Comparison of analytical performance and economic value of two biosurveillance methods for tracking SARS-COV-2 variants of concern.

The development of biosurveillance programs with strong analytical performance and economically accessible protocols is essential for monitoring viral pathogens. Throughout the COVID-19 pandemic, whole-genome sequencing (WGS) has been the prevailing technology for SARS-CoV-2 variant of concern (VOC) detection. While WGS offers benefits, it is a lengthy process, financially and technically straining for scalable viral tracking. The aim of this study was to compare the analytical performance and economic feasibility of WGS and PCR mutation panels for distinguishing six known VOCs: Alpha (B.1.1.7 and Q.4), Gamma (P.1), Delta (B.1.617.2 and AY.4.2), and Omicron. (B.1.1.529.1). In all, 78 SARS-CoV-2-positive samples were collected from April to December 2021 at Northeastern University (Cabot Testing Site, Boston, MA, USA) for genotyping PCR and WGS analysis. MagMax Viral/Pathogen II Nucleic Acid Isolation and TaqPath COVID-19 Combo Kits were used for RNA extraction and SARS-CoV-2 confirmation. VOC discrimination was assessed using two TaqMan SARS-CoV-2 single nucleotide polymorphism (SNP) assay layouts, and Ion Torrent WGS. In November 2021, the mutation panel demonstrated marked versatility by detecting the emerging Omicron variant reported by South Africa. SNP panel analysis yielded the following 78 VOC identifications: Alpha B.1.1.7 (N = 20), Alpha Q.4 (N = 3), Gamma P.1 (N = 1), Delta B.1.617.2 (N = 30), Delta AY.4.2 (N = 3), and Omicron B.1.1.529.1 (N = 20) with one undetermined (N = 1) sample. Genotyping mutation panels designated lineages in 77 of 78 samples, 46/78 were confirmed by WGS, while 32 samples failed WGS lineage assignment. RT-PCR genotyping panels offer pronounced throughput and sensitivity and provide an economically advantageous technique for SARS-CoV-2 biosurveillance.IMPORTANCEThe results presented in our manuscript demonstrate how the value of simplistic and reliable molecular assays coupled with the core scientific principle of standardization can be overlooked by the charm of more sophisticated assays and instrumentation. This effect can often be amplified during tumultuous public health events, such as the COVID-19 pandemic. By adapting standardized PCR mutation panels to detect prominently circulating SARS-CoV-2 variants, we were able to better assess the potential health impacts of rising positivity rates and transmission clusters within the Northeastern University population. While several literature publications utilizing genotyping PCR and NGS have a similar scope to ours, many investigations lack sufficiently standardized genotyping PCR and NGS bioinformatics inclusionary/exclusionary criteria for SARS-CoV-2 variant identification. Finally, the economic benefits of standardized PCR mutation panels would allow for global implementation of biosurveillance, rather than reserving biosurveillance to more economically developed nations.

Clinical performance evaluation of TAQPATH Enteric Bacterial Select Panel for the detection of common enteric bacterial pathogens in comparison to routine stool culture and other qPCR-based diagnostic tests.

IMPORTANCE: Enteric bacterial infections caused by Salmonella, Shigella, pathogenic Escherichia coli, and Campylobacter represent one of the most common causes of infectious enteritis worldwide. The timely and accurate diagnosis of pathogens causing gastroenteritis is crucial for patient care, public health, and disease surveillance. While stool culture has long been the standard and highly specific method for detecting enteric pathogens, it is labor-intensive and time-consuming with limited sensitivity. To improve patient outcomes, there is a need to implement new cost-effective approaches for the detection of bacterial enteric pathogens with higher sensitivity and faster time to result. This study shows that multiplex real-time polymerase chain reaction-based tests, such as the TAQPATH Enteric Bacterial Select Panel, are accurate and cost-effective diagnostic alternatives for the detection and differentiation of the most common enteric bacterial pathogens, offering quicker time to result and higher sensitivity compared to routine stool culture.

Cross-sectional Ct distributions from qPCR tests can provide an early warning signal for the spread of COVID-19 in communities.

