Identification and quantification of defective virus genomes in high throughput sequencing data using DVG-profiler, a novel post-sequence alignment processing algorithm.

Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective helper virus. DVGs, especially of the copyback type, frequently observed with paramyxoviruses, have been recognized to be important triggers of the antiviral innate immune response. DVGs have therefore gained interest for their potential to alter the attenuation and immunogenicity of vaccines. To investigate this potential, accurate identification and quantification of DVGs is essential. Conventional methods, such as RT-PCR, are labor intensive and will only detect primer sequence-specific species. High throughput sequencing (HTS) is much better suited for this undertaking. Here, we present an HTS-based algorithm called DVG-profiler to identify and quantify all DVG sequences in an HTS data set generated from a virus preparation. DVG-profiler identifies DVG breakpoints relative to a reference genome and reports the directionality of each segment from within the same read. The specificity and sensitivity of the algorithm was assessed using both in silico data sets as well as HTS data obtained from parainfluenza virus 5, Sendai virus and mumps virus preparations. HTS data from the latter were also compared with conventional RT-PCR data and with data obtained using an alternative algorithm. The data presented here demonstrate the high specificity, sensitivity, and robustness of DVG-profiler. This algorithm was implemented within an open source cloud-based computing environment for analyzing HTS data. DVG-profiler might prove valuable not only in basic virus research but also in monitoring live attenuated vaccines for DVG content and to assure vaccine lot to lot consistency.

Evidence that a polyhexameric genome length is preferred, but not strictly required, for efficient mumps virus replication.

Mumps virus (MuV) is postulated to adhere to the "rule of six" for efficient replication. To examine the requirement for MuV, minigenomes of nonpolyhexameric length (6n-1 and 6n+1) were analyzed. Expression of the reporter gene CAT was significantly reduced with minigenomes of nonpolyhexameric length compared to the wild type 6n genome, and reduction was more pronounced for the 6n-1 than for the 6n+1 minigenome. That 6n-1 genomes are impacted by nonconformance with the rule of six to a greater degree as compared to 6n+1 genomes was also suggested with MuV derived from cDNA coding for 6n+1 or 6n-1 genomes. While viruses recovered from 6n+1 cDNAs maintained a nonpolyhexameric genome length over multiple replication cycles, viruses rescued from the 6n-1 cDNAs acquired length correcting mutations rapidly following rescue. Our data indicate that polyhexameric genomes are the preferred template for the MuV RNA polymerase, but that this requirement is not absolute.

Generation and propagation of recombinant mumps viruses exhibiting an additional U residue in the homopolymeric U tract of the F gene-end signal.

As a member of the family paramyxoviridae, subfamily paramyxovirinae, the genome of mumps virus (MuV) is postulated to be polyhexameric in length in order to be able to replicate efficiently. While all natural MuV strains sequenced so far obey to this "rule of six," we describe here the isolation of recombinant MuVs that appeared to contain an additional U residue in the homopolymeric tract of the F gene-end signal, resulting in a genome length of 6n + 1. Sequencing of several plaque-purified viruses from these preparations did not reveal the existence of length-correcting mutations, suggesting that they are violators of the rule of six. Employing high-throughput sequencing technology, we provide evidence that the insertion of an additional U residue is mainly the result of the rescue system used that relies on T7 RNA polymerase. Limited in vitro and in vivo testing of the viruses did not reveal any significant impact of the longer genome on virus replication or virulence, suggesting that the rule of six is not a strict requirement for MuV replication.

Recent mumps outbreaks in vaccinated populations: no evidence of immune escape.

Recently, numerous large-scale mumps outbreaks have occurred in vaccinated populations. Clinical isolates sequenced from these outbreaks have invariably been of genotypes distinct from those of vaccine viruses, raising concern that certain mumps virus strains may escape vaccine-induced immunity. To investigate this concern, sera obtained from children 6 weeks after receipt of measles, mumps, and rubella (MMR) vaccine were tested for the ability to neutralize a carefully selected group of genetically diverse mumps virus strains. Although the geometric mean neutralizing antibody titer of the sera was lower against some virus strains than others, all viruses were readily neutralized, arguing against immune escape.

