Steven J. Conrad

Marek’s disease (MD) is caused by the highly contagious Marek’s disease virus (MDV), a tumorigenic alphaherpesvirus. The disease is presently managed through vaccination. Nevertheless, the dynamic nature of MDV presents challenges, as new virulent strains may arise that can potentially overcome existing vaccine-mediated immunity. To address this, new and more effective vaccines are crucial. The immune system’s first line of defense against infections is the innate immune response, where type I interferons (IFN-I) play a key role. As with other alphaherpesviruses, MDV encodes numerous genes capable of modulating IFN-I production during infection. Ablation of these genes in MDV could result in attenuation, thereby facilitating the creation of novel vaccine strains with augmented IFN-I induction during infection. Hence, a system to identify immunomodulatory MDV gene products that impede IFN-I production in avian innate immune cells is required. Herein, we present an innovative screening method that quantifies the expression of Interferon Omega 1 (IFNω1) in an avian macrophage-like cell line expressing MDV genes via recombinant lentivirus (rLV) integration. Five MDV gene products hypothesized to inhibit IFN-I production (Meq, US3, R-LORF4, UL46, and UL48) were assayed in our screening system. Stable expression of all MDV genes was obtained when rLV was utilized to insert these genes into HD11 cells. Additionally, we established the optimal conditions for activation of the IFN-I response in HD11 cells using a 2 kb double-stranded DNA segment delivered by transfection as a stimulant. With this approach, we demonstrated that these MDV gene products significantly decreased the gene expression of IFN-I when activated by transfection with double-stranded DNA.

Avian reovirus (ARVs) is an ubiquitous double-stranded RNA (dsRNA) virus that can infect both wild and domestic birds. Although many avian reovirus strains cause no clinical disease, pathogenic variants have been associated with a variety of clinical symptoms. Despite biocontainment measures and vaccination efforts, there has been an increase in pathogenic field isolates in the past two decades, placing a major burden on poultry producers. The increase in the number of ARV cases and outbreaks has prompted a deeper molecular characterization of this pathogen and the molecular mechanisms that drive ARV pathogenesis in poultry. TCID ₅₀ is the preferred method to quantify ARV, but it does not provide specific information about the number of genomes present in a sample. Therefore, there is a need for a molecular method to accurately quantify ARV genomes. Herein, we present a strand-specific quantitative polymerase chain reaction (SS-qPCR) assay to quantify viral genome copy numbers without interference from viral mRNAs and to more accurately characterize viral replication. The SS-qPCR can detect ARV even with as little as 5 × 10 ⁻⁷ ng of cDNA present in the sample, and the limit of detection was estimated to be 200 genome copies per PCR reaction. SS-qPCR was compared to a traditional qPCR assay in the quantification of ARV genomes during viral growth curves in chicken hepatocellular carcinoma (LMH) and quail fibrosarcoma (QM5), revealing the overestimation of genome counts observed with traditional qPCR. Surprisingly, during this process of validation, we noted distinct differences between ARV strains in their degree of cell association that can prompt further investigation in future experiments. IMPORTANCE: Avian reovirus (ARVs) is a pathogen of poultry and wild birds that have become a significant source of disruption in the poultry industry in recent years. Herein, we detail the validation of a new qPCR assay that quantitates only the negative-sense portion of the ARV genome, which allows for the accurate quantification of viral genomes even in the presence of viral gene expression. This assay will enable researchers to precisely quantitate the ARV genome without the overestimates provided by traditional qPCR methods.