Background: SARS-CoV-2 PCR testing data has been widely used for COVID-19 surveillance. Existing COVID-19 forecasting models mainly rely on case counts obtained from qPCR results, even though the binary PCR results provide a limited picture of the pandemic trajectory. Most forecasting models have failed to accurately predict the COVID-19 waves before they occur. Recently a model utilizing cross-sectional population cycle threshold (Ct-the number of cycles required for the fluorescent signal to cross the background threshold) values obtained from PCR tests (Ct-based model) was developed to overcome the limitations of using only binary PCR results. In this study, we aimed to improve on COVID-19 forecasting models using features derived from the Ct-based model, to detect epidemic waves earlier than case-based trajectories. Methods: PCR data was collected weekly at Northeastern University (NU) between August 2020 and January 2022. Campus and county epidemic trajectories were generated from case counts. A novel forecasting approach was developed by enhancing a recent deep learning model with Ct-based features and applied in Suffolk County and NU campus. For this, cross-sectional Ct values from PCR data were used to generate Ct-based epidemic trajectories, including effective reproductive rate (Rt) and incidence. The improvement in forecasting performance was compared using absolute errors and residual squared errors with respect to actual observed cases at the 7-day and 14-day forecasting horizons. The model was also tested prospectively over the period January 2022 to April 2022. Results: Rt curves estimated from the Ct-based model indicated epidemic waves 12 to 14 days earlier than Rt curves from NU campus and Suffolk County cases, with a correlation of 0.57. Enhancing the forecasting models with Ct-based information significantly decreased absolute error (decrease of 49.4 and 221.5 for the 7 and 14-day forecasting horizons) and residual squared error (40.6 and 217.1 for the 7 and 14-day forecasting horizons) compared to the original model without Ct features. Conclusion: Ct-based epidemic trajectories can herald an earlier signal for impending epidemic waves in the community and forecast transmission peaks. Moreover, COVID-19 forecasting models can be enhanced using these Ct features to improve their forecasting accuracy. In this study, we make the case that public health agencies should publish Ct values along with the binary positive/negative PCR results. Early and accurate forecasting of epidemic waves can inform public health policies and countermeasures which can mitigate spread.

Expanded PCR Panel Testing for Identification of Respiratory Pathogens and Coinfections in Influenza-like Illness.

While COVID-19 has dominated Influenza-like illness (ILI) over the past few years, there are many other pathogens responsible for ILI. It is not uncommon to have coinfections with multiple pathogens in patients with ILI. The goal of this study was to identify the different organisms in symptomatic patients presenting with ILI using two different high throughput multiplex real time PCR platforms. Specimens were collected from 381 subjects presenting with ILI symptoms. All samples (nasal and nasopharyngeal swabs) were simultaneously tested on two expanded panel PCR platforms: Applied Biosystems™ TrueMark™ Respiratory Panel 2.0, OpenArray™ plate (OA) (32 viral and bacterial targets); and Applied Biosystems™ TrueMark™ Respiratory Panel 2.0, TaqMan™ Array card (TAC) (41 viral, fungal, and bacterial targets). Results were analyzed for concordance between the platforms and for identification of organisms responsible for the clinical presentation including possible coinfections. Very good agreement was observed between the two PCR platforms with 100% agreement for 12 viral and 3 bacterial pathogens. Of 381 specimens, approximately 58% of the samples showed the presence of at least one organism with an important incidence of co-infections (~36-40% of positive samples tested positive for two and more organisms). S. aureus was the most prevalent detected pathogen (~30%) followed by SARS-CoV-2 (~25%), Rhinovirus (~15%) and HHV6 (~10%). Co-infections between viruses and bacteria were the most common (~69%), followed by viral-viral (~23%) and bacterial-bacterial (~7%) co-infections. These results showed that coinfections are common in RTIs suggesting that syndromic panel based multiplex PCR tests could enable the identification of pathogens contributing to coinfections, help guide patient management thereby improving clinical outcomes and supporting antimicrobial stewardship.

