Search Results (15)

Whole transcriptome amplification (WTA2) and sequence-independent single primer amplification (SISPA) are two widely used methods for combined metagenomic sequencing of RNA and DNA viruses. However, information on the reproducibility and bias of these methods on diverse viruses in faecal samples is currently lacking. A mock community (MC) of diverse viruses was developed and used to spike faecal samples at different concentrations. Virus-like particles (VLPs) were extracted, nucleic acid isolated, reverse-transcribed, and PCR amplified using either WTA2 or SISPA and sequenced for metagenomic analysis. A bioinformatics pipeline measured the recovery of MC viruses in replicates of faecal samples from three human donors, analysing the consistency of viral abundance measures and taxonomy. Viruses had different recovery levels with VLP extraction introducing variability between replicates, while WTA2 and SISPA produced comparable results. In comparing WTA2- and SISPA-generated libraries, WTA2 gave more uniform coverage depth profiles and improved assembly quality and virus identification. SISPA produced more consistent abundance, with a 50% difference between replicates occurring in ~20% and ~10% of sequences for WTA2 and SISPA, respectively. In conclusion, a bioinformatics pipeline has been developed to assess the methodological variability and bias of WTA2 and SISPA, demonstrating higher sensitivity with WTA2 and higher consistency with SISPA. Full article

Studies on the human virome based on the application of metagenomic approaches involve overcoming a series of challenges and limitations inherent not only to the biological features of viruses, but also to methodological pitfalls which different approaches have tried to minimize. These approaches fall into two main categories: bulk-metagenomes and virus-like particle (VLP) enrichment. In order to address issues associated with commonly used experimental procedures to assess the degree of reliability, representativeness, and reproducibility, we designed a comparative analysis applied to three experimental protocols, one based on bulk-metagenomes and two based on VLP enrichment. These protocols were applied to stool samples from 10 adult participants, including two replicas per protocol and subject. We evaluated the performances of the three methods, not only through the analysis of the resulting composition, abundance, and diversity of the virome via taxonomical classification and type of molecule (DNA versus RNA, single stranded vs. double stranded), but also according to how the a priori identical replicas differed from each other according to the extraction methods used. Our results highlight the strengths and weaknesses of each approach, offering valuable insights and tailored recommendations for drawing reliable conclusions based on specific research goals. Full article

A beneficial effect of corn-expressed phytase (CEP) on the growth performance of pigs fed with very low-protein (VLP) diets was previously shown. Little is known whether this improvement is related to alterations in the expression profiles of blood proteins and amino acids (AAs). The objective of this study was to investigate whether supplementation of VLP, low-calcium (Ca), and low-P diets with a CEP would alter the blood AAs and protein expression profiles in pigs. Forty-eight pigs were subjected to one of the following groups (n = 8/group) for 4 weeks: positive control (PC), negative control-reduced protein (NC), NC + low-dose CEP (LD), NC + high-dose CEP (HD), LD with reduced Ca/P (LDR), and HD with reduced Ca/P (HDR). Plasma leucine and phenylalanine concentrations were reduced in NC; however, the LD diet recovered the concentration of these AAs. Serum proteomics analysis revealed that proteins involved with growth regulation, such as selenoprotein P were upregulated while the IGF-binding proteins family proteins were differentially expressed in CEP-supplemented groups. Furthermore, a positive correlation was detected between growth and abundance of proteins involved in bone mineralization and muscle structure development. Taken together, CEP improved the blood profile of some essential AAs and affected the expression of proteins involved in the regulation of growth. Full article

