Discovery, Pathogenesis, and Complete Genome Characterization of Lates calcarifer Herpesvirus.

In 2015 and 2016, two Barramundi (Lates calcarifer) farms in Singapore reported a disease outbreak characterized by lethargic behavior, pronounced inappetence, generalized skin lesions, erosions of the fins and tail, and ultimately high mortality in their fish. Next-generation sequencing and PCR confirmed presence of a novel virus belonging to the Alloherpesviridae family, Lates calcarifer herpesvirus (LCHV), which was subsequently isolated and cultured. We characterize, for the first time, the complete genome of two cultured LCHV isolates. The genome contains a long unique region of approximately 105,000 bp flanked by terminal repeats of approximately 24,800 bp, of which the first 8.2 kb do not show any similarity to described genomes in the Alloherpesviridae family. The two cultured isolates share 89% nucleotide identity, and their closest relatives are the viruses belonging to the genus Ictalurivirus. Experimental infections using one of the cultured LCHV isolates resulted in identical clinical signs as originally described in the index farm, both in intraperitoneal-injection infected fish and cohabitant fish, with mortality in both groups. Histopathological analysis showed pronounced abnormalities in the gills. Virus culture and PCR analysis confirmed the replication of LCHV in the infected fish, and thus Koch's postulates were fulfilled.

Influenza-like illness symptoms due to endemic human coronavirus reinfections are not influenced by the length of the interval separating reinfections.

After 3 years of its introduction to humans, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been declared as endemic. Little is known about the severity of the disease manifestation that future infections may cause, especially when reinfections occur after humoral immunity from a previous infection or vaccination has waned. Such knowledge could inform policymakers regarding the frequency of vaccination. Reinfections by endemic human coronaviruses (HCoVs) can serve as a model system for SARS-CoV-2 endemicity. We monitored 44 immunocompetent male adults with blood sampling every 6 months (for 17 years), for the frequency of HCoV (re-)infections, using rises in N-antibodies of HCoV-NL63, HCoV-29E, HCoV-OC43, and HCoV-HKU1 as markers of infection. Disease associations during (re-)infections were examined by comparison of self-reporting records of influenza-like illness (ILI) symptoms, every 6 months, by all participants. During 8,549 follow-up months, we found 364 infections by any HCoV with a median of eight infections per person. Symptoms more frequently reported during HCoV infection were cough, sore throat, and myalgia. Two hundred fifty-one of the 364 infections were species-specific HCoV-reinfections, with a median interval of 3.58 (interquartile range 1.92-5.67) years. The length of the interval between reinfections-being either short or long-had no influence on the frequency of reporting ILI symptoms. All HCoV-NL63, HCoV-229E, HCoV-OC43, and HCoV-HKU1 (re-)infections are associated with the reporting of ILIs. Importantly, in immunocompetent males, these symptoms are not influenced by the length of the interval between reinfections. IMPORTANCE: Little is known about the disease following human coronavirus (HCoV) reinfection occurring years after the previous infection, once humoral immunity has waned. We monitored endemic HCoV reinfection in immunocompetent male adults for up to 17 years. We found no influence of reinfection interval length in the disease manifestation, suggesting that immunocompetent male adults are adequately protected against future HCoV infections.

The Impact of First-Time SARS-CoV-2 Infection on Human Anelloviruses.

Members of the Anelloviridae family dominate the blood virome, emerging early in life. The anellome, representing the variety of anelloviruses within an individual, stabilizes by adulthood. Despite their supposedly commensal nature, elevated anellovirus concentrations under immunosuppressive treatment indicate an equilibrium controlled by immunity. Here, we investigated whether anelloviruses are sensitive to the immune activation that accompanies a secondary infection. As a model, we investigated 19 health care workers (HCWs) with initial SARS-CoV-2 infection, with blood sampling performed pre and post infection every 4 weeks in a 3-month-follow-up during the early 2020 COVID-19 pandemic. A concurrently followed control group (n = 27) remained SARS-CoV-2-negative. Serum anellovirus loads were measured using qPCR. A significant decrease in anellovirus load was found in the first weeks after SARS-CoV-2 infection, whereas anellovirus concentrations remained stable in the uninfected control group. A restored anellovirus load was seen approximately 10 weeks after SARS-CoV-2 infection. For five subjects, an in-time anellome analysis via Illumina sequencing could be performed. In three of the five HCWs, the anellome visibly changed during SARS-CoV-2 infection and returned to baseline in two of these cases. In conclusion, anellovirus loads in blood can temporarily decrease upon an acute secondary infection.