Efficient SARS-CoV-2 Surveillance during the Pandemic-Endemic Transition Using PCR-Based Genotyping Assays.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC) pose an increased risk to public health due to higher transmissibility and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 10 selected real-time PCR (RT-PCR) genotyping assays compared to whole-genome sequencing (WGS) for identification of 5 VOC circulating in The Netherlands. SARS-CoV-2 positive samples (N = 664), collected during routine PCR screening (15 ≤ CT ≤ 32) between May-July 2021 and December 2021-January 2022, were selected and analyzed using the RT-PCR genotyping assays. VOC lineage was determined based on the detected mutation profile. In parallel, all samples underwent WGS with the Ion AmpliSeq SARS-CoV-2 research panel. Among 664 SARS-CoV-2 positive samples, the RT-PCR genotyping assays classified 31.2% as Alpha (N = 207); 48.9% as Delta (N = 325); 19.4% as Omicron (N = 129), 0.3% as Beta (N = 2), and 1 sample as a non-VOC. Matching results were obtained using WGS in 100% of the samples. RT-PCR genotyping assays enable accurate detection of SARS-CoV-2 VOC. Furthermore, they are easily implementable, and the costs and turnaround time are significantly reduced compared to WGS. For this reason, a higher proportion of SARS-CoV-2 positive cases in the VOC surveillance testing can be included, while reserving valuable WGS resources for identification of new variants. Therefore, RT-PCR genotyping assays would be a powerful method to include in SARS-CoV-2 surveillance testing. IMPORTANCE The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome changes constantly. It is estimated that there are thousands of variants of SARS-CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists, and public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, and develop counter measures like vaccines. The technique used for the pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of SARS-CoV-2. In this study, a new PCR method based on the detection of specific changes of those building blocks is presented. This method enables a fast, accurate and cheap determination of different SARS-CoV-2 VOC. Therefore, it would be a powerful method to include in SARS-CoV-2 surveillance testing.

Clinical Performance of Direct RT-PCR Testing of Raw Saliva for Detection of SARS-CoV-2 in Symptomatic and Asymptomatic Individuals.

RT-PCR tests based on RNA extraction from nasopharyngeal swabs (NPS) are promoted as the "gold standard" for SARS-CoV-2 detection. However, the use of saliva samples offers noninvasive self-collection more suitable for high-throughput testing. This study evaluated performance of the TaqPath COVID-19 Fast PCR Combo kit 2.0 assay for detection of SARS-CoV-2 in raw saliva relative to a lab-developed direct RT-PCR test (SalivaDirect-based PCR, SDB-PCR) and an RT-PCR test based on RNA extraction from NPS. Saliva and NPS samples were collected from symptomatic and asymptomatic individuals (N = 615). Saliva samples were tested for SARS-CoV-2 using the TaqPath COVID-19 Fast PCR Combo kit 2.0 and the SDB-PCR, while NPS samples were tested by RT-PCR in RNA extracts according to the Irish national testing system. TaqPath COVID-19 Fast PCR Combo kit 2.0 detected SARS-CoV-2 in 52 saliva samples, of which 51 were also positive with the SDB-PCR. Compared to the NPS "gold standard" biospecimen method, 49 samples displayed concordant results, while three samples (35

Global profiling reveals common and distinct N6-methyladenosine (m6A) regulation of innate immune responses during bacterial and viral infections.

N6-methyladenosine (m6A) is a dynamic post-transcriptional RNA modification influencing all aspects of mRNA biology. While m6A modifications during numerous viral infections have been described, the role of m6A in innate immune response remains unclear. Here, we examined cellular m6A epitranscriptomes during infections of Pseudomonas aeruginosa and herpes simplex virus type 1 (HSV-1), and lipopolysaccharide (LPS) stimulation to identify m6A-regulated innate immune response genes. We showed that a significant portion of cellular genes including many innate immune response genes underwent m6A modifications in 5'UTR and 3'UTR. We identified common and distinct m6A-modified genes under different stimulating conditions. Significantly, the expression of a subset of innate immune response genes was positively correlated with m6A level. Importantly, we identified genes that had significant enrichments of m6A peaks during P. aeruginosa infection following knockdown of m6A "eraser" ALKBH5, confirming the regulation of these genes by m6A and ALKBH5. Among them, we confirmed the association of m6A modification with gene expression in immune response genes TNFAIP3, IFIT1, IFIT2 and IFIH1. Taken together, our results revealed the vital role of m6A in regulating innate immunity against bacterial and viral infections. These works also provided rich resources for the scientific community.

Insights into the pathogenesis of varicella viruses.

