Moriah L. Szpara

Comparative genomic studies of Marek’s disease virus (MDV) conducted in the past two decades have proven powerful for identifying genomic loci driving historical shifts in virulence. However, past efforts were limited to comparing a few strains at a time, lacked access to certain regions of the MDV genome, and were restricted in their ability to characterize viral phenotypes. To address these limitations, we performed whole-genome sequencing on a collection of 65 MDV strains previously characterized using a standardized phenotyping assay. In addition to phylogenetic and recombination analyses, this approach enabled us to perform a genome-wide association study of virulence for this pathogen. In total, we identified 10 genome-wide significant loci associated with virulence, including a tandem repeat variant resulting from two alternative versions of a 132-bp repeating motif. Our findings support prior descriptions of virulence as a complex trait in MDV, and highlight the potential contribution of repeat-based and intergenic variants to the phenotypic diversity of herpesviruses.

Background: Herpes simplex virus (HSV) type 1 and 2 infect over 14,000 newborns each year globally, with most cases (55%) causing a severe outcome that can be fatal if untreated. Neonatal disease severity and long-term outcomes remain unpredictable, and elucidating viral factors that contribute to these outcomes remains a challenge. However, through viral genomic and phenotypic analysis of HSV isolated from neonatal patients, we can begin to connect aspects of viral diversity with clinical patient data. Methods: Here we have applied viral whole genome sequencing to a collection of residual clinical diagnostic samples from ten neonates who had either mild or severe HSV-1 infections. Samples from different body sites or across recurrences allowed for both within- and between-host comparisons. From 30 residual diagnostic samples, we successfully isolated 14 viral cultures. These cultured viruses enabled additional phenotypic analyses, including in vitro growth evaluation in multiple cell types and an in vivo murine model of infection. Results: These data highlight the diversity found in HSV-1 isolated from neonates. Through comparative genomics approaches, we demonstrate both within- and between-host diversity of these samples. Growth of cultured isolates in cell-based assays and a murine model of infection reveal additional aspects of within- and between-host differences. Conclusions: Overall, this study presents the first investigation into the viral diversity of neonatal HSV-1, which is now more common in the US than neonatal HSV-2. Through integration of phenotypic, genomic, and clinical data we characterized these neonatal viral samples to begin unraveling the complexity of neonatal HSV-1 infections.

We report the complete genome sequence of CGI, a strain of varicella-zoster virus (VZV) isolated from a 3-year-old immunocompetent patient with severe herpes zoster. The isolated strain exhibits an ORF62 genotype characteristic of vOka, in addition to a wild-type ORF0 gene and a novel methionine to valine substitution in ORF58.

We sequenced seven herpes simplex virus 2 (HSV2) clinical isolates circulating between 2000 and 2014 in Finland. Few HSV2 virus isolates have been sequenced from Europe, particularly from Nordic countries. These sequences expand our knowledge of the global sequence diversity of HSV2 sequences and help elucidate total variation between geographic regions.

Herpes simplex virus (HSV) is an endemic pathogen, infecting most adults world-wide. HSV infection can cause a wide spectrum of disease outcomes, ranging from asymptomatic infection or mild lesions to rare cases of infectious keratitis, encephalitis, and death. HSV genome sequences have been shown to differ between individual patients, as well as within individuals. To date, the vast majority of publicly available HSV genomic data has come from Europe and North America. Our current understanding of these patterns are missing data from under-sampled populations, particularly in South America, Africa, and Asia. Also missing have been HSV samples from non-industrial (e.g., agricultural, pastoral) populations, for which the natural environment plays a large role in health and disease dynamics. In this study, we capitalized on Whatman FTA card stabilization of DNA to develop a procedure for capturing oral and genital swabs from a geographically isolated pastoralist population in a desert region of northern Namibia. These are the first data to document HSV diversity in this type of remote setting. These are also the first HSV genomes from Namibia. These approaches may prove useful in broadening the accessibility of viral detection for these chronic pathogens, help improve diagnostics, and raise public health awareness about the burden of these pathogens in under-served populations.

Comparative genomic studies of Marek’s disease virus (MDV) have suggested that attenuated and virulent strains share >98% sequence identity. However, these estimates fail to account for variation in regions of the MDV genome harboring tandem repeats. To resolve these loci and enable assessments of intrapopulation diversity, we used a PacBio Sequel II platform to sequence MDV strains CVI988/Rispens (attenuated), HPRS-B14 (virulent), Md5 (very virulent) and 675A (very virulent plus). This approach enabled us to identify patterns of variation in tandem repeat regions that may contribute to the known phenotypic differences between these strains, including the proline-rich regions of the meq oncogene (Meq-PRR) and MDV049/UL36 (UL36-PRR), the multiple telomeric repeats (mTMR) region of the a-like sequence, the promoter region of the latency-associated transcript (LAT), and the MDV006.5/MDV075.2 transcripts. We also found CVI988/Rispens variants showing a 4.3-kb deletion in the Unique Short (US) region, resulting in the loss of SORF1, SORF2, US1, US10, SORF3, and US2. Despite the conventional wisdom that MDV harbors little genomic diversity even when compared to other herpesviruses, we found MDV tandem repeat regions to be highly variable both within individual samples and across strains. In addition to providing a foundation for future studies seeking to explore a potential link between MDV tandem repeats and phenotypic traits like virulence and attenuation, these findings provide detailed support for the premise that DNA viruses can harbor high levels of within-sample diversity in tandem repeat regions.