TMPRSS2 is a functional receptor for human coronavirus HKU1.

Four endemic seasonal human coronaviruses causing common colds circulate worldwide: HKU1, 229E, NL63 and OC43 (ref. 1). After binding to cellular receptors, coronavirus spike proteins are primed for fusion by transmembrane serine protease 2 (TMPRSS2) or endosomal cathepsins2-9. NL63 uses angiotensin-converting enzyme 2 as a receptor10, whereas 229E uses human aminopeptidase-N11. HKU1 and OC43 spikes bind cells through 9-O-acetylated sialic acid, but their protein receptors remain unknown12. Here we show that TMPRSS2 is a functional receptor for HKU1. TMPRSS2 triggers HKU1 spike-mediated cell-cell fusion and pseudovirus infection. Catalytically inactive TMPRSS2 mutants do not cleave HKU1 spike but allow pseudovirus infection. Furthermore, TMPRSS2 binds with high affinity to the HKU1 receptor binding domain (Kd 334 and 137 nM for HKU1A and HKU1B genotypes) but not to SARS-CoV-2. Conserved amino acids in the HKU1 receptor binding domain are essential for binding to TMPRSS2 and pseudovirus infection. Newly designed anti-TMPRSS2 nanobodies potently inhibit HKU1 spike attachment to TMPRSS2, fusion and pseudovirus infection. The nanobodies also reduce infection of primary human bronchial cells by an authentic HKU1 virus. Our findings illustrate the various evolution strategies of coronaviruses, which use TMPRSS2 to either directly bind to target cells or prime their spike for membrane fusion and entry.

Correction: Kosoltanapiwat et al. A Novel Simian Adenovirus Associating with Human Adenovirus Species G Isolated from Long-Tailed Macaque Feces. Viruses 2023, 15, 1371.

After publication of the article, the authors received comments from a member of the Viruses editorial board who is an expert in the field of adenovirus concerning figures and references that should be included in the paper [...].

Parasitic, bacterial, viral, immune-mediated, metabolic and nutritional factors associated with nodding syndrome.

Nodding syndrome is a neglected, disabling and potentially fatal epileptic disorder of unknown aetiology affecting thousands of individuals mostly confined to Eastern sub-Saharan Africa. Previous studies have identified multiple associations-including Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 deficiency and measles virus infection-yet, none is proven causal. We conducted a case-control study of children with early-stage nodding syndrome (symptom onset <1 year). Cases and controls were identified through a household survey in the Greater Mundri area in South Sudan. A wide range of parasitic, bacterial, viral, immune-mediated, metabolic and nutritional risk factors was investigated using conventional and state-of-the-art untargeted assays. Associations were examined by multiple logistic regression analysis, and a hypothetical causal model was constructed using structural equation modelling. Of 607 children with nodding syndrome, 72 with early-stage disease were included as cases and matched to 65 household- and 44 community controls. Mansonella perstans infection (odds ratio 7.04, 95% confidence interval 2.28-21.7), Necator americanus infection (odds ratio 2.33, 95% confidence interval 1.02-5.3), higher antimalarial seroreactivity (odds ratio 1.75, 95% confidence interval 1.20-2.57), higher vitamin E concentration (odds ratio 1.53 per standard deviation increase, 95% confidence interval 1.07-2.19) and lower vitamin B12 concentration (odds ratio 0.56 per standard deviation increase, 95% confidence interval 0.36-0.87) were associated with higher odds of nodding syndrome. In a structural equation model, we hypothesized that Mansonella perstans infection, higher vitamin E concentration and fewer viral exposures increased the risk of nodding syndrome while lower vitamin B12 concentration, Necator americanus and malaria infections resulted from having nodding syndrome. We found no evidence that Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 and other factors were associated with nodding syndrome. Our results argue against several previous causal hypotheses including Onchocerca volvulus. Instead, nodding syndrome may be caused by a complex interplay between multiple pathogens and nutrient levels. Further studies need to confirm these associations and determine the direction of effect.

A Novel Simian Adenovirus Associating with Human Adeno-virus Species G Isolated from Long-Tailed Macaque Feces.

