Bovine Respiratory Syncytial Virus (BRSV) Infection Detected in Exhaled Breath Condensate of Dairy Calves by Near-Infrared Aquaphotomics.

Bovine respiratory syncytial virus (BRSV) is a major contributor to respiratory disease in cattle worldwide. Traditionally, BRSV infection is detected based on non-specific clinical signs, followed by reverse transcriptase-polymerase chain reaction (RT-PCR), the results of which can take days to obtain. Near-infrared aquaphotomics evaluation based on biochemical information from biofluids has the potential to support the rapid identification of BRSV infection in the field. This study evaluated NIR spectra (n = 240) of exhaled breath condensate (EBC) from dairy calves (n = 5) undergoing a controlled infection with BRSV. Changes in the organization of the aqueous phase of EBC during the baseline (pre-infection) and infected (post-infection and clinically abnormal) stages were found in the WAMACS (water matrix coordinates) C1, C5, C9, and C11, likely associated with volatile and non-volatile compounds in EBC. The discrimination of these chemical profiles by PCA-LDA models differentiated samples collected during the baseline and infected stages with an accuracy, sensitivity, and specificity >93% in both the calibration and validation. Thus, biochemical changes occurring during BRSV infection can be detected and evaluated with NIR-aquaphotomics in EBC. These findings form the foundation for developing an innovative, non-invasive, and in-field diagnostic tool to identify BRSV infection in cattle.

Chikungunya in Mississippi: The Health Department Response to Imported Cases.

Chikungunya (CHIK), a newly recognized mosquito-borne disease in the Western Hemisphere, has resulted in well over a million cases since December 2013. Only about a dozen locally-acquired cases thus far have been reported in the U. S. (Florida), but approximately 1500 imported cases have been seen in returning travelers from the Caribbean and Central and South America. Public health officials are concerned that imported cases may lead to infection of local mosquitoes and, thus disease transmission. This paper documents 9 confirmed CHIK cases in Mississippi: 5 resulting from travel to the Dominican Republic, 2 from Haiti, 1 from Honduras, and 1 from Puerto Rico. In addition, the Mississippi State Department of Health response to those cases is presented and discussed.

TLR-TRIF pathway enhances the expression of KSHV replication and transcription activator.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human γ-herpesvirus. KSHV replication and transcription activator (RTA) is necessary and sufficient for KSHV reactivation from latency. Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, act through adaptors, and initiate innate and adaptive immune responses against pathogens. Toll/interleukin-1-receptor domain containing adaptor protein inducing interferon-ß (TRIF) is an adaptor associated with TLR3 and TLR4 signaling, and is closely related to antiviral signaling to activate type I interferon (IFN) production. We previously found that KSHV RTA degrades TRIF indirectly and blocks TLR3 pathways. In this report, we find that TRIF, as well as TLR3 activation, enhances KSHV RTA protein expression. The C-terminal region of the RTA is involved in the responding TRIF-mediated enhancement. The degradation of TRIF and the enhancement of RTA expression are using two different pathways. The enhancement by TLR-TRIF is at least partially via promoting translational efficiency of RTA mRNA. Finally, the receptor-interacting protein 1 (RIP1) may be involved in TRIF-mediated enhancement of RTA expression, but not in the RTA-mediated degradation of TRIF. Therefore, the activation of TLR-TRIF pathway enhances KSHV RTA protein expression, and KSHV RTA in turn degrades TRIF to block innate immunity. The putative KSHV-TLR-adaptor-interacting loop may be a critical element to evade and usurp host innate immunity in KSHV life-cycle.

Do bed bugs transmit human viruses, or do humans spread bed bugs and their viruses? A worldwide survey of the bed bug RNA virosphere.

