Microbial and human transcriptome in vaginal fluid at midgestation: Association with spontaneous preterm delivery

Background Intrauterine infection and inflammation caused by microbial transfer from the vagina are believed to be important factors causing spontaneous preterm delivery (PTD). Multiple studies have examined the relationship between the cervicovaginal microbiome and spontaneous PTD with divergent results. Most studies have applied a DNA-based assessment, providing information on the microbial composition but not transcriptional activity. A transcriptomic approach was applied to investigate differences in the active vaginal microbiome and human transcriptome at midgestation between women delivering spontaneously preterm versus those delivering at term. Methods Vaginal swabs were collected in women with a singleton pregnancy at 18 + 0 to 20 + 6 gestational weeks. For each case of spontaneous PTD (delivery <37 + 0 weeks) two term controls were randomized (39 + 0 to 40 + 6 weeks). Vaginal specimens were subject to sequencing of both human and microbial RNA. Microbial reads were taxonomically classified using Kraken2 and RefSeq as a reference. Statistical analyses were performed using DESeq2. GSEA and HUMAnN3 were used for pathway analyses. Results We found 17 human genes to be differentially expressed (false discovery rate, FDR < 0.05) in the preterm group (n = 48) compared to the term group (n = 96). Gene expression of kallikrein-2 (KLK2), KLK3 and four isoforms of metallothioneins 1 (MT1s) was higher in the preterm group (FDR < 0.05). We found 11 individual bacterial species to be differentially expressed (FDR < 0.05), most with a low occurrence. No statistically significant differences in bacterial load, diversity or microbial community state types were found between the groups. Conclusions In our mainly white population, primarily bacterial species of low occurrence were differentially expressed at midgestation in women who delivered preterm versus at term. However, the expression of specific human transcripts including KLK2, KLK3 and several isoforms of MT1s was higher in preterm cases. This is of interest, because these genes may be involved in critical inflammatory pathways associated with spontaneous PTD

Genetic variants of herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) virus causes oral as well as genital lesions, and, on rare occasions, focal encephalitis and severe neonatal infections. Due to the partly overlapping clinical manifestations of infection with HSV-1 and its closest relative among herpesviruses, HSV-2, diagnostic methods are required to discriminate between infections with these two viruses. To improve our understanding of the genetic basis for such diagnostic procedures, we have employed DNA sequencing to investigate the genetic variability of HSV-1 viruses derived from clinical isolates.The viral envelope glycoprotein G (gG-1) has been suggested as a prototype antigen for HSV-1-specific serodiagnosis, but the interstrain variability of this gene has not been investigated. In a large material of clinical HSV-1 isolates, we found a high degree of sequence variation, especially in the middle part of the gene encoding immunodominant antibody epitopes. By phylogenetic analysis, two main groups of this gene were discovered among the clinical isolates, one represented by laboratory strains Syn 17+ and F being reactive with an anti-gG-1 monoclonal antibody (MAb), and the other variant being unreactive to the same MAb and showing sequence similarity to strain KOS. Five isolates appeared to be recombinants of these two gG-1 variants. Furthermore, one isolate displayed a gG-1-negative phenotype due to a frameshift mutation in the form of an insertion of one cytosine nucleotide. The existence of two main genetic groups of gG-1 did not markedly affect the antibody response to this protein when IgG titers in hosts carrying either of the two variant viruses were compared in a gG-1-based ELISA.To investigate whether a genetic diversity in form of dichotomy into two genogroups also existed in other HSV-1 envelope genes, we sequenced parts or complete genes coding for gB, gI, and gE in a large proportion of the clinical isolates. Surprisingly, the gE/gI genes exhibited sequence diversity that clustered into three main groups as supported by high bootstrap values. Intra- and intergenic recombinants were demonstrated in the gE/gI gene complex. In contrast, phylogenetic analysis did not separate the gB sequences into main genogroups, although a relatively large sequence variability was documented. Our approach of sequencing selected genes or fragments thereof provided insight as regards evolution of investigated genes, while phylogeny of whole-virus genomes remains to be elucidated. Despite that only short regions of the genome were sequenced, the frequent finding of recombinants among our isolates indicate that, besides point and indel mutations, recombination stands out as a major source of HSV genetic variability.Lastly, we investigated the degree of heterogeneity within individual HSV-1 and HSV-2 isolates. The frequencies of negative phenotypes of genes encoding for gG-1, gG-2 and gC-1 were estimated by antibody-staining of viral plaques. DNA-sequencing defined the responsible genetic mechanisms either as frameshift mutations due to insertion or deletion of nt G or C in homopolymer runs, or point mutations within MAb epitopes. Finally, exploiting our previous finding of a dichotomy of the gG-1 gene, we developed a KOS-genovariant-specific PCR. In a large proportion of clinical isolates showing a Syn 17+-like sequence, KOS-like sequences were detected as minority populations. Our results demonstrate a hitherto unknown degree of inter- and intrastrain genetic diversity among HSV strains

