A Recombinant System and Reporter Viruses for Papiine Alphaherpesvirus 2

Primate simplex viruses, including Herpes simplex viruses 1 and 2, form a group of closely related herpesviruses, which establish latent infections in neurons of their respective host species. While neuropathogenic infections in their natural hosts are rare, zoonotic transmission of Macacine alphaherpesvirus 1 (McHV1) from macaques to humans is associated with severe disease. Human infections with baboon-derived Papiine alphaherpesvirus 2 (PaHV2) have not been reported, although PaHV2 and McHV1 share several biological properties, including neuropathogenicity in mice. The reasons for potential differences in PaHV2 and McHV1 pathogenicity are presently not understood, and answering these questions will require mutagenic analysis. Here, we report the development of a recombinant system, which allows rescue of recombinant PaHV2. In addition, we used recombineering to generate viruses carrying reporter genes (Gaussia luciferase or enhanced green fluorescent protein), which replicate with similar efficiency as wild-type PaHV2. We demonstrate that these viruses can be used to analyze susceptibility of cells to infection and inhibition of infection by neutralizing antibodies and antiviral compounds. In summary, we created a recombinant system for PaHV2, which in the future will be invaluable for molecular analyses of neuropathogenicity of PaHV2

Evaluating assembly and variant calling software for strain-resolved analysis of large DNA viruses

Infection with human cytomegalovirus (HCMV) can cause severe complications in immunocompromised individuals and congenitally infected children. Characterizing heterogeneous viral populations and their evolution by high-throughput sequencing of clinical specimens requires the accurate assembly of individual strains or sequence variants and suitable variant calling methods. However, the performance of most methods has not been assessed for populations composed of low divergent viral strains with large genomes, such as HCMV. In an extensive benchmarking study, we evaluated 15 assemblers and 6 variant callers on 10 lab-generated benchmark data sets created with two different library preparation protocols, to identify best practices and challenges for analyzing such data. Most assemblers, especially metaSPAdes and IVA, performed well across a range of metrics in recovering abundant strains. However, only one, Savage, recovered low abundant strains and in a highly fragmented manner. Two variant callers, LoFreq and VarScan2, excelled across all strain abundances. Both shared a large fraction of false positive variant calls, which were strongly enriched in T to G changes in a ‘G.G’ context. The magnitude of this context-dependent systematic error is linked to the experimental protocol. We provide all benchmarking data, results and the entire benchmarking workflow named QuasiModo, Quasispecies Metric determination on omics, under the GNU General Public License v3.0 (https://github.com/hzi-bifo/Quasimodo), to enable full reproducibility and further benchmarking on these and other data.Deutsches Zentrum für Infektionsforschun

A Fosmid-Based System for the Generation of Recombinant Cercopithecine Alphaherpesvirus 2 Encoding Reporter Genes

The transmission of Macacine alphaherpesvirus 1 (McHV-1) from macaques, the natural host, to humans causes encephalitis. In contrast, human infection with Cercopithecine alphaherpesvirus 2 (CeHV-2), a closely related alphaherpesvirus from African vervet monkeys and baboons, has not been reported and it is believed that CeHV-2 is apathogenic in humans. The reasons for the differential neurovirulence of McHV-1 and CeHV-2 have not been explored on a molecular level, in part due to the absence of systems for the production of recombinant viruses. Here, we report the generation of a fosmid-based system for rescue of recombinant CeHV-2. Moreover, we show that, in this system, recombineering can be used to equip CeHV-2 with reporter genes. The recombinant CeHV-2 viruses replicated with the same efficiency as uncloned, wt virus and allowed the identification of cell lines that are highly susceptible to CeHV-2 infection. Collectively, we report a system that allows rescue and genetic modification of CeHV-2 and likely other alphaherpesviruses. This system should aid future analysis of CeHV-2 biology

Human cytomegalovirus genome diversity in longitudinally collected breast milk samples

Reactivation and shedding of human cytomegalovirus (HCMV) in breast milk during lactation is highly frequent in HCMV-seropositive mothers. This represents a key transmission route for postnatal HCMV infection and can lead to severe disease in preterm neonates. Little is known about HCMV strain composition or longitudinal intrahost viral population dynamics in breast milk from immunocompetent women. We performed HCMV-specific target enrichment and high-throughput sequencing of 38 breast milk samples obtained in Germany between days 10 and 60 postpartum from 15 mothers with HCMV DNA-lactia, and assembled HCMV consensus sequences de novo. The genotype distribution and number of HCMV strains present in each sample were determined by quantifying genotype-specific sequence motifs in 12 hypervariable viral genes, revealing a wide range of genotypes (82/109) for these genes in the cohort and a unique, longitudinally stable strain composition in each mother. Reactivation of up to three distinct HCMV strains was detected in 8/15 of mothers, indicating that a representative subset of the woman’s HCMV reservoir might be locally reactivated early during lactation. As described previously, nucleotide diversity of samples with multiple strains was much higher than that of samples with single strains. Breast milk as a main source of postnatal mother-to-infant transmission may serve as a repository for viral diversity and thus play an essential role in the natural epidemiology of HCMV

Haploflow: strain-resolved de novo assembly of viral genomes

Fritz A, Bremges A, Deng Z-L, et al. Haploflow: strain-resolved de novo assembly of viral genomes. Genome Biology. 2021;22(1): 212

In vivo adenine base editing reverts C282Y and improves iron metabolism in hemochromatosis mice

Hemochromatosis is one of the most common inherited metabolic diseases among white populations and predominantly originates from a homozygous C282Y mutation in the HFE gene. The G > A transition at position c.845 of the gene causes misfolding of the HFE protein, ultimately resulting in its absence at the cell membrane. Consequently, the lack of interaction with the transferrin receptors 1 and 2 leads to systemic iron overload. We screened potential gRNAs in a highly precise cell culture assay and applied an AAV8 split-vector expressing the adenine base editor ABE7.10 and our candidate gRNA in 129-Hfetm.1.1Nca mice. Here we show that a single injection of our therapeutic vector leads to a gene correction rate of >10% and improved iron metabolism in the liver. Our study presents a proof-of-concept for a targeted gene correction therapy for one of the most frequent hereditary diseases affecting humans.ISSN:2041-172