BED BUGS: (Hemiptera: Cimicidae) are a globally distributed hematophagous pest that routinely feed on humans. Unlike many blood-sucking arthropods, they have never been linked to pathogen transmission in a natural setting, and despite increasing interest in their role as disease vectors, little is known about the viruses that bed bugs naturally harbor. Here, we present a global-scale survey of the bed bug RNA virosphere. We sequenced the metatranscriptomes of 22 individual bed bugs (Cimex lectularius and Cimex hemipterus) from 8 locations around the world. We detected sequences from two known bed bug viruses (Shuangao bedbug virus 1 and Shuangao bedbug virus 2) which extends their geographical range. We identified three novel bed bug virus sequences from a tenui-like virus (Bunyavirales), a toti-like virus (Ghabrivirales), and a luteo-like virus (Tolivirales). Interestingly, some of the bed bug viruses branch near to insect-transmitted plant-infecting viruses, opening questions regarding the evolution of plant virus infection. When we analyzed the viral sequences by their host's collection location, we found unexpected patterns of geographical diversity that may reflect humans' role in bed bug dispersal. Additionally, we investigated the effect that Wolbachia, the primary bed bug endosymbiont, may have on viral abundance and found that Wolbachia infection neither promotes nor inhibits viral infection. Finally, our results provide no evidence that bed bugs transmit any known human pathogenic viruses.

Evidence of a Protein-Coding Gene Antisense to the UL5 Gene in Bovine Herpesvirus I.

Bovine herpesvirus type 1 (BoHV-1) is an important agricultural pathogen that infects cattle and other ruminants worldwide. Though it was first sequenced and annotated over twenty years ago, the Cooper strain, used in this study, was sequenced as recently as 2012 and is currently said to encode 72 unique proteins. However, tandem mass spectrometry has identified several peptides produced during active infection that align with the BoHV-1 genome in unannotated regions. One of these abundant peptides, "ORF M", aligned antisense to the DNA helicase/primase protein UL5. This study characterizes the novel transcript and its protein product and provides evidence to support the existence of homolog protein-coding genes in other Herpesviruses.

NMR-based metabolomics of plasma from dairy calves infected with two primary causal agents of bovine respiratory disease (BRD).

Each year, bovine respiratory disease (BRD) results in significant economic loss in the cattle sector, and novel metabolic profiling for early diagnosis represents a promising tool for developing effective measures for disease management. Here, 1H-nuclear magnetic resonance (1H-NMR) spectra were used to characterize metabolites from blood plasma collected from male dairy calves (n = 10) intentionally infected with two of the main BRD causal agents, bovine respiratory syncytial virus (BRSV) and Mannheimia haemolytica (MH), to generate a well-defined metabolomic profile under controlled conditions. In response to infection, 46 metabolites (BRSV = 32, MH = 33) changed in concentration compared to the uninfected state. Fuel substrates and products exhibited a particularly strong effect, reflecting imbalances that occur during the immune response. Furthermore, 1H-NMR spectra from samples from the uninfected and infected stages were discriminated with an accuracy, sensitivity, and specificity ≥ 95% using chemometrics to model the changes associated with disease, suggesting that metabolic profiles can be used for further development, understanding, and validation of novel diagnostic tools.

Bioinformatic Surveillance Leads to Discovery of Two Novel Putative Bunyaviruses Associated with Black Soldier Fly.

The black soldier fly (Hermetia illucens, BSF) has emerged as an industrial insect of high promise because of its ability to convert organic waste into nutritious feedstock, making it an environmentally sustainable alternative protein source. As global interest rises, rearing efforts have also been upscaled, which is highly conducive to pathogen transmission. Viral epidemics have stifled mass-rearing efforts of other insects of economic importance, such as crickets, silkworms, and honeybees, but little is known about the viruses that associate with BSF. Although BSFs are thought to be unusually resistant to pathogens because of their expansive antimicrobial gene repertoire, surveillance techniques could be useful in identifying emerging pathogens and common BSF microbes. In this study, we used high-throughput sequencing data to survey BSF larvae and frass samples, and we identified two novel bunyavirus-like sequences. Our phylogenetic analysis grouped one in the family Nairoviridae and the other with two unclassified bunyaviruses. We describe these putative novel viruses as BSF Nairovirus-like 1 and BSF uncharacterized bunyavirus-like 1. We identified candidate segments for the full BSF Nairovirus-like 1 genome using a technique based on transcript co-occurrence and only a partial genome for BSF uncharacterized bunyavirus-like 1. These results emphasize the value of routine BSF colony surveillance and add to the number of viruses associated with BSF.

Interferon regulatory factor 4 (IRF-4) targets IRF-5 to regulate Epstein-Barr virus transformation.