Comparative genomic studies of Marek's disease virus (MDV) have suggested that attenuated and virulent strains share >98% sequence identity. However, these estimates fail to account for variation in regions of the MDV genome harboring tandem repeats. To resolve these loci and enable assessments of intrapopulation diversity, we used a PacBio Sequel II platform to sequence MDV strains CVI988/Rispens (attenuated), HPRS-B14 (virulent), Md5 (very virulent) and 675A (very virulent plus). This approach enabled us to identify patterns of variation in tandem repeat regions that are consistent with known phenotypic differences across these strains. We also found CVI988/Rispens variants showing a 4.3-kb deletion in the Unique Short (US) region, resulting in the loss of five genes. These findings support a potential link between MDV tandem repeats and phenotypic traits like virulence and attenuation, and demonstrate that DNA viruses can harbor high levels of intrapopulation diversity in tandem repeat regions.

The arrival of novel sequencing technologies throughout the past two decades has led to a paradigm shift in our understanding of herpesvirus genomic diversity. Previously, herpesviruses were seen as a family of DNA viruses with low genomic diversity. However, a growing body of evidence now suggests that herpesviruses exist as dynamic populations that possess standing variation and evolve at much faster rates than previously assumed. In this review, we explore how strategies such as deep sequencing, long-read sequencing, and haplotype reconstruction are allowing scientists to dissect the genomic composition of herpesvirus populations. We also discuss the challenges that need to be addressed before a detailed picture of herpesvirus diversity can emerge.

Current strategies to understand the molecular basis of Marek’s disease virus (MDV) virulence primarily consist of cataloging divergent nucleotides between strains with different phenotypes. However, most comparative genomic studies of MDV rely on previously published consensus genomes despite the confirmed existence of MDV strains as mixed viral populations. To assess the reliability of interstrain genomic comparisons relying on published consensus genomes of MDV, we obtained two additional consensus genomes of vaccine strain CVI988 (Rispens) and two additional consensus genomes of the very virulent strain Md5 by sequencing viral stocks and cultured field isolates. In conjunction with the published genomes of CVI988 and Md5, this allowed us to perform three-way comparisons between multiple consensus genomes of the same strain. We found that consensus genomes of CVI988 can vary in as many as 236 positions involving 13 open reading frames (ORFs). By contrast, we found that Md5 genomes varied only in 11 positions involving a single ORF. Notably, we were able to identify 3 single-nucleotide polymorphisms (SNPs) in the unique long region and 16 SNPs in the unique short (US) region of CVI988_GenBank.BAC that were not present in either CVI988_{Pirbright.lab} or CVI988_{USDA.PA.field}. Recombination analyses of field strains previously described as natural recombinants of CVI988 yielded no evidence of crossover events in the US region when either CVI988_{Pirbright.lab} or CVI988_{USDA.PA.field} were used to represent CVI988 instead of CVI988_GenBank.BAC. We were also able to confirm that both CVI988 and Md5 populations were mixed, exhibiting a total of 29 and 27 high-confidence minor variant positions, respectively. However, we did not find any evidence of minor variants in the positions corresponding to the 19 SNPs in the unique regions of CVI988_GenBank.BAC. Taken together, our findings suggest that continued reliance on the same published consensus genome of CVI988 may have led to an overestimation of genomic divergence between CVI988 and virulent strains and that multiple consensus genomes per strain may be necessary to ensure the accuracy of interstrain genomic comparisons.

Genomic rearrangements contribute to the enhancement of genetic diversity in populations. However, non-canonical rearrangements (NCRs) such as deletions, insertions, and inversions have the potential to trigger genomic instability. In the case of DNA viruses, NCRs can lead to generation of defective viral genomes (DVGs). To study NCRs in herpes simplex virus type 1 (HSV-1) genomes, we enriched DVGs formation by undiluted serial passaging on various cell types. We found that viral passaging on cell type that enables more viral genomes to initiate replication induces higher amplitude and frequency of cyclic patterns associated with DVGs formation. Despite differences in the rates of DVG accumulation, cell lines displayed comparable quantities of distinct NCRs, indicating that fluctuations caused by DVGs may impose bottlenecks on population genetic diversity. These findings propose additional roles for DVGs in modulating viral genetic diversity. Each cell type exhibited a unique population of NCRs, suggesting that NCRs accumulate in a cell type-specific manner. Interestingly, we identified a higher prevalence of short homologies and short reverse complementary in the parental sequences of NCR junction sites across all cell types. These shared sequence characteristics were also observed in NCRs identified in sequences obtained from clinical samples. The fundamental properties of HSV-1 NCR formation uncovered in this study may have broader implications for other DNA viruses. IMPORTANCE: Mutations and genetic rearrangements are the primary driving forces of evolution. Viruses provide valuable model systems for investigating these mechanisms due to their rapid evolutionary rates and vast genetic variability. To investigate genetic rearrangements in the double-stranded DNA genome of herpes simplex virus type 1, the viral population was serially passaged in various cell types. The serial passaging led to formation of defective genomes, resulted from cell-specific non-canonical rearrangements (NCRs). Interestingly, we discovered shared sequence characteristics underlying the formation of these NCRs across all cell types. Moreover, most NCRs identified in clinical samples shared these characteristics. Based on our findings, we propose a model elucidating the formation of NCRs during viral replication within the nucleus of eukaryotic cells.