Nota de investigación- Determinación de una línea celular aviar adecuada para la cuantificación del reovirus aviar mediante ensayos de placa. Las infecciones por reovirus aviares (ARV) representan una amenaza económica significativa para la producción avícola, a pesar de las medidas de biocontención y vacunación sistemática. Cuantificar la carga de reovirus aviares es esencial para comprender la dinámica de la infección. Existe la idea errónea generalizada de que los reovirus aviares en cultivos no producen placas contables, lo que lleva a la mayoría de los profesionales de campo a utilizar cuantificaciones de dosis infecciosas al 50% en cultivos de tejidos, menos útiles. En este trabajo, comparamos la idoneidad de dos líneas celulares aviares bien conocidas, el clon 5 de mioblastos de codorniz (QM5) y el carcinoma hepatocelular de machos Leghorn (LMH), para su uso en ensayos de placa para la cuantificación de reovirus aviares. Las células LMH, que presentan formación de sincitios tras la infección, resultaron inadecuadas para los ensayos de placa debido a su deficiente adherencia al sustrato, la tendencia a formar conglomerados celulares similares a sincitios, incluso sin infección, y la tendencia de las áreas del sustrato despejadas por el efecto citopático viral (ECP) a ocuparse rápidamente con el crecimiento celular. Por el contrario, las células QM5 demostraron una clara inhibición por contacto y un efecto citopático bien definido, lo que permitió la formación de placas distintivas y contables incluso con altos títulos virales. Por lo tanto, si bien las células LMH son ventajosas para la propagación viral, las células QM5 son más adecuadas para los ensayos de placa destinados a evaluar la infectividad de los reovirus aviares, ya que QM5 permite un seguimiento y una cuantificación más precisos del crecimiento viral.

Avian reovirus (ARV) remains a significant concern in the poultry industry due to its economic impact and genetic diversity. Three rounds of plaque purification are routinely employed to obtain clonal populations of a viral isolate for experimental purposes. However, the effectiveness of this approach in achieving viral homogeneity has not been evaluated. This study aimed to determine the purity of a plaque-purified ARV isolate (strain AL) using conventional PCR, Sanger sequencing, and whole genome sequencing (WGS). While conventional PCR targeting the sigma C (σC) gene failed to amplify the AL isolate using standard primers, de novo primers based on WGS successfully detected its presence. Sanger sequencing of the σC gene confirmed sequence divergence from the reference S1133 strain with only ~44% amino acid identity. This placed the AL isolate in genetic cluster GC4, which was phylogenetically farthest from the vaccine group GC1. WGS analysis revealed the presence of mixed viral populations despite three rounds of plaque purification on chicken embryo liver cells (CELi) and both S1133-like and divergent contigs were found in the assembled genome. These findings indicate that plaque purification in chicken embryo liver cells may not ensure clonal isolation of ARV.

Since viruses are obligate intracellular pathogens, sequencing their genomes results in metagenomic data from both the virus and the host. Virology researchers are constantly seeking new, cost-effective strategies and bioinformatic pipelines for the retrieval of complete viral genomes from these metagenomic samples. Avian orthoreoviruses (ARVs) pose a significant and growing threat to the poultry industry and frequently cause economic losses associated with disease in production birds. Currently available commercial vaccines are ineffective against new ARV variants and ARV outbreaks are increasing worldwide, requiring whole genome sequencing (WGS) to characterize strains that evade vaccines. This study compares the effectiveness of long-read and short-read sequencing technologies for obtaining ARV complete genomes. We used eight clinical isolates of ARV, each previously processed using our published viral genome enrichment protocol. Additionally, we evaluate three assembly methods to determine which provided the most complete and reliable whole genomes: De novo, reference-guided or hybrid. The results suggest that our ARV genome enrichment protocol caused some fragmentation of the viral cDNA that impacted the length of the long reads (but not the short reads) and, as a result, caused a failure to produce complete genomes via de novo assembly. Overall, we observed that regardless of the sequencing technology, the best quality assemblies were generated by mapping quality-trimmed reads to a custom reference genome. The custom reference genomes were in turn constructed with the publicly available ARV genomic segments that shared the highest sequence similarity with the contigs from short-read de novo assemblies. Hence, we conclude that short-read sequencing is the most suitable technology to combine with our ARV genome enrichment protocol.