Gene-specific contributions to mumps virus neurovirulence and neuroattenuation.

Mumps virus (MuV) is highly neurotropic and was the leading cause of aseptic meningitis in the Western Hemisphere prior to widespread use of live attenuated MuV vaccines. Due to the absence of markers of virus neuroattenuation and neurovirulence, ensuring mumps vaccine safety has proven problematic, as demonstrated by the occurrence of aseptic meningitis in recipients of certain vaccine strains. Here we examined the genetic basis of MuV neuroattenuation and neurovirulence by generating a series of recombinant viruses consisting of combinations of genes derived from a cDNA clone of the neurovirulent wild-type 88-1961 strain (r88) and from a cDNA clone of the highly attenuated Jeryl Lynn vaccine strain (rJL). Testing of these viruses in rats demonstrated the ability of several individual rJL genes and gene combinations to significantly neuroattenuate r88, with the greatest effect imparted by the rJL nucleoprotein/matrix protein combination. Interestingly, no tested combination of r88 genes, including the nucleoprotein/matrix protein combination, was able to convert rJL into a highly neurovirulent virus, highlighting mechanistic differences between processes involved in neuroattenuation and neurovirulence.

Presence of lysine at aa 335 of the hemagglutinin-neuraminidase protein of mumps virus vaccine strain Urabe AM9 is not a requirement for neurovirulence.

The recent global resurgence of mumps has drawn attention to the continued need for robust mumps immunization programs. Unfortunately, some vaccines derived from inadequately attenuated vaccine strains of mumps virus have caused meningitis in vaccinees, leading to withdrawal of certain vaccine strains from the market, public resistance to vaccination, or in some cases, cessation of national mumps vaccination programs. The most widely implicated mumps vaccine in cases of postvaccination meningitis is derived from the Urabe AM9 strain, which remains in use in some countries. The Urabe AM9 vaccine virus has been shown to exhibit a considerable degree of nucleotide and amino acid heterogeneity. Some studies have specifically implicated variants containing a lysine residue at amino acid position 335 in the hemagglutinin-neuraminidase (HN) protein with neurotoxicity, whereas a glutamic acid residue at this position was associated with attenuation. To test this hypothesis we generated two modified Urabe AM9 cDNA clones coding either for a lysine or a glutamic acid at position 335 in the HN gene. The two viruses were rescued by reverse genetics and characterized in vitro and in vivo. Both viruses exhibited similar growth kinetics in neuronal and non-neuronal cell lines and were of similar neurotoxicity when tested in rats, suggesting that amino acid 335 is not a crucial determinant of Urabe AM9 growth or neurovirulence.

Changes in mumps virus neurovirulence phenotype associated with quasispecies heterogeneity.

Mumps virus is a highly neurotropic virus with evidence of central nervous system invasion (CNS) in approximately half of all cases of infection. In countries where live attenuated mumps virus vaccines were introduced, the number of mumps cases declined dramatically; however, recently, the safety of some vaccine strains has been questioned. For example, one of the most widely used vaccines, the Urabe AM9 strain, was causally associated with meningitis, leading to the withdrawal of this product from the market in several countries. This highlights the need for a better understanding of the attenuation process and the identification of markers of attenuation. To this end, we further attenuated the Urabe AM9 strain by serial passage in cell culture and compared the complete nucleotide sequences of the parental and passaged viruses. Interestingly, despite a dramatic decrease in virus virulence (as assayed in rats), the only genomic changes were in the form of changes in the level of genetic heterogeneity at specific genome sites, i.e., either selection of one nucleotide variant at positions where the starting material exhibited nucleotide heterogeneity or the evolution of an additional nucleotide to create a heterogenic site. This finding suggests that changes in the level of genetic heterogeneity at specific genome sites can have profound neurovirulence phenotypic consequences and, therefore, caution should be exercised when evaluating genetic markers of virulence or attenuation based only on a consensus sequence.