PURPOSE OF REVIEW: Varicella zoster virus (VZV) is a highly contagious, neurotropic alpha herpes virus that causes varicella (chickenpox). VZV establishes lifelong latency in the sensory ganglia from which it can reactivate to induce herpes zoster (HZ), a painful disease that primarily affects older individuals and those who are immune-suppressed. Given that VZV infection is highly specific to humans, developing a reliable in vivo model that recapitulates the hallmarks of VZV infection has been challenging. Simian Varicella Virus (SVV) infection in nonhuman primates reproduces the cardinal features of VZV infections in humans and allows the study of varicella virus pathogenesis in the natural host. In this review, we summarize our current knowledge about genomic and virion structure of varicelloviruses as well as viral pathogenesis and antiviral immune responses during acute infection, latency and reactivation. We also examine the immune evasion mechanisms developed by varicelloviruses to escape the host immune responses and the current vaccines available for protecting individuals against chickenpox and herpes zoster. RECENT FINDINGS: Data from recent studies suggest that infected T cells are important for viral dissemination to the cutaneous sites of infection as well as site of latency and that a viral latency-associated transcript might play a role in the transition from lytic infection to latency and then reactivation. SUMMARY: Recent studies have provided exciting insights into mechanisms of varicelloviruses pathogenesis such as the critical role of T cells in VZV/SVV dissemination from the respiratory mucosa to the skin and the sensory ganglia; the ability of VZV/SVV to interfere with host defense; and the identification of VLT transcripts in latently infected ganglia. However, our understanding of these phenomena remains poorly understood. Therefore, it is critical that we continue to investigate host-pathogen interactions during varicelloviruses infection. These studies will lead to a deeper understanding of VZV biology as well as novel aspects of cell biology.

Molecular Biology of KSHV in Relation to HIV/AIDS-Associated Oncogenesis.

Discovered in 1994, Kaposi's sarcoma-associated herpesvirus (KSHV) has been associated with four human malignancies including Kaposi's sarcoma, primary effusion lymphoma, a subset of multicentric Castleman's disease, and KSHV inflammatory cytokine syndrome. These malignancies mostly occur in immunocompromised patients including patients with acquired immunodeficiency syndrome and often cause significant mortality because of the lack of effective therapies. Significant progresses have been made to understand the molecular basis of KSHV infection and KSHV-induced oncogenesis in the last two decades. This chapter provides an update on the recent advancements focusing on the molecular events of KSHV primary infection, the mechanisms regulating KSHV life cycle, innate and adaptive immunity, mechanism of KSHV-induced tumorigenesis and inflammation, and metabolic reprogramming in KSHV infection and KSHV-transformed cells.

Varicella Virus-Host Interactions During Latency and Reactivation: Lessons From Simian Varicella Virus.

Varicella zoster virus (VZV) is a neurotropic alphaherpesvirus and the causative agent of varicella (chickenpox) in humans. Following primary infection, VZV establishes latency in the sensory ganglia and can reactivate to cause herpes zoster, more commonly known as shingles, which causes significant morbidity, and on rare occasions mortality, in the elderly. Because VZV infection is highly restricted to humans, the development of a reliable animal model has been challenging, and our understanding of VZV pathogenesis remains incomplete. As an alternative, infection of rhesus macaques with the homologous simian varicella virus (SVV) recapitulates the hallmarks of VZV infection and thus constitutes a robust animal model to provide critical insights into VZV pathogenesis and the host antiviral response. In this model, SVV infection results in the development of varicella during primary infection, generation of an adaptive immune response, establishment of latency in the sensory ganglia, and viral reactivation upon immune suppression. In this review, we discuss our current knowledge about host and viral factors involved in the establishment of SVV latency and reactivation as well as the important role played by T cells in SVV pathogenesis and antiviral immunity.

Viral and cellular N6-methyladenosine and N6,2'-O-dimethyladenosine epitranscriptomes in the KSHV life cycle.