The fecal virome has been reported to be associated with CRC. However, little is known about the mucosal virome signature in CRC. This study aimed to determine the viral community within CRC tissues and their contributions to colorectal carcinogenesis. Colonic mucosal biopsies were harvested from patients with CRC (biopsies of both neoplasia and adjacent normal tissue (CRC-A)) and healthy controls (HC). The shot-gun metagenomic sequencing of virus-like particles (VLPs) was performed on the biopsies. Viral community, functional pathways, and their correlations to clinical data were analyzed. Fluorescence in situ hybridizations (FISH) for the localization of viruses in the intestine was performed, as well as quantitative PCR for the detection of Torque teno virus load in human mucosal VLP DNA. A greater number and proportion of core species were found in CRC tissues than in CRC-A and HC tissues. The diversity of the mucosal virome in CRC tissues was significantly increased compared to that in HC and CRC-A tissues. The mucosal virome signature of CRC tissues were significantly different from those of HC and CRC-A tissues at the species level. The abundances of eukaryotic viruses from the Anelloviridae family and its sub-species Torque teno virus (TTV) were significantly higher in CRC patients than in HC. Furthermore, increased levels of TTV in the intestinal lamina propria were found in the CRC group. Multiple viral functions of TTV associated with carcinogenesis were enriched in CRC tissues. We revealed for the first time that the mucosal virobiota signature of CRC is characterized by a higher diversity and more eukaryotic viruses. The enrichment of TTV species in CRC tissues suggests that they may play an oncogenic role in CRC. Targeting eukaryotic viruses in the gut may provide novel strategies for the prevention and treatment of CRC. Full article

Virome research is a rapidly growing area in the microbiome field that is increasingly associated with human diseases, such as inflammatory bowel disease (IBD). Although substantial progress has been made, major methodological challenges limit our understanding of the virota. In this review, we describe challenges that must be considered to accurately report the virome composition and the current knowledge on the virome in health and IBD. First, the description of the virome shows strong methodological biases related to wetlab (e.g., VLP enrichment) and bioinformatics approaches (viral identification and classification). Second, IBD patients show consistent viral imbalances characterized by a high relative abundance of phages belonging to the Caudovirales and a low relative abundance of phages belonging to the Microviridae. Simultaneously, a sporadic contraction of CrAss-like phages and a potential expansion of the lysogenic potential of the intestinal virome are observed. Finally, despite numerous studies that have conducted diversity analysis, it is difficult to draw firm conclusions due to methodological biases. Overall, we present the many methodological and environmental factors that influence the virome, its current consensus in health and IBD, and a contributing hypothesis called the “positive inflammatory feedback loop” that may play a role in the pathophysiology of IBD. Full article

A mixture of valine (Val) and isoleucine (Ile) not only decreases the negative impact of very low protein (VLP) diets on the growth of pigs, but also influences the nitrogen (N) balance and lipid metabolism; however, the underlying pathways are not well understood. This study aimed to investigate the effect of dietary Val and Ile on lipogenesis, lipolysis, and ureagenesis under protein restriction. After one week of acclimation, forty three-week-old pigs were randomly assigned to following dietary treatments (n = 8/group) for 5 weeks: positive control (PC): normal protein diet; negative control (NC): VLP diet; HV: NC supplemented with Val; HI: NC supplemented with Ile; and HVI: NC supplemented with both Val and Ile. HVI partially improved the body weight and completely recovered the feed intake (FI) of pigs fed with NC. HVI increased thermal radiation and improved the glucose clearance. HVI had a lower blood triglyceride than PC and blood urea N than NC. NC and HV promoted lipogenesis by increasing the transcript of fatty acid synthase (FAS) in the liver and lipoprotein lipase (LPL) in adipose tissue but reducing hormone-sensitive lipase (HSL) in the liver. HVI reduced the increased rate of lipogenesis induced by the NC group through normalizing the mRNA abundance of hepatic FAS, sterol regulatory element binding transcription factor 1, and HSL and LPL in adipose tissue. NC, HV, HI, and HVI reduced the ureagenesis by decreasing the protein abundance of carbamoyl phosphate synthetase I, ornithine transcarboxylase, and arginosuccinate lyase in the liver. Overall, HVI improved the growth, FI, and glucose clearance, and decreased the rate of lipogenesis induced by VLP diets. Full article

