Rapid genome assembly and comparison decode intrastrain variation in human alphaherpesviruses.

UNLABELLED: Herpes simplex virus (HSV) is a widespread pathogen that causes epithelial lesions with recurrent disease that manifests over a lifetime. The lifelong aspect of infection results from latent viral infection of neurons, a reservoir from which the virus reactivates periodically. Recent work has demonstrated the breadth of genetic variation in globally distributed HSV strains. However, the amount of variation or capacity for mutation within one strain has not been well studied. Here we developed and applied a streamlined new approach for assembly and comparison of large DNA viral genomes such as HSV-1. This viral genome assembly (VirGA) workflow incorporates a combination of de novo assembly, alignment, and annotation strategies to automate the generation of draft genomes for large viruses. We applied this approach to quantify the amount of variation between clonal derivatives of a common parental virus stock. In addition, we examined the genetic basis for syncytial plaque phenotypes displayed by a subset of these strains. In each of the syncytial strains, we found an identical DNA change, affecting one residue in the gB (UL27) fusion protein. Since these identical mutations could have appeared after extensive in vitro passaging, we applied the VirGA sequencing and comparison approach to two clinical HSV-1 strains isolated from the same patient. One of these strains was syncytial upon first culturing; its sequence revealed the same gB mutation. These data provide insight into the extent and origin of genome-wide intrastrain HSV-1 variation and present useful methods for expansion to in vivo patient infection studies. IMPORTANCE: Herpes simplex virus (HSV) infects more than 70% of adults worldwide, causing epithelial lesions and recurrent disease that manifests over a lifetime. Prior work has demonstrated that HSV strains vary from country to country and between individuals. However, the amount of variation within one strain has not been well studied. To address this, we developed a new approach for viral genome assembly (VirGA) and analysis. We used this approach to quantify the amount of variation between sister clones of a common parental virus stock and to determine the basis of a unique fusion phenotype displayed by several variants. These data revealed that while sister clones of one HSV stock are more than 98% identical, these variants harbor enough genetic differences to change their observed characteristics. Comparative genomics approaches will allow us to explore the impacts of viral inter- and intrastrain diversity on drug and vaccine efficacy.

Genome Sequence of the Anterograde-Spread-Defective Herpes Simplex Virus 1 Strain MacIntyre.

We used paired-end Illumina deep sequencing and de novo assembly to determine the genome sequence of herpes simplex virus 1 (HSV-1) strain MacIntyre (aka McIntyre). The MacIntyre strain originated from the brain of a patient with lethal HSV encephalitis and has a unique limitation in its neuronal spread, moving solely in the retrograde direction.

A wide extent of inter-strain diversity in virulent and vaccine strains of alphaherpesviruses.

Alphaherpesviruses are widespread in the human population, and include herpes simplex virus 1 (HSV-1) and 2, and varicella zoster virus (VZV). These viral pathogens cause epithelial lesions, and then infect the nervous system to cause lifelong latency, reactivation, and spread. A related veterinary herpesvirus, pseudorabies (PRV), causes similar disease in livestock that result in significant economic losses. Vaccines developed for VZV and PRV serve as useful models for the development of an HSV-1 vaccine. We present full genome sequence comparisons of the PRV vaccine strain Bartha, and two virulent PRV isolates, Kaplan and Becker. These genome sequences were determined by high-throughput sequencing and assembly, and present new insights into the attenuation of a mammalian alphaherpesvirus vaccine strain. We find many previously unknown coding differences between PRV Bartha and the virulent strains, including changes to the fusion proteins gH and gB, and over forty other viral proteins. Inter-strain variation in PRV protein sequences is much closer to levels previously observed for HSV-1 than for the highly stable VZV proteome. Almost 20% of the PRV genome contains tandem short sequence repeats (SSRs), a class of nucleic acids motifs whose length-variation has been associated with changes in DNA binding site efficiency, transcriptional regulation, and protein interactions. We find SSRs throughout the herpesvirus family, and provide the first global characterization of SSRs in viruses, both within and between strains. We find SSR length variation between different isolates of PRV and HSV-1, which may provide a new mechanism for phenotypic variation between strains. Finally, we detected a small number of polymorphic bases within each plaque-purified PRV strain, and we characterize the effect of passage and plaque-purification on these polymorphisms. These data add to growing evidence that even plaque-purified stocks of stable DNA viruses exhibit limited sequence heterogeneity, which likely seeds future strain evolution.

Preparation of viral DNA from nucleocapsids.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.(1,2,3) This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.(4,5) The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD(260/280;) of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 µg or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 10(6) PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 10(8) plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.(6) For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 µl of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate). Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.