High genetic stability of co-circulating human adenovirus type 31 lineages over 59 years.

Type 31 of human adenovirus species A (HAdV-A31) is a significant pathogen primarily associated with diarrhoea in children but also with life-threatening disseminated disease in allogeneic haematopoietic stem cell transplant (HSCT) recipients. Nosocomial outbreaks of HAdV-A31 have been frequently described. However, the evolution of HAdV-A31 has not been studied in detail. The evolution of other HAdV types is driven either by intertypic recombination, where different types exchange genome regions, or by immune escape selection of neutralisation determinants. Complete genomic HAdV-A31 sequences from sixty diagnostic specimens of the past 18 years (2003-21) were generated, including fourteen specimens of a presumed outbreak on two HSCT wards. Additionally, twenty-three complete genomes from GenBank were added to our phylogenetic analysis as well as in silico generated and previously published restriction fragment polymorphism (RFLP) data. Phylogenetic analysis of eighty-three genomes indicated that HAdV-A31 evolved slowly with six lineages co-circulating. The two major lineages were lineage 1, which included the prototype from 1962 and nine recent isolates, and lineage 2, which split into four sublineages and included most isolates from 2003 to 2021. The average nucleotide identity within lineages was high (99.8 per cent) and identity between lineages was 98.7 and 99.2 per cent. RFLP data allowed the construction of a lower-resolution phylogeny with two additional putative lineages. Surprisingly, regions of higher diversity separating lineages were found in gene regions coding for non-structural and minor capsid proteins. Intertypic recombinations were not observed, but the phylogeny of lineage 3 was compatible with an interlineage recombination event in the fibre gene. Applying the phylogenetic analysis to the presumed nosocomial outbreak excluded two suspected transmission events and separated it into two different, simultaneous outbreaks caused by different sublineages of lineage 2. However, due to the high nucleotide identity within HAdV-A31 lineages, the proof of infection chains remains debatable. This in-depth study on the molecular phylogeny of HAdV-A31 highlights the high genetic stability of co-circulating HAdV-A31 lineages over almost six decades. It also supports the epidemiological hypothesis that HAdV-A31 circulates as an etiological agent of a childhood disease infecting immunologically naive patients without strong positive selection of immune escape variants and recombinants.

Molecular phylogeny of human adenovirus type 41 lineages.

Type 41 of human adenovirus species F (HAdV-F41) is a frequent aetiology of gastroenteritis in children, and nosocomial as well as kindergarten outbreaks have been frequently described. In contrast to other HAdV types, HAdV-F41 was not associated with a life-threatening disseminated disease in allogeneic haematopoietic stem cell transplant (HSCT) recipients or any severe organ infections so far. Due to the limited clinical significance, the evolution of HAdV-F41 has not been studied in detail. Recently, HAdV-F41 has been associated with severe hepatitis in young children, and interest in HAdV-F41 has skyrocketed, although the aetiology of hepatitis has not been resolved. Complete genomic HAdV-F41 sequences from thirty-two diagnostic specimens of the past 11 years (2011-22) were generated, all originating from gastroenteritis patients. Additionally, thirty-three complete HAdV-F41 genomes from GenBank were added to our phylogenetic analysis. Phylogenetic analysis of sixty-five genomes indicated that HAdV-F41 evolved with three lineages co-circulating. Lineage 1 included the prototype 'Tak' from 1973 and six isolates from 2007 to 2017 with an average nucleotide identity of 99.3 per cent. Lineage 2 included 53 isolates from 2000 to 2022, had an average nucleotide identity of 99.8 per cent, and split into two sublineages. Lineage 3, probably described for the first time in 2009, had a 45-nucleotide deletion in the long fibre gene and had evolved significantly in the short fibre and E3 region. Moreover, a recent Lineage 3 isolate from 2022 had a recombinant phylogeny of the short fibre gene. Fibres interact with cellular receptors and determine cellular tropism, whereas E3 gene products interfere with the immune recognition of HAdV-infected cells. This in-depth study on the phylogeny of HAdV-F41 discovered significant evolution of recently described Lineage 3 of HAdV-F41, possibly resulting in altered cellular tropism, virulence, and pathophysiology.