N6-methyladenosine (m6A) and N6,2'-O-dimethyladenosine (m6Am) modifications (m6A/m) of messenger RNA mediate diverse cellular functions. Oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) has latent and lytic replication phases that are essential for the development of KSHV-associated cancers. To date, the role of m6A/m in KSHV replication and tumorigenesis is unclear. Here, we provide mechanistic insights by examining the viral and cellular m6A/m epitranscriptomes during KSHV latent and lytic infection. KSHV transcripts contain abundant m6A/m modifications during latent and lytic replication, and these modifications are highly conserved among different cell types and infection systems. Knockdown of YTHDF2 enhanced lytic replication by impeding KSHV RNA degradation. YTHDF2 binds to viral transcripts and differentially mediates their stability. KSHV latent infection induces 5' untranslated region (UTR) hypomethylation and 3'UTR hypermethylation of the cellular epitranscriptome, regulating oncogenic and epithelial-mesenchymal transition pathways. KSHV lytic replication induces dynamic reprogramming of epitranscriptome, regulating pathways that control lytic replication. These results reveal a critical role of m6A/m modifications in KSHV lifecycle and provide rich resources for future investigations.

The Critical Role of Genome Maintenance Proteins in Immune Evasion During Gammaherpesvirus Latency.

Gammaherpesviruses are important pathogens that establish latent infection in their natural host for lifelong persistence. During latency, the viral genome persists in the nucleus of infected cells as a circular episomal element while the viral gene expression program is restricted to non-coding RNAs and a few latency proteins. Among these, the genome maintenance protein (GMP) is part of the small subset of genes expressed in latently infected cells. Despite sharing little peptidic sequence similarity, gammaherpesvirus GMPs have conserved functions playing essential roles in latent infection. Among these functions, GMPs have acquired an intriguing capacity to evade the cytotoxic T cell response through self-limitation of MHC class I-restricted antigen presentation, further ensuring virus persistence in the infected host. In this review, we provide an updated overview of the main functions of gammaherpesvirus GMPs during latency with an emphasis on their immune evasion properties.

Macavirus latency-associated protein evades immune detection through regulation of protein synthesis in cis depending upon its glycin/glutamate-rich domain.

Alcelaphine herpesvirus 1 (AlHV-1) is a γ-herpesvirus (γ-HV) belonging to the macavirus genus that persistently infects its natural host, the wildebeest, without inducing any clinical sign. However, cross-transmission to other ruminant species causes a deadly lymphoproliferative disease named malignant catarrhal fever (MCF). AlHV-1 ORF73 encodes the latency-associated nuclear antigen (LANA)-homolog protein (aLANA). Recently, aLANA has been shown to be essential for viral persistence in vivo and induction of MCF, suggesting that aLANA shares key properties of other γ-HV genome maintenance proteins. Here we have investigated the evasion of the immune response by aLANA. We found that a glycin/glutamate (GE)-rich repeat domain was sufficient to inhibit in cis the presentation of an epitope linked to aLANA. Although antigen presentation in absence of GE was dependent upon proteasomal degradation of aLANA, a lack of GE did not affect protein turnover. However, protein self-synthesis de novo was downregulated by aLANA GE, a mechanism directly associated with reduced antigen presentation in vitro. Importantly, codon-modification of aLANA GE resulted in increased antigen presentation in vitro and enhanced induction of antigen-specific CD8+ T cell responses in vivo, indicating that mRNA constraints in GE rather than peptidic sequence are responsible for cis-limitation of antigen presentation. Nonetheless, GE-mediated limitation of antigen presentation in cis of aLANA was dispensable during MCF as rabbits developed the disease after virus infection irrespective of the expression of full-length or GE-deficient aLANA. Altogether, we provide evidence that inhibition in cis of protein synthesis through GE is likely involved in long-term immune evasion of AlHV-1 latent persistence in the wildebeest natural host, but dispensable in MCF pathogenesis.

Genomic duplication and translocation of reactivation transactivator and bZIP-homolog genes is a conserved event in alcelaphine herpesvirus 1.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces malignant catarrhal fever (MCF), a fatal lymphoproliferative disease of ruminants, including cattle. The strain C500 has been cloned as an infectious, pathogenic bacterial artificial chromosome (BAC) that is used to study MCF. Although AlHV-1 infection can be established in cell culture, multiple passages in vitro cause a loss of virulence associated with rearrangements of the viral genome. Here, sequencing of the BAC clone showed that the long unique region (LUR) of the genome is nearly identical to that of the previously sequenced strain from which the BAC was derived, and identified the duplication and translocation of a region from within LUR, containing the entire coding sequences of ORF50-encoding reactivation transactivator Rta and A6-encoding bZIP protein genes. The duplicated region was further located to a position within the terminal repeat (TR) and its deletion resulted in lower ORF50 expression levels and reduced viral fitness. Finally, the presence of a similar but not identical duplication and translocation containing both genes was found in AlHV-1 strain WC11. These results indicate that selection pressure for enhanced viral fitness may drive the duplication of ORF50 and A6 in AlHV-1.