The cellular interferon regulatory factor-4 (IRF-4), which is a member of IRF family, is involved in the development of multiple myeloma and Epstein-Barr virus (EBV)-mediated transformation of B lymphocytes. However, the molecular mechanism of IRF-4 in cellular transformation is unknown. We have found that knockdown of IRF-4 leads to high expression of IRF-5, a pro-apoptotic member in the IRF family. Overexpression of IRF-4 represses IRF-5 expression. Reduction of IRF-4 leads to growth inhibition, and the restoration of IRF-4 by exogenous plasmids correlates with the growth recovery and reduces IRF-5 expression. In addition, IRF-4 negatively regulates IRF-5 promoter reporter activities and binds to IRF-5 promoters in vivo and in vitro. Knockdown of IRF-5 rescues IRF-4 knockdown-mediated growth inhibition, and IRF-5 overexpression alone is sufficient to induce cellular growth inhibition of EBV-transformed cells. Therefore, IRF-5 is one of the targets of IRF-4, and IRF-4 regulates the growth of EBV-transformed cells partially through IRF-5. This work provides insight on how IRFs interact with one another to participate in viral pathogenesis and transformation.

Kaposi sarcoma-associated herpesvirus degrades cellular Toll-interleukin-1 receptor domain-containing adaptor-inducing beta-interferon (TRIF).

Kaposi sarcoma-associated herpesvirus (KSHV) is a human γ-herpesvirus associated with several human malignancies. The replication and transcription activator (RTA) is necessary and sufficient for the switch from KSHV latency to lytic replication. Toll-interleukin-1 receptor (TIR) domain-containing adaptor-inducing ß-interferon (TRIF, also called TIR-domain-containing adaptor molecule-1 (TICAM-1)) is a signaling adaptor molecule that is critically involved in the Toll-like receptor 3 (TLR-3) and TLR-4 signaling pathways for type I interferon (IFN) production, a key component of innate immunity against microbial infection. In this report, we find a new mechanism by which RTA blocks innate immunity by targeting cellular TRIF. RTA specifically degrades TRIF by shortening the half-life of TRIF protein. This RTA-mediated degradation is at least partially mediated through the ubiquitin-proteasome pathway because proteasome inhibitors as well as knockdown of cellular ubiquitin expression alleviate the degradation. RTA may not directly interact with TRIF and may activate TRIF degradation indirectly through an unknown mediator(s). RTA targets multiple regions of TRIF and may use its ubiquitin ligase domain for the degradation. In addition, physiological levels of TRIF protein are down-regulated during KSHV lytic replication when RTA is expressed. Finally, RTA down-regulates double-stranded RNA-initiated activation of TLR-3 pathway, in the absence of degradation of IFN regulatory factor 7 (IRF-7). Taken together, these data suggest that KSHV employs a novel mechanism to block the innate immunity by degrading TRIF protein. This work may contribute to our understandings on how KSHV evades host immunity for its survival in vivo.

Dengue virus type 3 isolated from a fatal case with visceral complications induces enhanced proinflammatory responses and apoptosis of human dendritic cells.

A recent (2007 to 2009) dengue outbreak caused by dengue virus (DENV) in Paraguay presented unusual severe clinical outcomes associated with 50% mortality rates. Although it has been reported that inflammatory responses influence the severity of dengue virus infection (T. Pang, M. J. Cardosa, and M. G. Guzman, Immunol. Cell Biol. 85:43-45, 2007), there remains a paucity of information on virus-innate immunity interactions influencing clinical outcome. Using human dendritic cells from a major innate immune cell population as an in vitro model, we have investigated signature cytokine responses as well as infectivity-replicative profiles of DENV clinical isolates from either a nonfatal case of classical dengue fever (strain DENV3/290; isolated in Brazil in 2002) or a fatal case of dengue fever with visceral complications isolated in Paraguay in 2007 (strain DENV3/5532). Strain DENV3/5532 was found to display significantly higher replicative ability than DENV3/290 in monocyte-derived dendritic cells (mdDCs). In addition, compared to DENV3/290 results, mdDCs exposed to DENV3/5532 showed increased production of proinflammatory cytokines associated with higher rates of programmed cell death, as shown by annexin V staining. The observed phenotype was due to viral replication, and tumor necrosis factor alpha (TNF-α) appears to exert a protective effect on virus-induced mdDC apoptosis. These results suggest that the DENV3/5532 strain isolated from the fatal case replicates within human dendritic cells, modulating cell survival and synthesis of inflammatory mediators.