Metagenomics has demonstrated its capability in outbreak investigations and pathogen surveillance and discovery. With high-throughput and effective bioinformatics, many disease-causing agents, as well as novel viruses of humans and animals, have been identified using metagenomic analysis. In this study, a VIDISCA metagenomics workflow was used to identify potential unknown viruses in 33 fecal samples from asymptomatic long-tailed macaques (Macaca fascicularis) in Ratchaburi Province, Thailand. Putatively novel astroviruses, enteroviruses, and adenoviruses were detected and confirmed by PCR analysis of long-tailed macaque fecal samples collected from areas in four provinces, Ratchaburi, Kanchanaburi, Lopburi, and Prachuap Khiri Khan, where humans and monkeys live in proximity (total n = 187). Astroviruses, enteroviruses, and adenoviruses were present in 3.2%, 7.5%, and 4.8% of macaque fecal samples, respectively. One adenovirus, named AdV-RBR-6-3, was successfully isolated in human cell culture. Whole-genome analysis suggested that it is a new member of the species Human adenovirus G, closely related to Rhesus adenovirus 53, with evidence of genetic recombination and variation in the hexon, fiber, and CR1 genes. Sero-surveillance showed neutralizing antibodies against AdV-RBR-6-3 in 2.9% and 11.2% of monkeys and humans, respectively, suggesting cross-species infection of monkeys and humans. Overall, we reported the use of metagenomics to screen for possible new viruses, as well as the isolation and molecular and serological characterization of the new adenovirus with cross-species transmission potential. The findings emphasize that zoonotic surveillance is important and should be continued, especially in areas where humans and animals interact, to predict and prevent the threat of emerging zoonotic pathogens.

Anellovirus evolution during long-term chronic infection.

Human anelloviruses (AVs) are extremely genetically diverse, are widespread in the human population, and cause chronic infections. However, the evolutionary dynamics of AVs within single hosts is currently unknown, and it is unclear whether these changes have an implication on the long-term persistence of AVs in the host. Here, we assessed the evolutionary dynamics of six AV lineages during 30 years of chronic infection at single host resolution. The total number of substitutions and the number of variable sites increased over time. However, not all substitutions reached population fixation, showing that AV lineages form heterogeneous swarms within the host. Most substitutions occurred within a hypervariable region (HVR) located between nucleotide positions 800 and 1,300 of ORF1, which is known to be located within the spike domain. Different regions of the ORF1 gene undergo either positive or negative selection pressure. Sites under strong diversifying selection pressure were detected in the HVR, while the majority of the sites under purifying selection were detected outside this region. The HVR may play the role of an immunological decoy that prevents antibodies from binding to more vulnerable parts of ORF1. Moreover, the frequent substitutions in this region may increase the chances of AV particles escaping immune recognition.

Viral, Bacterial, Metabolic, and Autoimmune Causes of Severe Acute Encephalopathy in Sub-Saharan Africa: A Multicenter Cohort Study.

OBJECTIVES: To assess whether viral, bacterial, metabolic, and autoimmune diseases are missed by conventional diagnostics among children with severe acute encephalopathy in sub-Saharan Africa. STUDY DESIGN: One hundred thirty-four children (6 months to 18 years) presenting with nontraumatic coma or convulsive status epilepticus to 1 of 4 medical referral centers in Uganda, Malawi, and Rwanda were enrolled between 2015 and 2016. Locally available diagnostic tests could be supplemented in 117 patients by viral, bacterial, and 16s quantitative polymerase chain reaction testing, metagenomics, untargeted metabolomics, and autoimmune immunohistochemistry screening. RESULTS: Fourteen (12%) cases of viral encephalopathies, 8 (7%) cases of bacterial central nervous system (CNS) infections, and 4 (4%) cases of inherited metabolic disorders (IMDs) were newly identified by additional diagnostic testing as the most likely cause of encephalopathy. No confirmed cases of autoimmune encephalitis were found. Patients for whom additional diagnostic testing aided causal evaluation (aOR 3.59, 90% CI 1.57-8.36), patients with a viral CNS infection (aOR 7.91, 90% CI 2.49-30.07), and patients with an IMD (aOR 9.10, 90% CI 1.37-110.45) were at increased risk for poor outcome of disease. CONCLUSIONS: Viral and bacterial CNS infections and IMDs are prevalent causes of severe acute encephalopathy in children in Uganda, Malawi, and Rwanda that are missed by conventional diagnostics and are associated with poor outcome of disease. Improved diagnostic capacity may increase diagnostic yield and might improve outcome of disease.