MicroRNAs in large herpesvirus DNA genomes: recent advances.

MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that regulate gene expression. They alter mRNA translation through base-pair complementarity, leading to regulation of genes during both physiological and pathological processes. Viruses have evolved mechanisms to take advantage of the host cells to multiply and/or persist over the lifetime of the host. Herpesviridae are a large family of double-stranded DNA viruses that are associated with a number of important diseases, including lymphoproliferative diseases. Herpesviruses establish lifelong latent infections through modulation of the interface between the virus and its host. A number of reports have identified miRNAs in a very large number of human and animal herpesviruses suggesting that these short non-coding transcripts could play essential roles in herpesvirus biology. This review will specifically focus on the recent advances on the functions of herpesvirus miRNAs in infection and pathogenesis.

Small RNA deep sequencing identifies viral microRNAs during malignant catarrhal fever induced by alcelaphine herpesvirus 1.

Alcelaphine herpesvirus 1 (AlHV-1) is a c-herpesvirus (c-HV) carried asymptomatically by wildebeest. Upon cross-species transmission, AlHV-1 induces a fatal lymphoproliferative disease named malignant catarrhal fever (MCF) in many ruminants, including cattle, and the rabbit model. Latency has been shown to be essential for MCF induction. However, the mechanisms causing the activation and proliferation of infected CD8+T cells are unknown. Many c-HVs express microRNAs (miRNAs). These small non-coding RNAs can regulate expression of host or viral target genes involved in various pathways and are thought to facilitate viral infection and/or mediate activation and proliferation of infected lymphocytes. The AlHV-1 genome has been predicted to encode a large number of miRNAs. However, their precise contribution in viral infection and pathogenesis in vivo remains unknown. Here, using cloning and sequencing of small RNAs we identified 36 potential miRNAs expressed in a lymphoblastoid cell line propagated from a calf infected with AlHV-1 and developing MCF. Among the sequenced candidate miRNAs, 32 were expressed on the reverse strand of the genome in two main clusters. The expression of these 32 viral miRNAs was further validated using Northern blot and quantitative reverse transcription PCR in lymphoid organs of MCF developing calves or rabbits. To determine the concerted contribution in MCF of 28 viralmiRNAs clustered in the non-protein-coding region of the AlHV-1 genome, a recombinant virus was produced. The absence of these 28 miRNAs did not affect viral growth in vitro or MCF induction in rabbits, indicating that the AlHV-1 miRNAs clustered in this non-protein-coding genomic region are dispensable for MCF induction.

Viral semaphorin inhibits dendritic cell phagocytosis and migration but is not essential for gammaherpesvirus-induced lymphoproliferation in malignant catarrhal fever.