Mannheimia haemolytica Negatively Affects Bovine Herpesvirus Type 1.1 Replication Capacity In Vitro.

Bovine Respiratory Disease (BRD) is a multifactorial condition affecting cattle worldwide resulting in high rates of morbidity and mortality. The disease can be triggered by Bovine Herpesvirus-1 (BoHV-1) infection, stress, and the subsequent proliferation and lung colonization by commensal bacteria such as Mannheimia haemolytica, ultimately inducing severe pneumonic inflammation. Due to its polymicrobial nature, the study of BRD microbes requires co-infection models. While several past studies have mostly focused on the effects of co-infection on host gene expression, we focused on the relationship between BRD pathogens during co-infection, specifically on M. haemolytica's effect on BoHV-1 replication. This study shows that M. haemolytica negatively impacts BoHV-1 replication in a dose-dependent manner in different in vitro models. The negative effect was observed at very low bacterial doses while increasing the viral dose counteracted this effect. Viral suppression was also dependent on the time at which each microbe was introduced to the cell culture. While acidification of the culture medium did not grossly affect cell viability, it significantly inhibited viral replication. We conclude that M. haemolytica and BoHV-1 interaction is dose and time-sensitive, wherein M. haemolytica proliferation induces significant viral suppression when the viral replication program is not fully established.

In-Depth Temporal Transcriptome Profiling of an Alphaherpesvirus Using Nanopore Sequencing.

In this work, a long-read sequencing (LRS) technique based on the Oxford Nanopore Technology MinION platform was used for quantifying and kinetic characterization of the poly(A) fraction of bovine alphaherpesvirus type 1 (BoHV-1) lytic transcriptome across a 12-h infection period. Amplification-based LRS techniques frequently generate artefactual transcription reads and are biased towards the production of shorter amplicons. To avoid these undesired effects, we applied direct cDNA sequencing, an amplification-free technique. Here, we show that a single promoter can produce multiple transcription start sites whose distribution patterns differ among the viral genes but are similar in the same gene at different timepoints. Our investigations revealed that the circ gene is expressed with immediate-early (IE) kinetics by utilizing a special mechanism based on the use of the promoter of another IE gene (bicp4) for the transcriptional control. Furthermore, we detected an overlap between the initiation of DNA replication and the transcription from the bicp22 gene, which suggests an interaction between the two molecular machineries. This study developed a generally applicable LRS-based method for the time-course characterization of transcriptomes of any organism.

Relative Roles of Blow Flies (Diptera: Calliphoridae) and Invasive Fire Ants (Hymenoptera: Formicidae: Solenopsis spp.) in Carrion Decomposition.

Fire ants (Solenopsis spp.) have increasingly been reported from carrion in the southeastern United States and are now a part of the normal succession community. There have been previous observations of these ants altering carrion and preying on other carrion-attendant fauna; however, the overall effects of these activities on carrion decomposition rates, community composition, and blow fly larval development are poorly understood. Alteration of these ecological processes by fire ants could affect the forensic interpretation of entomological data. We conducted a study in Mississippi and Florida whereby portions of the succession fauna were excluded from access to pig carrion to study the relative effects of fire ants and blow flies on carrion decomposition and succession: a control with all fauna having access, a second treatment where fire ants and other geophilic taxa were excluded, and a third treatment in which blow flies and other large organisms were excluded. Fire ants inflicted lesions in the carrion, buried portions that touched the ground, and preyed on some members of the succession fauna. Their exclusion did not affect carrion decomposition rates that were measured but slightly affected the overall carrion community, and strongly affected the oviposition and development of blow flies. Despite the presence of fire ants early in the control, blow flies were eventually able to overcome predation of eggs and larvae, continue colonization, and complete development; however, the delay in the colonization of blow flies on carrion could affect the determination of postmortem intervals when development rates of blow flies are considered in the calculation.

Gaeumannomyces nanograminis, sp. nov., a hyphopodiate fungus identified from diseased roots of ultradwarf bermudagrass in the United States.

