Nádia Conceição-Neto

Modular approach to customise sample preparation procedures for viral metagenomics: a reproducible protocol for virome analysis.

A major limitation for better understanding the role of the human gut virome in health and disease is the lack of validated methods that allow high throughput virome analysis. To overcome this, we evaluated the quantitative effect of homogenisation, centrifugation, filtration, chloroform treatment and random amplification on a mock-virome (containing nine highly diverse viruses) and a bacterial mock-community (containing four faecal bacterial species) using quantitative PCR and next-generation sequencing. This resulted in an optimised protocol that was able to recover all viruses present in the mock-virome and strongly alters the ratio of viral versus bacterial and 16S rRNA genetic material in favour of viruses (from 43.2% to 96.7% viral reads and from 47.6% to 0.19% bacterial reads). Furthermore, our study indicated that most of the currently used virome protocols, using small filter pores and/or stringent centrifugation conditions may have largely overlooked large viruses present in viromes. We propose NetoVIR (Novel enrichment technique of VIRomes), which allows for a fast, reproducible and high throughput sample preparation for viral metagenomics studies, introducing minimal bias. This procedure is optimised mainly for faecal samples, but with appropriate concentration steps can also be used for other sample types with lower initial viral loads.

Complete genome sequence of a porcine epidemic diarrhea virus from a novel outbreak in belgium, january 2015.

ABSTRACT Porcine epidemic diarrhea virus (PEDV) is a member of the family Coronaviridae and can cause severe outbreaks of diarrhea in piglets from different age groups. Here, we report the complete genome sequence (28,028 nt) of a PEDV strain isolated during a novel outbreak in Belgium.

Rotavirus Surveillance in Kisangani, the Democratic Republic of the Congo, Reveals a High Number of Unusual Genotypes and Gene Segments of Animal Origin in Non-Vaccinated Symptomatic Children

Group A rotavirus (RVA) infections form a major public health problem, especially in low-income countries like the Democratic Republic of the Congo (COD). However, limited data on RVA diversity is available from sub-Saharan Africa in general and the COD in particular. Therefore, the first aim of this study was to determine the genetic diversity of 99 RVAs detected during 2007–2010 in Kisangani, COD. The predominant G-type was G1 (39%) and the most predominant P-type was P[6] (53%). A total of eight different G/P-combinations were found: G1P[8] (28%), G8P[6] (26%), G2P[4] (14%), G12P[6] (13%), G1P[6] (11%), G9P[8] (4%), G4P[6] (2%) and G8P[4] (1%). The second aim of this study was to gain insight into the diversity of P[6] RVA strains in the COD. Therefore, we selected five P[6] RVA strains in combination with the G1, G4, G8 (2x) or G12 genotype for complete genome analysis. Complete genome analysis showed that the genetic background of the G1P[6] and G12P[6] strains was entirely composed of genotype 1 (Wa-like), while the segments of the two G8P[6] strains were identified as genotype 2 (DS-1-like). Interestingly, all four strains possessed a NSP4 gene of animal origin. The analyzed G4P[6] RVA strain was found to possess the unusual G4-P[6]-I1-R1-C1-M1-A1-N1-T7-E1-H1 constellation. Although the majority of its genes (if not all), were presumably of porcine origin, this strain was able to cause gastro-enteritis in humans. The high prevalence of unusual RVA strains in the COD highlights the need for continued surveillance of RVA diversity in the COD. These results also underline the importance of complete genetic characterization of RVA strains and indicate that reassortments and interspecies transmission among human and animal RVAs strains occur regularly. Based on these data, RVA vaccines will be challenged with a wide variety of different RVA strain types in the COD.

Fecal virome analysis of three carnivores reveals a novel nodavirus and multiple gemycircularviruses.