Avian reovirus (ARV) is an emerging pathogen which causes significant economic challenges to the chicken and turkey industry in the USA and globally, yet the molecular characterization of most ARV strains is restricted to a single particular gene, the sigma C gene. The genome of arthrogenic reovirus field isolates (R18-37308 and R18-38167), isolated from broiler chickens in North Carolina (NC), USA in 2018, was sequenced using long-read next-generation sequencing (NGS). The isolates were genotyped based on the amino acid sequence of sigma C (σC) followed by phylogenetic and amino acid analyses of the other 11 genomically encoded proteins for whole genomic constellation and genetic variation detection. The genomic length of the NC field strains was 23,494 bp, with 10 dsRNA segments ranging from 3959 bp (L1) to 1192 bp (S4), and the 5′ and 3′ untranslated regions (UTRs) of all the segments were found to be conserved. R18-37308 and R18-38167 were found to belong to genotype (G) VI based on the σC analysis and showed nucleotide and amino acid sequence identity ranging from 84.91–98.47% and 83.43–98.46%, respectively, with G VI strains. Phylogenetic analyses of individual genes of the NC strains did not define a single common ancestor among the available completely sequenced ARV strains. Nevertheless, most sequences supported the Chinese strain LY383 as a probable ancestor of these isolates. Moreover, amino acid analysis revealed multiple amino acid substitution events along the entirety of the genes, some of which were unique to each strain, which suggests significant divergence owing to the accumulation of point mutations. All genes from R18-37308 and R18-38167 were found to be clustered within genotypic clusters that included only ARVs of chicken origin, which negates the possibility of genetic pooling or host variation. Collectively, this study revealed sequence divergence between the NC field strains and reference ARV strains, including the currently used vaccine strains could help updating the vaccination regime through the inclusion of these highly divergent circulating indigenous field isolates.

To enhance the efficacy of the current Newcastle disease vaccine, we have selected potential adjuvants that target well-characterized pattern recognition receptors: the toll-like receptors (TLRs). Imiquimod is a small-molecule activator of TLR7, which is a sensor of dsDNA. ODN-1826 is a mimetic of CpG DNA and ligates TLR21 (a chicken homologue of TLR9 in mammals). The activation of TLRs leads to antiviral responses, including the induction of type I interferons (IFNs). In this study, birds were vaccinated intranasally with a live LaSota strain with or without imiquimod or ODN-1826 (50 µg/bird). Two weeks after vaccination, the birds were challenged with a virulent Newcastle disease virus (chicken/CA/212676/2002). Both adjuvants (imiquimod or ODN-1826) induced higher and more uniform antibody titers among vaccinated birds compared with the live vaccine-alone group. In addition, adjuvanted vaccines demonstrated greater protective efficacy in terms of the reduction in virus-shedding titer and the number of birds shedding the challenge virus at 2 and 4 days post-challenge. A differential expression of antiviral and immune-related genes was observed among groups from tissues (Harderian gland, trachea, cecal tonsil, and spleen) collected 1 and 3 days after treatment. These results demonstrate the potential of TLR-targeted adjuvants as mucosal vaccine enhancers and warrant a further characterization of immune correlates and optimization for efficacy.

Whole-genome sequencing (WGS) is becoming an essential tool to characterize the genomes of avian reovirus (ARV), a viral disease of economic significance to poultry producers. The current strategies and procedures used to obtain the complete genome sequences of ARV isolates are not cost-effective because most of the genetic material data resulting from next-generation sequencing belong to the host and cannot be used to assemble the viral genome. The purpose of this study was to develop a workflow to enrich the ARV genomic content in a sample before subjecting it to next-generation sequencing (NGS). Herein, we compare four different ARV purification and enrichment approaches at the virion, RNA and cDNA levels to determine which treatment or treatment combination would provide a higher proportion of ARV-specific reads after WGS. Seven ARV isolates were subjected to different combinations of virion purification via ultracentrifugation in sucrose density gradient or Capto Core 700 resin with or without a subsequent Benzonase treatment, followed by a chicken rRNA depletion step after RNA extraction and a final ARV cDNA amplification step using a single-primer amplification assay. Our results show that the combination of Capto Core 700 resin, Chicken rRNA depletion and cDNA amplification is the most cost-effective strategy to obtain ARV whole genomes after short-read sequencing.