile HIV-1 gp160 envelope glycoprotein was converted into a stable disulfide-linked oligomer. Although proteolytic cleavage into gp120 and gp41 glycoproteins was largely blocked, the disulfide-linked oligomer was efficiently transported to the cell surface and was recognized by a series of conformationally dependent antibodies. The pattern of hetero-oligomer formation between this construct and an analogous construct lacking portions of the gp120 variable loops and of the gp41 cytoplasmic tail demonstrates that these oligomers are trimers. These results support the relevance of the proposed gp41 structure and intersubunit contacts to the native, complete HIV-1 envelope glycoprotein. Disulfide-mediated stabilization of the labile HIV-1 envelope glycoprotein oligomer, which has been suggested to possess advantages as an immunogen, may assist attempts to develop vaccines.

Nucleotide sequence and genetic analysis of the leader region of Canadian, American and European equine arteritis virus isolates.

The extreme 5' end, the entire leader sequence of the Arvac vaccine strain, and 10 equine arteritis virus (EAV) isolates, including the ATCC Bucyrus reference strain and 5 Canadian field isolates, were determined and compared at the primary nucleotide and secondary structure levels. The leader sequence of eight EAV isolates, including the Bucyrus reference strain, and the leader sequence of the Arvac vaccine strain was determined to be 206 nt in length (not including the putative 5' cap structure-associated nucleotide) whereas those of the 86AB-A1 and 86NY-A1 isolates were found to be 205 and 207 nt in length, respectively. The sequence identity of the leader sequences, between the different isolates and the Bucyrus reference strain, ranged from 94.2 to 98.5%. Phylogenetic analysis and estimation of genetic distances, based on the leader nucleic acid sequences, showed that all EAV isolates/strains are likely to represent a large phylogenetically-related group. An AUG start codon found at position 14 in all EAV isolates/strains could initiate an open reading frame (ORF) that could produce a polypeptide of 37 amino acids, except for the 86NY-A1 isolate where the intraleader polypeptide would contain 54 amino acids. Computer-predicted RNA secondary structures were identified in the 11 EAV leader regions analyzed. All EAV isolates/strains showed 3 conserved stem-loops (designated A, B and C). An additional conserved stem-loop (D) was observed in 7 EAV isolates, including the Bucyrus reference strain. The leader region distal to stem-loop D did not contain conserved sequences or stem-loop structures common to the EAV isolates/strains.