Bacterial ribosomal RNA detection in cerebrospinal fluid using a viromics approach.

BACKGROUND: In patients with central nervous system (CNS) infections identification of the causative pathogen is important for treatment. Metagenomic next-generation sequencing techniques are increasingly being applied to identify causes of CNS infections, as they can detect any pathogen nucleic acid sequences present. Viromic techniques that enrich samples for virus particles prior to sequencing may simultaneously enrich ribosomes from bacterial pathogens, which are similar in size to small viruses. METHODS: We studied the performance of a viromic library preparation technique (VIDISCA) combined with low-depth IonTorrent sequencing (median ~ 25,000 reads per sample) for detection of ribosomal RNA from common pathogens, analyzing 89 cerebrospinal fluid samples from patients with culture proven bacterial meningitis. RESULTS: Sensitivity and specificity to Streptococcus pneumoniae (n = 24) before and after optimizing threshold parameters were 79% and 52%, then 88% and 90%. Corresponding values for Neisseria meningitidis (n = 22) were 73% and 93%, then 67% and 100%, Listeria monocytogenes (n = 24) 21% and 100%, then 27% and 100%, and Haemophilus influenzae (n = 18) 56% and 100%, then 71% and 100%. A higher total sequencing depth, no antibiotic treatment prior to lumbar puncture, increased disease severity, and higher c-reactive protein levels were associated with pathogen detection. CONCLUSION: We provide proof of principle that a viromic approach can be used to correctly identify bacterial ribosomal RNA in patients with bacterial meningitis. Further work should focus on increasing assay sensitivity, especially for problematic species (e.g. L. monocytogenes), as well as profiling additional pathogens. The technique is most suited to research settings and examination of idiopathic cases, rather than an acute clinical setting.

Host prediction for disease-associated gastrointestinal cressdnaviruses.

Metagenomic techniques have facilitated the discovery of thousands of viruses, yet because samples are often highly biodiverse, fundamental data on the specific cellular hosts are usually missing. Numerous gastrointestinal viruses linked to human or animal diseases are affected by this, preventing research into their medical or veterinary importance. Here, we developed a computational workflow for the prediction of viral hosts from complex metagenomic datasets. We applied it to seven lineages of gastrointestinal cressdnaviruses using 1,124 metagenomic datasets, predicting hosts of four lineages. The Redondoviridae, strongly associated to human gum disease (periodontitis), were predicted to infect Entamoeba gingivalis, an oral pathogen itself involved in periodontitis. The Kirkoviridae, originally linked to fatal equine disease, were predicted to infect a variety of parabasalid protists, including Dientamoeba fragilis in humans. Two viral lineages observed in human diarrhoeal disease (CRESSV1 and CRESSV19, i.e. pecoviruses and hudisaviruses) were predicted to infect Blastocystis spp. and Endolimax nana respectively, protists responsible for millions of annual human infections. Our prediction approach is adaptable to any virus lineage and requires neither training datasets nor host genome assemblies. Two host predictions (for the Kirkoviridae and CRESSV1 lineages) could be independently confirmed as virus-host relationships using endogenous viral elements identified inside host genomes, while a further prediction (for the Redondoviridae) was strongly supported as a virus-host relationship using a case-control screening experiment of human oral plaques.

Control of Human Anelloviruses by Cytosine to Uracil Genome Editing.