BackgroundMore knowledge about viral populations in wild animals is needed in order to better understand and assess the risk of zoonotic diseases. In this study we performed viral metagenomic analysis of fecal samples from three healthy carnivores: a badger (Meles meles), a mongoose (Herpestes ichneumon) and an otter (Lutra lutra) from Portugal.ResultsWe detected the presence of novel highly divergent viruses in the fecal material of the carnivores analyzed, such as five gemycircularviruses. Four of these gemycircularviruses were found in the mongoose and one in the badger. In addition we also identified an RNA-dependent RNA polymerase gene from a putative novel member of the Nodaviridae family in the fecal material of the otter.ConclusionsTogether these results underline that many novel viruses are yet to be discovered and that fecal associated viruses are not always related to disease. Our study expands the knowledge of viral species present in the gut, although the interpretation of the true host species of such novel viruses needs to be reviewed with great caution.

Antibodies to hepatitis E in Portuguese mothers and their newborns

The high mortality of pregnant women is one of the main features of hepatitis E virus (HEV) infection in endemic countries. With the emergence of genotype 3 HEV in industrialized countries, attention should be given to pregnant women from these regions since the effect of genotype 3 HEV in pregnancy is not yet known. In this study, archived sera dating from 1993 from mothers with no history of traveling to HEV endemic countries and their newborns were tested for anti‐HEV IgG antibodies. Four of the 12 maternal sera were positive for IgG anti‐HEV, indicating exposure to this virus. Their newborns were also positive, showing higher anti‐HEV IgG levels. Overall, these findings demonstrate not only an efficient transplacental transport of anti‐HEV IgG, but also circulation of autochthonous HEV in Portugal in the early 1990s. J. Med. Virol. 85:1377–1378, 2013.

Experimental feline enteric coronavirus infection reveals an aberrant infection pattern and shedding of mutants with impaired infectivity in enterocyte cultures.

Feline infectious peritonitis (FIP) results from mutations in the viral genome during a common feline enteric coronavirus (FECV) infection. Since many virological and immunological data on FECV infections are lacking, the present study investigated these missing links during experimental infection of three SPF cats with FECV strain UCD. Two cats showed mild clinical signs, faecal shedding of infectious virus from 4 dpi, a cell-associated viraemia at inconsistent time points from 5 dpi, a highly neutralising antibody response from 9 dpi, and no major abnormalities in leukocyte numbers. Faecal shedding lasted for 28–56 days, but virus shed during this stage was less infectious in enterocyte cultures and affected by mutations. Remarkably, in the other cat neither clinical signs nor acute shedding were seen, but virus was detected in blood cells from 3 dpi, and shedding of non-enterotropic, mutated viruses suddenly occurred from 14 dpi onwards. Neutralising antibodies arose from 21 dpi. Leukocyte numbers were not different compared to the other cats, except for the CD8+ regulatory T cells. These data indicate that FECV can infect immune cells even in the absence of intestinal replication and raise the hypothesis that the gradual adaptation to these cells can allow non-enterotropic mutants to arise.

Highly diverse population of Picornaviridae and other members of the Picornavirales , in Cameroonian fruit bats

BackgroundThe order Picornavirales represents a diverse group of positive-stranded RNA viruses with small non-enveloped icosahedral virions. Recently, bats have been identified as an important reservoir of several highly pathogenic human viruses. Since many members of the Picornaviridae family cause a wide range of diseases in humans and animals, this study aimed to characterize members of the order Picornavirales in fruit bat populations located in the Southwest region of Cameroon. These bat populations are frequently in close contact with humans due to hunting, selling and eating practices, which provides ample opportunity for interspecies transmissions.ResultsFecal samples from 87 fruit bats (Eidolon helvum and Epomophorus gambianus), were combined into 25 pools and analyzed using viral metagenomics. In total, Picornavirales reads were found in 19 pools, and (near) complete genomes of 11 picorna-like viruses were obtained from 7 of these pools. The picorna-like viruses possessed varied genomic organizations (monocistronic or dicistronic), and arrangements of gene cassettes. Some of the viruses belonged to established families, including the Picornaviridae, whereas others clustered distantly from known viruses and most likely represent novel genera and families. Phylogenetic and nucleotide composition analyses suggested that mammals were the likely host species of bat sapelovirus, bat kunsagivirus and bat crohivirus, whereas the remaining viruses (named bat iflavirus, bat posalivirus, bat fisalivirus, bat cripavirus, bat felisavirus, bat dicibavirus and bat badiciviruses 1 and 2) were most likely diet-derived.ConclusionThe existence of a vast genetic variability of picorna-like viruses in fruit bats may increase the probability of spillover infections to humans especially when humans and bats have direct contact as the case in this study site. However, further screening for these viruses in humans will fully indicate their zoonotic potential.

