Seyed Mohammad Jazaeri Farsani

Autologous antibody capture to enrich immunogenic viruses for viral discovery.

Discovery of new viruses has been boosted by novel deep sequencing technologies. Currently, many viruses can be identified by sequencing without knowledge of the pathogenicity of the virus. However, attributing the presence of a virus in patient material to a disease in the patient can be a challenge. One approach to meet this challenge is identification of viral sequences based on enrichment by autologous patient antibody capture. This method facilitates identification of viruses that have provoked an immune response within the patient and may increase the sensitivity of the current virus discovery techniques. To demonstrate the utility of this method, virus discovery deep sequencing (VIDISCA-454) was performed on clinical samples from 19 patients: 13 with a known respiratory viral infection and 6 with a known gastrointestinal viral infection. Patient sera was collected from one to several months after the acute infection phase. Input and antibody capture material was sequenced and enrichment was assessed. In 18 of the 19 patients, viral reads from immunogenic viruses were enriched by antibody capture (ranging between 1.5x to 343x in respiratory material, and 1.4x to 53x in stool). Enriched reads were also determined in an identity independent manner by using a novel algorithm Xcompare. In 16 of the 19 patients, 21% to 100% of the enriched reads were derived from infecting viruses. In conclusion, the technique provides a novel approach to specifically identify immunogenic viral sequences among the bulk of sequences which are usually encountered during virus discovery metagenomics.

Metagenomic analysis of a sample from a patient with respiratory tract infection reveals the presence of a γ-papillomavirus.

Previously unknown or unexpected pathogens may be responsible for that proportion of respiratory diseases in which a causative agent cannot be identified. The application of broad-spectrum, sequence independent virus discovery techniques may be useful to reduce this proportion and widen our knowledge about respiratory pathogens. Thanks to the availability of high-throughput sequencing (HTS) technology, it became today possible to detect viruses which are present at a very low load, but the clinical relevance of those viruses must be investigated. In this study we used VIDISCA-454, a restriction enzyme based virus discovery method that utilizes Roche 454 HTS system, on a nasal swab collected from a subject with respiratory complaints. A γ-papillomavirus was detected (complete genome: 7142 bp) and its role in disease was investigated. Respiratory samples collected both during the acute phase of the illness and 2 weeks after full recovery contained the virus. The patient presented antibodies directed against the virus but there was no difference between IgG levels in blood samples collected during the acute phase and 2 weeks after full recovery. We therefore concluded that the detected γ-papillomavirus is unlikely to be the causative agent of the respiratory complaints and its presence in the nose of the patient is not related to the disease. Although HTS based virus discovery techniques proved their great potential as a tool to clarify the etiology of some infectious diseases, the obtained information must be subjected to cautious interpretations. This study underlines the crucial importance of performing careful investigations on viruses identified when applying sensitive virus discovery techniques, since the mere identification of a virus and its presence in a clinical sample are not satisfactory proofs to establish a causative link with a disease.

The first complete genome sequences of clinical isolates of human coronavirus 229E

Human coronavirus 229E has been identified in the mid-1960s, yet still only one full-genome sequence is available. This full-length sequence has been determined from the cDNA-clone Inf-1 that is based on the lab-adapted strain VR-740. Lab-adaptation might have resulted in genomic changes, due to insufficient pressure to maintain gene integrity of non-essential genes. We present here the first full-length genome sequence of two clinical isolates. Each encoded gene was compared to Inf-1. In general, little sequence changes were noted, most could be attributed to genetic drift, since the clinical isolates originate from 2009 to 2010 and VR740 from 1962. Hot spots of substitutions were situated in the S1 region of the Spike, the nucleocapsid gene, and the non-structural protein 3 gene, whereas several deletions were detected in the 3′UTR. Most notable was the difference in genome organization: instead of an ORF4A and ORF4B, an intact ORF4 was present in clinical isolates.

Identification of a new genotype of Torque Teno Mini virus

BackgroundAlthough human torque teno viruses (TTVs) were first discovered in 1997, still many associated aspects are not clarified yet. The viruses reveal a remarkable heterogeneity and it is possible that some genotypes are more pathogenic than others. The identification of all genotypes is essential to confirm previous pathogenicity data, and an unbiased search for novel viruses is needed to identify TTVs that might be related to disease.MethodThe virus discovery technique VIDISCA-454 was used to screen serum of 55 HIV-1 positive injecting drug users, from the Amsterdam Cohort Studies, in search for novel blood-blood transmittable viruses which are undetectable via normal diagnostics or panvirus-primer PCRs.ResultsA novel torque teno mini virus (TTMV) was identified in two patients and the sequence of the full genomes were determined. The virus is significantly different from the known TTMVs (< 40% amino acid identity in ORF1), yet it contains conserved characteristics that are also present in other TTMVs. The virus is chronically present in both patients, and these patients both suffered from a pneumococcal pneumonia during follow up and had extremely low B-cells counts.ConclusionWe describe a novel TTMV which we tentatively named TTMV-13. Further research is needed to address the epidemiology and pathogenicity of this novel virus.

Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma

Cancer is one of the leading health concerns for human and animal health. Since the tumorigenesis process is not completely understood and it is known that some viruses can induce carcinogenesis, it is highly important to identify novel oncoviruses and extensively study underlying oncogenic mechanisms. Here, we investigated a case of diffuse histiocytic sarcoma in a 22 year old slow loris (Nycticebus coucang), using a broad spectrum virus discovery technique. A novel parvovirus was discovered and the phylogenetic analysis performed on its fully sequenced genome demonstrated that it represents the first member of a novel genus. The possible causative correlation between this virus and the malignancy was further investigated and 20 serum and 61 organ samples from 25 animals (N. coucang and N. pygmaeus) were screened for the novel virus but only samples collected from the originally infected animal were positive. The virus was present in all tested organs (intestine, liver, spleen, kidneys, and lungs) and in all banked serum samples collected up to 8 years before death. All attempts to identify a latent viral form (integrated or episomal) were unsuccessful and the increase of variation in the viral sequences during the years was consistent with absence of latency. Since it is well known that parvoviruses are dependent on cell division to successfully replicate, we hypothesized that the virus could have benefitted from the constantly dividing cancer cells and may not have been the cause of the histiocytic sarcoma. It is also possible to conjecture that the virus had a role in delaying the tumor progression and this report might bring new exciting opportunities in recognizing viruses to be used in cancer virotherapy.

Influenza and Other Respiratory Viruses Involved in Severe Acute Respiratory Disease in Northern Italy during the Pandemic and Postpandemic Period (2009–2011)

Since 2009 pandemic, international health authorities recommended monitoring severe and complicated cases of respiratory disease, that is, severe acute respiratory infection (SARI) and acute respiratory distress syndrome (ARDS). We evaluated the proportion of SARI/ARDS cases and deaths due to influenza A(H1N1)pdm09 infection and the impact of other respiratory viruses during pandemic and postpandemic period (2009–2011) in northern Italy; additionally we searched for unknown viruses in those cases for which diagnosis remained negative. 206 respiratory samples were collected from SARI/ARDS cases and analyzed by real-time RT-PCR/PCR to investigate influenza viruses and other common respiratory pathogens; also, a virus discovery technique (VIDISCA-454) was applied on those samples tested negative to all pathogens. Influenza A(H1N1)pdm09 virus was detected in 58.3% of specimens, with a case fatality rate of 11.3%. The impact of other respiratory viruses was 19.4%, and the most commonly detected viruses were human rhinovirus/enterovirus and influenza A(H3N2). VIDISCA-454 enabled the identification of one previously undiagnosed measles infection. Nearly 22% of SARI/ARDS cases did not obtain a definite diagnosis. In clinical practice, great efforts should be dedicated to improving the diagnosis of severe respiratory disease; the introduction of innovative molecular technologies, as VIDISCA-454, will certainly help in reducing such “diagnostic gap.”

A Novel Astrovirus-Like RNA Virus Detected in Human Stool

Several novel clades of astroviruses have recently been identified in human faecal samples. Here, we describe a novel astrovirus-like RNA virus detected in human stools, which we have tentatively named bastrovirus. The genome of this novel virus consists of 6,300 nucleotides organized in three open reading frames. Several sequence divergent strains were detected sharing 67–93 per cent nucleotide identity. Bastrovirus encodes a putative structural protein that is homologous to the capsid protein found in members of the Astroviridae family (45% amino acid identity). The virus also encodes a putative non-structural protein that is genetically distant from astroviruses but shares some homology to the non-structural protein encoded by members of the Hepeviridae family (28% amino acid identity). This novel bastrovirus is present in 8.7 per cent (35/400) of faecal samples collected from 300 HIV-1-positive and 100 HIV-1-negative individuals suggesting common occurrence of the virus. However, whether the source of the virus is infected human cells or other, for example, dietary, remains to be determined.

Reduced maternal levels of common viruses during pregnancy predict offspring psychosis: potential role of enhanced maternal immune activity?

Viral infections during the prenatal or early childhood periods are one of the environmental factors which might play an etiological role in psychoses. Several studies report higher antibody levels against viruses during pregnancy in blood of mothers of offspring with psychotic disorders, but the presence of such viruses has never been demonstrated. The goal of this study was to investigate the potential association between viral infections during pregnancy and progeny with psychotic disorders and, for this purpose, we performed a nested case-control study involving pregnant mothers of offspring with schizophrenia or bipolar disorder with psychotic features (cases, N=43) and pregnant women with healthy offspring (controls, N=95). Since several potential viral candidates have been suggested in prior work, a broad-spectrum virus detection system was necessary. A metagenomic analysis performed with the virus discovery method VIDISCA-454 revealed only common blood-associated viruses in all cohorts. However, a significantly lower viral prevalence was detected in the group of cases and in the sub-population of pregnant mothers of offspring with schizophrenia (p<0.05). Consistent with the existing inverse correlation between the level of these viruses and the immunocompetence of an individual, we hypothesized the presence of a higher immune activity during pregnancy in mothers whose offspring later develop a psychotic disorder as compared to controls. Combining our results with previously available literature data on antibody levels during the gestation period suggests that a more prominent maternal immune activity can be considered a risk factor for developing psychosis.

Culturing of respiratory viruses in well-differentiated pseudostratified human airway epithelium as a tool to detect unknown viruses.

Currently, virus discovery is mainly based on molecular techniques. Here, we propose a method that relies on virus culturing combined with state‐of‐the‐art sequencing techniques. The most natural ex vivo culture system was used to enable replication of respiratory viruses.

Identification of a Novel Human Rhinovirus C Type by Antibody Capture VIDISCA-454

Causative agents for more than 30 percent of respiratory infections remain unidentified, suggesting that unknown respiratory pathogens might be involved. In this study, antibody capture VIDISCA-454 (virus discovery cDNA-AFLP combined with Roche 454 high-throughput sequencing) resulted in the discovery of a novel type of rhinovirus C (RV-C). The virus has an RNA genome of at least 7054 nt and carries the characteristics of rhinovirus C species. The gene encoding viral protein 1, which is used for typing, has only 81% nucleotide sequence identity with the closest known RV-C type, and, therefore, the virus represents the first member of a novel type, named RV-C54.