Anelloviruses are the most common viruses infecting humans. Every human carries a nonpathogenic personal anellovirus virome (anellome), yet it is unknown which mechanisms contribute to its stability. Here, we assessed the dynamics and impact of a host antiviral defense mechanism-cytidine deaminase activity leading to C to U editing in anelloviruses-on the stability of the anellome. We investigated anellome sequence data obtained from serum samples collected every 6 months from two healthy subjects followed for more than 30 years. The subjects were infected by a total of 64 anellovirus lineages. Minus-stranded C to U editing was observed in lineages belonging to the Alpha-, Beta-, and Gammatorquevirus genera. The edited genomes were present within virus particles, therefore editing must have occurred at the late stages of the virus life cycle. Editing was favored by 5'-TC contexts in the virus genome, indicating that apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like, catalytic subunit 3 or A3 (APOBEC3) proteins are involved. Within a lineage, mutational dynamics varied over time and few fixations of mutations were detected, indicating that C to U editing is a dead end for a virus genome. We detected an editing coldspot in the GC-rich regions, suggesting that the GC-rich region is crucial for genome packaging, since only packaged virus particles were included in the analysis. Finally, we noticed a lineage-specific reduced concentration after an editing event, yet no clearance. In conclusion, cytidine deaminase activity does not clear anelloviruses, nor does it play a major role in virus evolution, but it does contribute to the stability of the anellome. IMPORTANCE Despite significant attention on anellovirus research, the interaction between the anellovirus virome and the human host remains unknown. We show the dynamics of APOBEC3-mediated cytidine deaminase activity on anelloviruses during a 30-year period of chronic infection and postulate that this antiviral mechanism controls anelloviruses. These results expand our knowledge of anellovirus-host interactions, which may be important for the design of gene therapies.

Transmission of anelloviruses to HIV-1 infected children.

Anelloviruses (AVs) are widespread in the population and infect humans at the early stage of life. The mode of transmission of AVs is still unknown, however, mother-to-child transmission, e.g., via breastfeeding, is one of the likely infection routes. To determine whether the mother-to-child transmission of AVs may still occur despite the absence of natural birth and breastfeeding, 29 serum samples from five HIV-1-positive mother and child pairs were Illumina-sequenced. The Illumina reads were mapped to an AV lineage database "Anellometrix" containing 502 distinct ORF1 sequences. Although the majority of lineages from the mother were not shared with the child, the mother and child anellomes did display a significant similarity. These findings suggest that AVs may be transmitted from mothers to their children via different routes than delivery or breastfeeding.

Human Clinical Isolates of Pathogenic Fungi Are Host to Diverse Mycoviruses.

Fungi host viruses from many families, and next-generation sequencing can be used to discover previously unknown genomes. Some fungus-infecting viruses (mycoviruses) confer hypovirulence on their pathogenic hosts, raising the possibility of therapeutic application in the treatment of fungal diseases. Though all fungi probably host mycoviruses, many human pathogens have none documented, implying the mycoviral catalogue remains at an early stage. Here, we carried out virus discovery on 61 cultures of pathogenic fungi covering 27 genera and at least 56 species. Using next-generation sequencing of total nucleic acids, we found no DNA viruses but did find a surprising RNA virus diversity of 11 genomes from six classified families and two unclassified lineages, including eight genomes likely representing new species. Among these was the first jivivirus detected in a fungal host (Aspergillus lentulus). We separately utilized rolling circle amplification and next-generation sequencing to identify ssDNA viruses specifically. We identified 13 new cressdnaviruses across all libraries, but unlike the RNA viruses, they could not be confirmed by PCR in either the original unamplified samples or freshly amplified nucleic acids. Their distributions among sequencing libraries and inconsistent detection suggest low-level contamination of reagents. This highlights both the importance of validation assays and the risks of viral host prediction on the basis of highly amplified sequencing libraries. Meanwhile, the detected RNA viruses provide a basis for experimentation to characterize possible hypovirulent effects, and hint at a wealth of uncharted viral diversity currently frozen in biobanks. IMPORTANCE Fungal pathogens of humans are a growing global health burden. Viruses of fungi may represent future therapeutic tools, but for many fungal pathogens there are no known viruses. Our study examined the viral content of diverse human-pathogenic fungi in a clinical biobank, identifying numerous viral genomes, including one lineage previously not known to infect fungi.

Diversity and Long-Term Dynamics of Human Blood Anelloviruses.

Anelloviruses (AVs) are commensal members of the human blood virome. Even though it was estimated that over 90% of the human population carries AVs, the dynamics of the AV virome ("anellome") are unknown. We investigated the dynamics of blood anellomes in two healthy people followed up for more than 30 years. Both subjects were positive for AVs in the majority of samples. Alphatorquevirus (torque teno virus [TTV]) was the most common genus in both subjects, followed by Betatorquevirus (torque teno minivirus [TTMV]) and Gammatorquevirus (torque teno midivirus [TTMDV]). Almost five times more lineages were found in subject 1 than in subject 2, and the anellomes differed phylogenetically. Both anellomes remained compositionally stable, and 9 out of 64 AV lineages were detected in over half of the time points. We confirmed the long-term and short-term persistence of 13 lineages by specific quantitative PCR (qPCR). AV lineages were detected in blood for over 30 years. Noticeable differences in anellome richness were found between the tested subjects, but both anellomes remained compositionally stable over time. These findings demonstrate that the human blood anellome is personal and that AV infection is chronic and potentially commensal. IMPORTANCE Knowledge of the persistence of AVs in humans is crucial to our understanding of the nature of AV infection (chronic or acute) and the role of AV in the host. We therefore investigated the dynamics of anellovirus infection in two healthy people followed up for 30 years. Our findings suggest that the human blood anellovirus virome (anellome) remains stable and personal for decades.