Dichotomy of Glycoprotein G Gene in Herpes Simplex Virus Type 1 Isolates

Herpes simplex virus type 1 (HSV-1) encodes 11 envelope glycoproteins, of which glycoprotein G-1 (gG-1) induces a type-specific antibody response. Variability of the gG-1 gene among wild-type strains may be a factor of importance for a reliable serodiagnosis and typing of HSV-1 isolates. Here, we used a gG-1 type-specific monoclonal antibody (MAb) to screen for mutations in the immunodominant region of this protein in 108 clinical HSV-1 isolates. Of these, 42 isolates showed no reactivity to the anti-gG-1 MAb. One hundred five strains were further examined by DNA sequencing of the middle part of the gG-1 gene, encompassing 106 amino acids including the immunodominant region and epitope of the anti-gG-1 MAb. By phylogenetic comparisons based on the sequence data, we observed two (main) genetic variants of the gG-1 gene among the clinical isolates corresponding to reactivity or nonreactivity to the anti-gG-1 MAb. Furthermore, four strains appeared to be recombinants of the two gG-1 variants. In addition, one strain displayed a gG-1-negative phenotype due to a frameshift mutation, in the form of insertion of a cytosine nucleotide. When immunoglobulin G reactivity to HSV-1 in sera from patients infected with either of the two variants was investigated, no significant differences were found between the two groups, either in a type-common enzyme-linked immunosorbent assay (ELISA) or in a type-specific gG-1 antigen-based ELISA. Despite the here-documented existence of two variants of the gG-1 gene affecting the immunodominant region of the protein, other circumstances, such as early phase of infection, might be sought for explaining the seronegativity to gG-1 commonly found in a proportion of the HSV-1-infected patients

Phylogenetic Analysis of Clinical Herpes Simplex Virus Type 1 Isolates Identified Three Genetic Groups and Recombinant Viruses

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen which establishes lifelong infections. In the present study, we determined the sequence diversity of the complete genes coding for glycoproteins G (gG), I (gI), and E (gE), comprising 2.3% of the HSV-1 genome and located within the unique short (US) region, for 28 clinical HSV-1 isolates inducing oral lesions, genital lesions, or encephalitis. Laboratory strains F and KOS321 were sequenced in parallel. Phylogenetic analysis, including analysis of laboratory strain 17 (GenBank), revealed that the sequences were separated into three genetic groups. The identification of different genogroups facilitated the detection of recombinant viruses by using specific nucleotide substitutions as recombination markers. Seven of the isolates and strain 17 displayed sequences consistent with intergenic recombination, and at least four isolates were intragenic recombinants. The observed frequency of recombination based on an analysis of a short stretch of the US region suggests that most full-length HSV-1 genomes consist of a mosaic of segments from different genetic groups. Polymorphic tandem repeat regions, consisting of two to eight blocks of 21 nucleotides in the gI gene and seven to eight repeats of 3 nucleotides in the gG gene, were also detected. Laboratory strain KOS321 displayed a frameshift mutation in the gI gene with a subsequent alteration of the deduced intracellular portion of the protein. The presence of polymorphic tandem repeat regions and the different genogroup identities can be used for molecular epidemiology studies and for further detection of recombination in the HSV-1 genome

Genetic Copy Number Variations in Colon Mucosa Indicating Risk for Colorectal Cancer.

Background: Sporadic colorectal tumors probably carry genetic alterations that may be related to familiar clusters according to risk loci visualized by SNP arrays on normal tissues. The aim of the present study was therefore to search for DNA regions (copy number variations, CNVs) as biomarkers associated to genetic susceptibility for early risk predictions of colorectal cancer. Such sequence alterations could provide additional information on phenotypic grouping of patients. Material and Methods: High resolution 105K oligonucleotide microarrays were used in search for CNV loci in DNA from tumor-free colon mucosa at primary operations for colon cancer in 60 unselected patients in comparison to DNA in buffy coat cells from 44 confirmed tumor-free and healthy blood donors. Array-detected CNVs were confirmed by Multiplex ligation-dependent probe amplification (MLPA). Results: A total number of 205 potential CNVs were present in DNA from colon mucosa. 184 (90%) of the 205 potential CNVs had been identified earlier in mucosa DNA from healthy individuals as reported to the Database of Genomic Variants. Remaining 21 (10%) CNVs were potentially novel sites. Two CNVs (3q23 and 10q21.1) were significantly related to colon cancer, but not confirmed in buffy coat DNA from the cancer patients. Conclusion: Our study reveals two CNVs that indicate increased risk for colon cancer; these DNA alterations may have been acquired by colon stem cells with subsequent appearance among epithelial mucosa cells. Impact: Certain mucosa CNV alterations may indicate individual susceptibility for malignant transformation in relationship to intestinal toxins and bacterial growth