This study is aimed at the effects of low-protein diets with four amino acids balanced on serum biochemical parameters and colonic microflora of finishing pigs. Fifty-four healthy (Duroc × Landrace × Yorkshire) hybrid barrows with an average body weight of 70.12 ± 4.03 kg were randomly assigned to one of three dietary treatments with three barrows per pen and six pens per treatment. The barrows were fed a normal protein diet (NP), a low-protein diet (LP), and a very low-protein diet (VLP). Compared with the NP diet, reduced dietary protein did not influence serum biochemical parameters (p > 0.05). The valeric acid was significantly increased with the VLP diet (p < 0.05). Compared with the NP diets, the abundance of Terrisporobacter (13.37%) Clostridium_sensu_stricto_1 (23.37%) and Turicibacter (2.57%) increased to 21.04, 33.42 and 13.68% in LP diets and 16.72, 43.71 and 14.61% in VLP diets, while the abundance of Lactobacillus (9.30%) and Streptococcus (25.26%) decreased to 3.57 and 14.50% in LP diets and 1.86 and 4.07% in VLP diets. Turicibacter and Clostridium_sensu_stricto_6 had a powerful negative correlation with the content of valeric acid (p < 0.01), while Peptococcus and Clostridia_UCG-014 had a very solid positive correlation (p < 0.01). In conclusion, reducing dietary protein level can improve colon microbiota composition, especially reducing the abundance of bacteria related to nitrogen metabolism, but has no significant effect on SCFA except valeric acid. In addition, reduction in the dietary protein level by 5.48% had more different flora than that of 2.74% reduction in dietary CP level. Full article

Little is known whether a combination Ile and added Val improves the growth of pigs offered very low protein (VLP) diets through changes in nutrients digestibility and gut microbiota. The objective of this study was to investigate the effect of a mixture of Val above and Ile at NRC levels on growth, nutrient digestibility and gut microbiota in pigs fed with VLP diets. Forty, weaned piglets were assigned to: positive control: normal-protein-diet; negative control (NC): VLP diet supplemented with first four limiting amino acids; VA: NC with Val above NRC; IL: NC with Ile at NRC level; VAIL: NC with Val above and Ile at NRC levels. While both VAIL and VA groups completely recovered the inhibitory effects of VLP diets on feed intake, only VAIL partially recovered the negative effects of VLP diets on growth performance. VAIL and VA increased the thermal radiation and decreased the digestibility of nitrogen. NC increased the relative abundance of Pasteurellaceae and Enterobacteriaceae in the colon. VAIL had a higher abundance of colonic Actinobacteria, Enterococcus, and Brevibacillus and the colon content of VA was more enriched with Mogibacterium. Overall, VAIL partially improved the growth performance which is likely linked with alterations in gut microbiota composition. Full article

Hypersensitivity to a contact allergen is one of the most abundant forms of inflammatory skin disease. Today, more than 20% of the general population are sensitized to one or more contact allergens, making this disease an important healthcare issue, as re-exposure to the allergen can initiate the clinical disease termed allergic contact dermatitis (ACD). The current standard treatment using corticosteroids is effective, but it has side effects when used for longer periods. Therefore, there is a need for new alternative therapies for severe ACD. In this study, we used the versatile Tag/Catcher AP205 capsid virus-like particle (cVLP) vaccine platform to develop an IL-1β-targeted vaccine and to assess the immunogenicity and in vivo efficacy of the vaccine in a translational mouse model of ACD. We show that vaccination with cVLPs displaying full-length murine IL-1β elicits high titers of neutralizing antibodies, leading to a significant reduction in local IL-1β levels as well as clinical symptoms induced by treatment with 1-Fluoro-2,4-dinitrobenzene (DNFB). Moreover, we show that a single amino acid mutation in muIL-1β reduces the biological activity while maintaining the ability to induce neutralizing antibodies. Collectively, the data suggest that a cVLP-based vaccine displaying full-length IL-1β represents a promising vaccine candidate for use as an alternative treatment modality against severe ACD. Full article