Early-Life Colonization by Anelloviruses in Infants.

Anelloviruses (AVs) are found in the vast majority of the human population and are most probably part of a healthy virome. These viruses infect humans in the early stage of life, however, the characteristics of the first colonizing AVs are still unknown. We screened a collection of 107 blood samples from children between 0.4 and 64.8 months of age for the presence of three AV genera: the Alpha-, Beta- and Gammatorquevirus. The youngest child that was positive for AV was 1.2 months old, and a peak in prevalence (100% of samples positive) was reached between the twelfth and eighteenth months of life. Intriguingly, the beta- and gammatorqueviruses were detected most at the early stage of life (up to 12 months), whereas alphatorqueviruses, the most common AVs in adults, increased in prevalence in children older than 12 months. To determine whether that order of colonization may be related to oral transmission and unequal presence of AV genera in breast milk, we examined 63 breast milk samples. Thirty-two percent of the breast milk samples were positive in a qPCR detecting beta- and gammatorqueviruses, while alphatorqueviruses were detected in 10% of the samples, and this difference was significant (p = 0.00654). In conclusion, we show that beta- and gammatorqueviruses colonize humans in the first months of life and that breastfeeding could play a role in AV transmission.

Divergent Rhabdovirus Discovered in a Patient with New-Onset Nodding Syndrome.

A divergent rhabdovirus was discovered in the bloodstream of a 15-year-old girl with Nodding syndrome from Mundri West County in South Sudan. Nodding syndrome is a progressive degenerative neuropathy of unknown cause affecting thousands of individuals in Sub-Saharan Africa. The index case was previously healthy until she developed head-nodding seizures four months prior to presentation. Virus discovery by VIDISCA-NGS on the patient's plasma detected multiple sequence reads belonging to a divergent rhabdovirus. The viral load was 3.85 × 103 copies/mL in the patient's plasma and undetectable in her cerebrospinal fluid. Further genome walking allowed for the characterization of full coding sequences of all the viral proteins (N, P, M, U1, U2, G, U3, and L). We tentatively named the virus "Mundri virus" (MUNV) and classified it as a novel virus species based on the high divergence from other known viruses (all proteins had less than 43% amino acid identity). Phylogenetic analysis revealed that MUNV forms a monophyletic clade with several human-infecting tibroviruses prevalent in Central Africa. A bioinformatic machine-learning algorithm predicted MUNV to be an arbovirus (bagged prediction strength (BPS) of 0.9) transmitted by midges (BPS 0.4) with an artiodactyl host reservoir (BPS 0.9). An association between MUNV infection and Nodding syndrome was evaluated in a case-control study of 72 patients with Nodding syndrome (including the index case) matched to 65 healthy households and 48 community controls. No subject, besides the index case, was positive for MUNV RNA in their plasma. A serological assay detecting MUNV anti-nucleocapsid found, respectively, in 28%, 22%, and 16% of cases, household controls and community controls to be seropositive with no significant differences between cases and either control group. This suggests that MUNV commonly infects children in South Sudan yet may not be causally associated with Nodding syndrome.

Dynamics of the Enteric Virome in a Swine Herd Affected by Non-PCV2/PRRSV Postweaning Wasting Syndrome.