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.

IFN-ß Deficiency Results in Fatal or Demyelinating Disease in C57BL/6 Mice Infected With Theiler's Murine Encephalomyelitis Viruses.

Type I Interferons (IFN-I) are important inducers of the antiviral immune response and immune modulators. IFN-ß is the most highly expressed IFN-I in the central nervous system (CNS). The infection of SJL mice with the BeAn or the DA strain of Theiler's murine encephalomyelitis virus (TMEV) results in a progressive demyelinating disease. C57BL/6 mice are usually resistant to TMEV-induced demyelination and eliminate these strains from the CNS within several weeks. Using C57BL/6 IFN-ß knockout (IFN-ß-/-) mice infected with TMEV, we evaluated the role of IFN-ß in neuroinfection. Despite the resistance of C57BL/6 wild type (WT) mice to TMEV infection, DA-infected IFN-ß-/- mice had to be killed at 7 to 8 days post infection (dpi) due to severe clinical disease. In contrast, BeAn-infected IFN-ß-/- mice survived until 98 dpi. Nevertheless at 14 dpi, BeAn-infected IFN-ß-/- mice showed a stronger encephalitis and astrogliosis, higher viral load as well as higher mRNA levels of Isg15, Eif2ak2 (PKR), Tnfa, Il1b, Il10, Il12 and Ifng in the cerebrum than BeAn-infected WT mice. Moreover, the majority of IFN-ß-/- mice did not clear the virus from the CNS and developed mild demyelination in the spinal cord at 98 dpi, whereas virus and lesions were absent in the spinal cord of WT mice. Persistently infected IFN-ß-/- mice also had higher Isg15, Eif2ak1, Tnfa, Il1a, Il1b and Ifng mRNA levels in the spinal cord at 98 dpi than their virus-negative counterparts indicating an activation of IFN-I signaling and ongoing inflammation. Most importantly, BeAn-infected NesCre+/- IFN-ßfl/fl mice, which do not express IFN-ß in neurons, astrocytes and oligodendrocytes, only developed mild brain lesions similar to WT mice. Consequently, IFN-ß produced by neuroectodermal cells does not seem to play a critical role in the resistance of C57BL/6 mice against fatal and demyelinating disease induced by TMEV strains.

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.

Diagnostic Yield and Therapeutic Consequences of Targeted Next-Generation Sequencing in Sporadic Primary Immunodeficiency.

INTRODUCTION: Primary immunodeficiencies (PIDs) are a heterogeneous group of disorders characterized by increased susceptibility to infections, immune dysregulation, and/or malignancy. Genetic studies, especially during the last decade, led to a better understanding of the pathogenesis of primary immunodeficiencies and contributed to their classification into distinct monogenic disorders falling under one of the >430 currently known inborn errors of immunity (IEI). The growing availability of molecular genetic testing resulted in the increasing identification of patients with IEI. Here, we evaluated the diagnostic yield and the clinical consequences of targeted next-generation sequencing (tNGS) in a cohort of 294 primary immunodeficiency patients, primarily consisting of cases with sporadic primary antibody deficiency. METHOD: We have custom designed a tNGS panel to sequence a cohort of PID patients. Agilent's HaloPlex Target Enrichment System for Illumina was used for DNA target enrichment. RESULTS: tNGS identified a definite or predicted pathogenic variant in 15.3% of patients. The highest diagnostic rate was observed among patients with combined immunodeficiency or immune dysregulation, for whom genetic diagnosis may affect therapeutic decision-making. CONCLUSION: Next-generation sequencing has changed diagnostic assignment and paved the way for targeted therapeutic intervention with agents directed at reverting the disease-causing molecular abnormality or its pathophysiological consequences. Therefore, such targeted therapies and identifying the genetic basis of PID can be essential for patients with manifested immune dysregulation as conventional immunomodulatory regimens may exert an immunosuppressive effect, aggravating their immunodeficiency or may only inadequately control autoimmune or lymphoproliferative manifestations.

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.

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.

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.