UNLABELLED: Viral semaphorins are semaphorin 7A (sema7A) mimics found in pox- and herpesviruses. Among herpesviruses, semaphorins are encoded by gammaherpesviruses of the Macavirus genus only. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus that persistently infects wildebeest asymptomatically but induces malignant catarrhal fever (MCF) when transmitted to several species of susceptible ruminants and the rabbit model. MCF is caused by the activation/proliferation of latently infected T lymphocytes. Viral semaphorins have been suggested to mediate immune evasion mechanisms and/or directly alter host T cell function. We studied AlHV-sema, the viral semaphorin encoded by the A3 gene of AlHV-1. Phylogenetic analyses revealed independent acquisition of pox- and herpesvirus semaphorins, suggesting that these proteins might have distinct functions. AlHV-sema showed a predicted three-dimensional structure very similar to sema7A and conserved key residues in sema7A-plexinC1 interaction. Expression analyses revealed that AlHV-sema is a secreted 93-kDa glycoprotein expressed during the early phase of virus replication. Purified AlHV-sema was able to bind to fibroblasts and dendritic cells and induce F-actin condensation and cell retraction through a plexinC1 and Rho/cofilin-dependent mechanism. Cytoskeleton rearrangement was further associated with inhibition of phagocytosis by dendritic cells and migration to the draining lymph node. Next, we generated recombinant viruses and demonstrated that the lack of A3 did not significantly affect virus growth in vitro and did not impair MCF induction and associated lymphoproliferative lesions. In conclusion, AlHV-sema has immune evasion functions through mechanisms similar to poxvirus semaphorin but is not directly involved in host T cell activation during MCF. IMPORTANCE: Whereas most poxviruses encode viral semaphorins, semaphorin-like genes have only been identified in few gammaherpesviruses belonging to the Macavirus genus. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus carried asymptomatically by wildebeest but induces a latency-associated lymphoproliferative disease of T lymphocytes in various ruminant species, namely, malignant catarrhal fever (MCF). Viral semaphorins have been hypothesized to have immune evasion functions and/or be involved in activating latently infected T cells. We present evidence that the viral semaphorin AlHV-sema inhibits dendritic cell phagocytosis and migration to the draining lymph node, both being indispensable mechanisms for protective antiviral responses. Next, we engineered recombinant viruses unable to express AlHV-sema and demonstrated that this protein is dispensable for the induction of MCF. In conclusion, this study suggests that herpesvirus and poxvirus semaphorins have independently evolved similar functions to thwart the immune system of the host while AlHV-sema is not directly involved in MCF-associated T-cell activation.

An essential role for γ-herpesvirus latency-associated nuclear antigen homolog in an acute lymphoproliferative disease of cattle.

Wildebeests carry asymptomatically alcelaphine herpesvirus 1 (AlHV-1), a γ-herpesvirus inducing malignant catarrhal fever (MCF) to several ruminant species (including cattle). This acute and lethal lymphoproliferative disease occurs after a prolonged asymptomatic incubation period after transmission. Our recent findings with the rabbit model indicated that AlHV-1 infection is not productive during MCF. Here, we investigated whether latency establishment could explain this apparent absence of productive infection and sought to determine its role in MCF pathogenesis. First, whole-genome cellular and viral gene expression analyses were performed in lymph nodes of MCF-developing calves. Whereas a severe disruption in cellular genes was observed, only 10% of the entire AlHV-1 genome was expressed, contrasting with the 45% observed during productive infection in vitro. In vivo, the expressed viral genes included the latency-associated nuclear antigen homolog ORF73 but none of the regions known to be essential for productive infection. Next, genomic conformation analyses revealed that AlHV-1 was essentially episomal, further suggesting that MCF might be the consequence of a latent infection rather than abortive lytic infection. This hypothesis was further supported by the high frequencies of infected CD8(+) T cells during MCF using immunodetection of ORF73 protein and single-cell RT-PCR approaches. Finally, the role of latency-associated ORF73 was addressed. A lack of ORF73 did not impair initial virus replication in vivo, but it rendered AlHV-1 unable to induce MCF and persist in vivo and conferred protection against a lethal challenge with a WT virus. Together, these findings suggest that a latent infection is essential for MCF induction.

La forme africaine du coryza gangreneux est une maladie lymphoproliférative létale causée par l'herpèsvirus alcélaphin 1.

Gammaherpesviruses include viruses able to induce lymphoproliferative diseases and tumors. These viruses can also establish a long and persistent asymptomatic infection in their natural host. Among gammaherpesviruses, AlHV-1 belongs to the Macavirus genus and infects asymptomatically its natural host, the wildebeest (Connochaetes spp.). However, this virus induces a lethal lymphoproliferative disease named the African form of malignant catarrhal fever (MCF) when transmitted to a large number of susceptible species belonging to the Artiodactyla order. Control of MCF in regions where AlHV-1 is endemic is of great importance and directly depends on the understanding of the pathogenic mechanisms responsible for the induction of MCF. The goal of the present review is to summarize current knowledge on AlHV-1 and MCF with a particular interest on the mechanisms used by AlHV-1 to induce the disease. Amongst different models of pathogenesis, the particular role of viral latency will be addressed based on current data. Finally, the evolutionary relationship between the wildebeest, AlHV-1 and the susceptible species to MCF will be discussed.