A commercial pig farm with no history of porcine circovirus 2 (PCV2) or porcine reproductive and respiratory syndrome virus (PRRSV) repeatedly reported a significant reduction in body weight gain and wasting symptoms in approximately 20-30% of the pigs in the period between three and six weeks after weaning. As standard clinical interventions failed to tackle symptomatology, viral metagenomics were used to describe and monitor the enteric virome at birth, 3 weeks, 4 weeks, 6 weeks, and 9 weeks of age. The latter four sampling points were 7 days, 3 weeks, and 6 weeks post weaning, respectively. Fourteen distinct enteric viruses were identified within the herd, which all have previously been linked to enteric diseases. Here we show that wasting is associated with alterations in the enteric virome of the pigs, characterized by: (1) the presence of enterovirus G at 3 weeks of age, followed by a higher prevalence of the virus in wasting pigs at 6 weeks after weaning; (2) rotaviruses at 3 weeks of age; and (3) porcine sapovirus one week after weaning. However, the data do not provide a causal link between specific viral infections and the postweaning clinical problems on the farm. Together, our results offer evidence that disturbances in the enteric virome at the preweaning stage and early after weaning have a determining role in the development of intestinal barrier dysfunctions and nutrient uptake in the postweaning growth phase. Moreover, we show that the enteric viral load sharply increases in the week after weaning in both healthy and wasting pigs. This study is also the first to report the dynamics and co-infection of porcine rotavirus species and porcine astrovirus genetic lineages during the first 9 weeks of the life of domestic pigs.

Rectal bacteriome and virome signatures and clinical outcomes in community-acquired pneumonia: An exploratory study.

Background Bacterial intestinal communities interact with the immune system and may contribute to protection against community-acquired pneumonia (CAP). Intestinal viruses are closely integrated with these bacterial communities, yet the composition and clinical significance of these communities in CAP patients are unknown. The aims of this exploratory study were to characterise the composition of the rectal bacteriome and virome at hospital admission for CAP, and to determine if microbiota signatures correlate with clinical outcomes. Methods We performed a prospective observational cohort study in CAP patients, admitted to a university or community hospital in the Netherlands between October 2016 and July 2018, and controls. Rectal bacteriome and virome composition were characterised using 16S ribosomal RNA gene sequencing and virus discovery next-generation sequencing, respectively. Unsupervised multi-omics factor analysis was used to assess the co-variation of bacterial and viral communities, which served as primary predictor. The clinical outcomes of interest were the time to clinical stability and the length of hospital stay. Findings 64 patients and 38 controls were analysed. Rectal bacterial alpha (p = 0•0015) and beta diversity (r2 =0•023, p = 0•004) of CAP patients differed from controls. Bacterial and viral microbiota signatures correlated with the time to clinical stability (hazard ratio 0•43, 95% confidence interval 0•20-0•93, p = 0•032) and the length of hospital stay (hazard ratio 0•37, 95% confidence interval 0•17-0•81, p = 0•012), although only the latter remained significant following p-value adjustment for examining multiple candidate cut-points (p = 0•12 and p = 0•046, respectively). Interpretation This exploratory study provides preliminary evidence that intestinal bacteriome and virome signatures could be linked with clinical outcomes in CAP. Such exploratory data, when validated in independent cohorts, could inform the development of a microbiota-based diagnostic panel used to predict clinical outcomes in CAP. Funding Netherlands Organization for Scientific Research and Netherlands Organization for Health Research and Development.

A Versatile Processing Workflow to Enable Pathogen Detection in Clinical Samples from Organs Using VIDISCA.

In recent years, refined molecular methods coupled with powerful high throughput sequencing technologies have increased the potential of virus discovery in clinical samples. However, host genetic material remains a complicating factor that interferes with discovery of novel viruses in solid tissue samples as the relative abundance of the virus material is low. Physical enrichment processing methods, although usually complicated, labor-intensive, and costly, have proven to be successful for improving sensitivity of virus detection in complex samples. In order to further increase detectability, we studied the application of fast and simple high-throughput virus enrichment methods on tissue homogenates. Probe sonication in high EDTA concentrations, organic extraction with Vertrel™ XF, or a combination of both, were applied prior to chromatography-like enrichment using Capto™ Core 700 resin, after which effects on virus detection sensitivity by the VIDISCA method were determined. Sonication in the presence of high concentrations of EDTA showed the best performance with an increased proportion of viral reads, up to 9.4 times, yet minimal effect on the host background signal. When this sonication procedure in high EDTA concentrations was followed by organic extraction with Vertrel™ XF and two rounds of core bead chromatography enrichment, an increase up to 10.5 times in the proportion of viral reads in the processed samples was achieved, with reduction of host background sequencing. We present a simple and semi-high-throughput method that can be used to enrich homogenized tissue samples for viral reads.