Novel highly divergent reassortant bat rotaviruses in Cameroon, without evidence of zoonosis

Bats are an important reservoir for zoonotic viruses. To date, only three RVA strains have been reported in bats in Kenya and China. In the current study we investigated the genetic diversity of RVAs in fecal samples from 87 straw-colored fruit bats living in close contact with humans in Cameroon using viral metagenomics. Five (near) complete RVA genomes were obtained. A single RVA strain showed a partial relationship with the Kenyan bat RVA strain, whereas the other strains were completely novel. Only the VP7 and VP4 genes showed significant variability, indicating the occurrence of frequent reassortment events. Comparing these bat RVA strains with currently used human RVA screening primers indicated that most of the novel VP7 and VP4 segments would not be detected in routine epidemiological screening studies. Therefore, novel consensus screening primers were developed and used to screen samples from infants with gastroenteritis living in close proximity with the studied bat population. Although RVA infections were identified in 36% of the infants, there was no evidence of zoonosis. This study identified multiple novel bat RVA strains, but further epidemiological studies in humans will have to assess if these viruses have the potential to cause gastroenteritis in humans.

Pet veterinarians have no increased risk of hepatitis E compared to the general population

The discovery of autochthonous hepatitis E in industrialized countries associated to genotype 3 has changed substantially the understanding about hepatitis E virus (HEV) circulation. In these countries transmission has been associated to the consumption and direct contact to swine, a well‐known reservoir for HEV. More animal reservoirs for HEV might exist since antibodies to HEV have been detected in other animal species, such as dogs and cats. Given the intimate contact between these pets and humans, the potential zoonotic HEV transmission from dogs and cats deserves to be explored. To address this issue, 493 sera from pet veterinarians (373) and matched general population (120) were tested for the presence of anti‐HEV IgG using a commercial ELISA. Antibodies to HEV were found in 9.9% veterinarians and 13.3% general population. No statistically significant difference was found between these two groups showing that pet veterinarians have no increased risk to hepatitis E compared to the general population. J. Med. Virol. 86:954–956, 2014.

Characterization of a genetically heterogeneous porcine rotavirus C, and other viruses present in the fecal virome of a non-diarrheic Belgian piglet

Abstract Next-generation sequencing (NGS) technologies are becoming increasingly accessible, leading to an expanded interest in the composition of the porcine enteric virome. In the present study, the fecal virome of a non-diarrheic Belgian piglet was determined. Although the virome of only a single piglet was analyzed, some interesting data were obtained, including the second complete genome of a pig group C rotavirus (RVC). This Belgian strain was only distantly related to the only other completely characterized pig RVC strain, Cowden. Its relatedness to RVC strains from other host species was also analyzed and the porcine strain found in our study was only distantly related to RVCs detected in humans and cows. The gene encoding the outer capsid protein VP7 belonged to the rare porcine G3 genotype, which might be serologically distinct from most other pig RVC strains. A putative novel RVC VP6 genotype was identified as well. A group A rotavirus strain also present in this fecal sample contained the rare pig genotype combination G11P[27], but was only partially characterized. Typical pig RVA genotypes I5, A8, and T7 were found for the viral proteins VP6, NSP1, and NSP3, respectively. Interestingly, the fecal virome of the piglet also contained an astrovirus and an enterovirus, of which the complete genomes were characterized. Results of the current study indicate that many viruses may be present simultaneously in fecal samples of non-diarrheic piglets. In this study, these viruses could not be directly associated with any disease, but still they might have had a potential subclinical impact on pig growth performance. The fast evolution of NGS will be a powerful tool for future diagnostics in veterinary practice. Its application will certainly lead to better insights into the relevance of many (sub)clinical enteric viral infections, that may have remained unnoticed using traditional diagnostic techniques. This will stimulate the development of new and durable prophylactic measures to improve pig health and production.