The genus Gaeumannomyces (Magnaporthaceae, Magnaporthales, Sordariomycetes, Ascomycota) includes root-infecting pathogens, saprobes, and endophytes. Morphological, biological, and phylogenetic analyses were employed to identify fungal isolates derived from turfgrass roots colonized with ectotrophic, dark runner hyphae. Phylogenetic trees for partial sequences of the 18S nuc rDNA, ITS1-5.8S-ITS2 nuc rDNA internal transcribed spacer, and 28S nuc rDNA regions and of the minichromosome maintenance complex 7 (MCM7), largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-alpha (TEF1) genes were obtained via maximum likelihood and Bayesian methods. Our isolates consistently formed a distinct and highly supported clade within Gaeumannomyces. Common and distinctive biological and morphological characters reinforced these findings. Additionally, we conducted pathogenicity evaluations and demonstrated the ability of this fungus to colonize roots of ultradwarf bermudagrass (Cynodon dactylon (L.) Pers. × C. transvaalensis Burtt-Davey), its native host, via ectotrophic, dark runner hyphae, causing disease symptoms including root discoloration and reduced root and shoot mass. Altogether, our discoveries enabled recognition and description of a new species, Gaeumannomyces nanograminis, associated with rotted roots of ultradwarf bermudagrass.

Profiling Mannheimia haemolytica infection in dairy calves using near infrared spectroscopy (NIRS) and multivariate analysis (MVA).

Bovine respiratory disease (BRD) linked with Mannheimia haemolytica is the principal cause of pneumonia in cattle. Diagnosis of BRD traditionally relies on visual assessment, which can be untimely, insensitive, and nonspecific leading to inadequate treatment and further spread of disease. Near Infrared Spectroscopy (NIRS) is a rapid acquisition vibrational spectroscopy that can profile changes in biofluids, and when used in combination with multivariate analysis, has potential for disease diagnosis. This study characterizes the NIR spectral profile of blood plasma from dairy calves infected with M. haemolytica and validates the spectral biochemistry using standardized clinical and hematological reference parameters. Blood samples were collected for four days prior to (baseline), and 23 days after, a controlled intrabronchial challenge. NIR spectral profiles of blood plasma discriminated and predicted Baseline and Infected states of animal disease progression with accuracy, sensitivity, and specificity ≥ 90% using PCA-LDA models. These results show that physiological and biochemical changes occurring in the bloodstream of dairy calves during M. haemolytica infection are reflected in the NIR spectral profiles, demonstrating the potential of NIRS as a diagnostic and monitoring tool of BRD over time.

Time course profiling of host cell response to herpesvirus infection using nanopore and synthetic long-read transcriptome sequencing.

Third-generation sequencing is able to read full-length transcripts and thus to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. In this study, we report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. Additionally, we report a large number of novel bovine transcript isoforms identified by nanopore and synthetic long-read sequencing. This study demonstrates that viral infection causes differential expression of host transcript isoforms. We could not detect an increased rate of transcriptional readthroughs as described in another alphaherpesvirus. According to our knowledge, this is the first report on the use of LoopSeq for the analysis of eukaryotic transcriptomes. This is also the first report on the application of nanopore sequencing for the kinetic characterization of cellular transcriptomes. This study also demonstrates the utility of nanopore sequencing for the characterization of dynamic transcriptomes in any organisms.

Magnaporthiopsis cynodontis, a novel turfgrass pathogen with widespread distribution in the United States.

The genus Magnaporthiopsis of Magnaporthaceae (Magnaporthales, Sordariomycetes, Ascomycota) contains species that are predominantly necrotrophic pathogens, often producing simple hyphopodia and dark, ectotrophic runner hyphae on plant roots and stems during colonization. Fungal isolates from turfgrass roots with dark and ectotrophic runner hyphae were examined and identified based on morphological, biological, and phylogenetic analyses. Maximum likelihood and Bayesian methods were implemented to obtain phylogenetic trees for partial sequences of the 18S nuc rDNA, ITS1-5.8S-ITS2 nuc rDNA internal transcribed spacer, and 28S nuc rDNA regions, and of the minichromosome maintenance complex 7 (MCM7), largest subunit of RNA polymerase II (RPB1), and translation elongation factor 1-alpha (TEF1) genes. Our isolates consistently formed a distinct and highly supported clade within Magnaporthiopsis. These findings were reinforced by common and distinctive biological and morphological characters. Additionally, we conducted pathogenicity evaluations and demonstrated the ability of this fungus to colonize roots of ultradwarf bermudagrass, one of its native hosts, via ectotrophic, dark runner hyphae, causing disease symptoms including root discoloration and reduced root and shoot mass. Altogether, our discoveries enabled recognition and description of a new species, Magnaporthiopsis cynodontis, which has widespread distribution in the United States.