Valine (Val) alone or in combination with isoleucine (Ile) improves the growth under severe protein restriction; however, the underlying mechanisms remain unknown. In this study, we assessed whether Val/Ile-induced growth in protein-restricted pigs is associated with changes in gut development, hepatic insulin-like growth factor 1 (IGF-1) production, and blood metabolomics. Forty piglets were assigned to five dietary groups: positive control (PC) with standard protein content; low protein (LP) with very low protein content; and LP supplemented with Val (LPV), Ile (LPI), and Val and Ile (LPVI). LPVI reversed the negative effects of VLP diets on growth and gut morphology. Both LPV and LPVI restored the reduced transcript of IGF-1 while decreasing the transcript of insulin-like growth factor binding protein 1 (IGFBP1) in the liver. LPV and LPVI recovered the reduced plasma Val, glycine, and leucine concentrations, which were positively correlated with improved gut morphology and the hepatic IGF-1 gene expression and negatively correlated with hepatic IGFBP1 mRNA abundance. In conclusion, supplementation with a combination of Val and Ile into the VLP diets restored the decreased growth performance of pigs fed with these diets likely through improved gut development, hepatic IGF-1 expression and bioavailability, and plasma metabolomics profile. Full article

Virus-like particles (VLPs) are thought to increase the dissolved organic carbon by releasing the contents of the host cell, which, in turn, can affect bacterial growth in natural aquatic environments. Yet, experimental tests have shown that the effect of VLPs on the bacterial growth rate at different depths has seldom been studied. Bacteria–VLP interaction and the effect of VLPs on bacterial growth rate in the sunlit surface (3 m) and dark, deep ocean (130 m) environments were first explored at a test site in the southern East China Sea of the northwest Pacific. Our experimental results indicated that bacterial and virus-like particle (VLP) abundance decreased with depth from 0.8 ± 0.3 × 10⁵ cells mL⁻¹ and 1.8 ± 0.4 × 10⁶ VLPs mL⁻¹ at 3 m to 0.4 ± 0.1 × 10⁵ cells mL⁻¹ and 1.4 ± 0.3 × 10⁶ VLPs mL⁻¹ at 130 m. We found that the abundance of VLPs to Bacteria Ratio (VBR) in the dark deep ocean (VBR = 35.0 ± 5.6) was higher than in the sunlit surface environment (VBR = 22.5 ± 2.1). The most interesting finding is that in the dark, deep ocean region the bacterial growth rate in the presence of VLPs was higher (0.05 h⁻¹) than that in virus-diluted treatments (0.01 h⁻¹). However, there was no significant difference in the bacterial growth rates between the treatments in the sunlit surface ocean region. Deep-sea ecosystems are dark and extreme environments that lack primary photosynthetic production, and our estimates imply that the contribution of recycled carbon by viral lysis is highly significant for bacterial growth in the dark, deep ocean environment. Further work for more study sites is needed to identify the relationship of VLPs and their hosts to enable us to understand the role of VLPs at different depths in the East China Sea. Full article

Meteorus pulchricornis (Ichneumonoidea, Braconidae) is an endoparasitoid wasp of lepidopteran caterpillars. Its parasitic success relies on vesicles (named M. pulchricornis Virus-Like Particles or MpVLPs) that are synthesized in the venom gland and injected into the parasitoid host along with the venom during oviposition. In order to define the content and understand the biogenesis of these atypical vesicles, we performed a transcriptome analysis of the venom gland and a proteomic analysis of the venom and purified MpVLPs. About half of the MpVLPs and soluble venom proteins identified were unknown and no similarity with any known viral sequence was found. However, MpVLPs contained a large number of proteins labelled as metalloproteinases while the most abundant protein family in the soluble venom was that of proteins containing the Domain of Unknown Function DUF-4803. The high number of these proteins identified suggests that a large expansion of these two protein families occurred in M. pulchricornis. Therefore, although the exact mechanism of MpVLPs formation remains to be elucidated, these vesicles appear to be “metalloproteinase bombs” that may have several physiological roles in the host including modifying the functions of its immune cells. The role of DUF4803 proteins, also present in the venom of other braconids, remains to be clarified. Full article

