A murine herpesvirus closely related to ubiquitous human herpesviruses causes T-cell depletion

ABSTRACT The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4 + T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and clinical ramifications of roseolovirus infection in humans have been hypothesized, but studies showing definitive causative relationships between infection and disease susceptibility are lacking. Here we show that MRV infects the thymus and causes T-cell depletion, suggesting that other roseoloviruses may have similar properties. </jats:p

Murine roseolovirus does not accelerate amyloid-β pathology and human roseoloviruses are not over-represented in Alzheimer disease brains

BACKGROUND: The role of viral infection in Alzheimer Disease (AD) pathogenesis is an area of great interest in recent years. Several studies have suggested an association between the human roseoloviruses, HHV-6 and HHV-7, and AD. Amyloid-β (Aβ) plaques are a hallmark neuropathological finding of AD and were recently proposed to have an antimicrobial function in response to infection. Identifying a causative and mechanistic role of human roseoloviruses in AD has been confounded by limitations in performing in vivo studies. Recent -omics based approaches have demonstrated conflicting associations between human roseoloviruses and AD. Murine roseolovirus (MRV) is a natural murine pathogen that is highly-related to the human roseoloviruses, providing an opportunity to perform well-controlled studies of the impact of roseolovirus on Aβ deposition. METHODS: We utilized the 5XFAD mouse model to test whether MRV induces Aβ deposition in vivo. We also evaluated viral load and neuropathogenesis of MRV infection. To evaluate Aβ interaction with MRV, we performed electron microscopy. RNA-sequencing of a cohort of AD brains compared to control was used to investigate the association between human roseolovirus and AD. RESULTS: We found that 5XFAD mice were susceptible to MRV infection and developed neuroinflammation. Moreover, we demonstrated that Aβ interacts with viral particles in vitro and, subsequent to this interaction, can disrupt infection. Despite this, neither peripheral nor brain infection with MRV increased or accelerated Aβ plaque formation. Moreover, -omics based approaches have demonstrated conflicting associations between human roseoloviruses and AD. Our RNA-sequencing analysis of a cohort of AD brains compared to controls did not show an association between roseolovirus infection and AD. CONCLUSION: Although MRV does infect the brain and cause transient neuroinflammation, our data do not support a role for murine or human roseoloviruses in the development of Aβ plaque formation and AD

Detailed phenotypic and molecular analyses of genetically modified mice generated by CRISPR-Cas9-mediated editing.

The bacterial CRISPR-Cas9 system has been adapted for use as a genome editing tool. While several recent reports have indicated that successful genome editing of mice can be achieved, detailed phenotypic and molecular analyses of the mutant animals are limited. Following pronuclear micro-injection of fertilized eggs with either wild-type Cas9 or the nickase mutant (D10A) and single or paired guide RNA (sgRNA) for targeting of the tyrosinase (Tyr) gene, we assessed genome editing in mice using rapid phenotypic readouts (eye and coat color). Mutant mice with insertions or deletions (indels) in Tyr were efficiently generated without detectable off-target cleavage events. Gene correction of a single nucleotide by homologous recombination (HR) could only occur when the sgRNA recognition sites in the donor DNA were modified. Gene repair did not occur if the donor DNA was not modified because Cas9 catalytic activity was completely inhibited. Our results indicate that allelic mosaicism can occur following -Cas9-mediated editing in mice and appears to correlate with sgRNA cleavage efficiency at the single-cell stage. We also show that larger than expected deletions may be overlooked based on the screening strategy employed. An unbiased analysis of all the deleted nucleotides in our experiments revealed that the highest frequencies of nucleotide deletions were clustered around the predicted Cas9 cleavage sites, with slightly broader distributions than expected. Finally, additional analysis of founder mice and their offspring indicate that their general health, fertility, and the transmission of genetic changes were not compromised. These results provide the foundation to interpret and predict the diverse outcomes following CRISPR-Cas9-mediated genome editing experiments in mice

Primer Sequences.

<p>Primer sequences for sgRNA synthesis, sequencing, and mutagenesis are shown.</p><p>Primer Sequences.</p

Successful genome editing of <i>Tyr</i> is evident on the day of birth and confirmed by DNA sequencing.

<p><b>(a)</b> Phenotypic results of guide B editing of B6xAB6 fertilized eggs (Experiment 1) resulted in loss of eye pigmentation in two of three pups photographed on day 1 after birth, whereas one pups appeared unaltered (middle pup). Since sequencing analysis was not pursued in these pups, a disruptive indel on the AB6 allele and/or an in-frame indel on the B6 allele could also result in the same phenotypic finding. The mice in Experiment 1 did not survive because the cage housing the pups from guide B was flooded while pups from guide D were cannibalized by the mother. <b>(b)</b> Sequencing results of guide D editing from Experiment 1 are shown here. The <i>Tyr</i> alleles from guide B targeting were not analyzed. The B6 allele is indicated with the position of guide D as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g001" target="_blank">Fig. 1</a>. Mice are numbered with the following convention: (experiment#.mouse#.allele#). Mouse 1.1 (in-frame deletion on the B6 allele) and mouse 1.4 (no indels observed) had black eyes while the remaining mice had pink eyes at birth. Sequencing results from unaltered <i>Tyr</i> alleles are not shown. Deleted nt (-). Inserted nt in brackets.</p

Generation of indels and donor DNA insertion following sgRNA guide D cleavage.

<p>The results of single guide D editing in AB6 mice (Experiment 5) with a donor template are depicted. Mice are numbered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g002" target="_blank">Fig. 2b</a>. The B6 allele is indicated with the positions of guides B and D as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g001" target="_blank">Fig. 1</a>. Sequencing results from unaltered <i>Tyr</i> alleles are not shown (5.4.2, 5.5.2, 5.7.2). Deleted nt (-). Inserted nt in brackets.</p

Clustered distribution of deleted nucleotides by CRISPR-Cas9-mediated genome editing.

<p>The number of times a particular nt was found to be deleted upon sequence analysis is represented by the bar above the specific nt. The DNA sequence indicates the PAM sites in red with the sgRNA recognition site for guides B and D in a box, with the guide identity indicated by the arrow below it. For each guide, the predicted cleavage site of Cas9 is indicated as the site between the two red bars. <b>(a)</b> An analysis of all deleted regions from sequence alleles containing one or two disrupted sites without the loss of the intervening residues. <b>(b)</b> As in <b>(a)</b>, but with all alleles (including intervening deletions) plotted. The data represent sequence results of 47 alleles from the 29 genome edited mice with indels.</p

Successful gene repair of point mutation by phenotype and DNA sequencing.

<p><b>(a)</b> Phenotypic results of guide B and D editing of AB6xAB6 fertilized eggs (Experiment 3) resulted in a gain of black coat color in one of three pups photographed on day 5 after birth. Mice 3.1 (black pup), 3.2, and 3.3 are shown. <b>(b)</b> The results of combined guide B and D editing in AB6 mice with a donor template are depicted. When a modified donor was used, the mutations are indicated by (*) above the lowercase base substitutions. Mice are numbered as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g002" target="_blank">Fig. 2b</a>. The AB6 allele is indicated with the positions of guides B and D as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116484#pone.0116484.g001" target="_blank">Fig. 1</a>. Sequencing results from the unaltered <i>Tyr</i> allele 3.19.2 is not shown. Mouse 3.3 is a genetic but not phenotypic mosaic. Underlined alleles were from a D10A Cas9 injection. Deleted nt (-). Inserted nt in brackets. The introduction of a novel <i>Sal</i>I restriction enzyme site is identified by a box for allele 3.1.1.</p