Time-course profiling of bovine alphaherpesvirus 1.1 transcriptome using multiplatform sequencing.

Long-read sequencing (LRS) has become a standard approach for transcriptome analysis in recent years. Bovine alphaherpesvirus 1 (BoHV-1) is an important pathogen of cattle worldwide. This study reports the profiling of the dynamic lytic transcriptome of BoHV-1 using two long-read sequencing (LRS) techniques, the Oxford Nanopore Technologies MinION, and the LoopSeq synthetic LRS methods, using multiple library preparation protocols. In this work, we annotated viral mRNAs and non-coding transcripts, and a large number of transcript isoforms, including transcription start and end sites, as well as splice variants of BoHV-1. Our analysis demonstrated an extremely complex pattern of transcriptional overlaps.

The cellular transcription factor, CCAAT enhancer-binding protein alpha (C/EBP-alpha), has the potential to activate the bovine herpesvirus 1 immediate-early transcription unit 1 promoter.

Following acute infection, bovine herpesvirus-1 (BHV-1) establishes a lifelong latent infection in sensory neurons of trigeminal ganglia. BHV-1 periodically reactivates from latency and is shed as infectious virus. The latency-related (LR) gene is abundantly expressed in trigeminal ganglia of infected calves, and proteins encoded by the LR gene are necessary for reactivation from latency. We previously demonstrated that a novel LR protein interacts with a host transcription factor, CCAAT enhancer-binding protein alpha (C/EBPalpha). C/EBPalpha increases plaque-forming efficiency when cotransfected with BHV-1 DNA and its expression is induced in neurons during reactivation from latency (Meyer et al, 2007, J Virol 81: 59-67). The ability of C/EBPalpha to bind DNA is necessary for stimulating productive infection, suggesting C/EBPalpha stimulates viral transcription. We tested whether C/EBPalpha could trans-activate the BHV-1 immediate early transcription unit 1 (IEtu1) promoter because the IEtu1 promoter activates expression of two viral genes (bICP0 and bICP4) that stimulate producitve infection. In the current study, We demonstrate that C/EBPalpha and the BHV-1 trans-inducing factor (bTIF) synergistically trans-activate IEtu1 promoter activity. However, bICP0 and C/EBPalpha did not synergistically trans-activate IEtu1 promoter activity. Deletion of IEtu1 promoter sequences demonstrated that C/EBPalpha by itself could trans-activate a truncated IEtu1 promoter, suggesting sequences in the distal region of the IEtu1 promoter negatively regulate C/EBPalpha activtiy. These studies suggest that C/EBPalpha stimulates productive infection and reactivation from latency, in part, by cooperating with bTIF to activate IEtu1 promoter activity.

Proteogenomic Identification of a Novel Protein-Encoding Gene in Bovine Herpesvirus 1 That Is Expressed during Productive Infection.

Bovine herpesvirus 1 (BoHV-1) is one of several microbes that contributes to the development of the bovine respiratory disease (BRD) and can also induce abortions in cattle. As other alpha-herpesvirinae subfamily members, BoHV-1 efficiently replicates in many cell types and subsequently establishes a life-long latent infection in sensory neurons. BoHV-1 encodes more than 70 proteins that are expressed in a well-defined manner during productive infection. However, in silico open reading frame (ORF) prediction of the BoHV-1 genome suggests that the virus may encode more than one hundred proteins. In this study we used mass spectrometry followed by proteogenomic mapping to reveal the existence of 92 peptides that map to previously un-annotated regions of the viral genome. Twenty-one of the newly termed "intergenic peptides" were predicted to have a viable ORF around them. Twelve of these produced an mRNA transcript as demonstrated by strand-specific RT-PCR. We further characterized the 5' and 3' termini of one mRNA transcript, ORF-A, and detected a 55 kDa protein produced during active infection using a custom-synthesized antibody. We conclude that the coding potential of BoHV-1 is underestimated.