Conjunctivitis in swine is a common finding, usually considered to be a secondary symptom of respiratory or viral systemic disease, or a result of irritation by dust or ammonia, or of local infections with Mycoplasma (M.) hyorhinis or chlamydia. In three unrelated swine farms in Germany with a high prevalence of conjunctivitis, a novel mycoplasma species, tentatively named Mycoplasma sp. 1654_15, was isolated from conjunctival swabs taken from affected pigs. Although 16S rRNA gene sequences shared highest nucleotide similarities with M. hyorhinis, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, partial rpoB sequencing, and comparative whole genome analyses indicated the identification of a novel species within genus Mycoplasma. Noticeable differences between Mycoplasma sp. 1654_15 and M. hyorhinis were the lack of a vlp locus and the presence of a myo-inositol pathway in the genome of strain 1654_15. Since myo-inositol might be used as an alternative energy source by this pathogen on the conjunctival surface, robust colonization by outcompeting other bacteria could be the consequence. In summary, abundant isolation of Mycoplasma sp. 1654_15 from the conjunctiva of affected pigs, its close relationship to M. hyorhinis, and identification of a panel of coding sequences (CDSs) potentially associated with virulence and pathogenicity suggested a local eye disease caused by a so far unknown, highly specialized mycoplasma species. Full article

Viruses are widely distributed in various ecosystems and have important impacts on microbial evolution, community structure and function and nutrient cycling in the environment. Viral abundance, diversity and distribution are important for a better understanding of ecosystem functioning and have often been investigated in marine, soil, and other environments. Though microbes have proven useful in oil recovery under extreme conditions, little is known about virus community dynamics in such systems. In this study, injection water and production fluids were sampled in two blocks of the Daqing oilfield limited company where water flooding and microbial flooding were continuously used to improve oil recovery. Virus-like particles (VLPs) and bacteria in these samples were extracted and enumerated with epifluorescence microscopy, and viromes of these samples were also sequenced with Illumina Hiseq PE150. The results showed that a large number of viruses existed in the oil reservoir, and VLPs abundance of production wells was 3.9 ± 0.7 × 10⁸ mL⁻¹ and virus to bacteria ratio (VBR) was 6.6 ± 1.1 during water flooding. Compared with water flooding, the production wells of microbial flooding had relative lower VLPs abundance (3.3 ± 0.3 × 10⁸ mL⁻¹) but higher VBR (7.9 ± 2.2). Assembled viral contigs were mapped to an in-house virus reference data separate from the GenBank non-redundant nucleotide (NT) database, and the sequences annotated as virus accounted for 35.34 and 55.04% of total sequences in samples of water flooding and microbial flooding, respectively. In water flooding, 7 and 6 viral families were identified in the injection and production wells, respectively. In microbial flooding, 6 viral families were identified in the injection and production wells. The total number of identified viral species in the injection well was higher than that in the production wells for both water flooding and microbial flooding. The Shannon diversity index was higher in the production well of water flooding than in the production well of microbial flooding. These results show that viruses are very abundant and diverse in the oil reservoir’s ecosystem, and future efforts are needed to reveal the potential function of viral communities in this extreme environment. Full article

Though microbial processes in the oxygen minimum zones (OMZs) of the Arabian Sea (AS) are well documented, prokaryote-virus interactions are less known. The present study was carried out to determine the potential physico-chemical factors influencing viral abundances and their life strategies (lytic and lysogenic) along the vertical gradient in the OMZ of the AS (southwest coast of India). Water samples were collected during the southwest monsoon (SWM) season in two consecutive years (2015 and 2016) from different depths, namely, the surface layer, secondary chlorophyll a maxima (~30–40 m), oxycline (~70–80 m), and hypoxic/suboxic layers (~200–350 m). The high viral abundances observed in oxygenated surface waters (mean ± SD = 6.1 ± 3.4 × 10⁶ viral-like particles (VLPs) mL⁻¹), drastically decreased with depth in the oxycline region (1.2 ± 0.5 × 10⁶ VLPs mL⁻¹) and hypoxic/suboxic waters (0.3 ± 0.3 × 10⁶ VLPs mL⁻¹). Virus to prokaryote ratio fluctuated in the mixed layer (~10) and declined significantly (p < 0.001) to 1 in the hypoxic layer. Viral production (VP) and frequency of virus infected cells (FIC) were maximum in the surface and minimum in the oxycline layer, whereas the viral lysis was undetectable in the suboxic/hypoxic layer. The detection of a high percentage of lysogeny in suboxic (48%) and oxycline zones (9–24%), accompanied by undetectable rates of lytic viral infection support the hypothesis that lysogeny may represent the major survival strategy for viruses in unproductive or harsh nutrient/host conditions in deoxygenated waters. Full article