Axel Mauroy

A Review of Known and Hypothetical Transmission Routes for Noroviruses

Human noroviruses (NoVs) are considered a worldwide leading cause of acute non-bacterial gastroenteritis. Due to a combination of prolonged shedding of high virus levels in feces, virus particle shedding during asymptomatic infections, and a high environmental persistence, NoVs are easily transmitted pathogens. Norovirus (NoV) outbreaks have often been reported and tend to affect a lot of people. NoV is spread via feces and vomit, but this NoV spread can occur through several transmission routes. While person-to-person transmission is without a doubt the dominant transmission route, human infective NoV outbreaks are often initiated by contaminated food or water. Zoonotic transmission of NoV has been investigated, but has thus far not been demonstrated. The presented review aims to give an overview of these NoV transmission routes. Regarding NoV person-to-person transmission, the NoV GII.4 genotype is discussed in the current review as it has been very successful for several decades but reasons for its success have only recently been suggested. Both pre-harvest and post-harvest contamination of food products can lead to NoV food borne illness. Pre-harvest contamination of food products mainly occurs via contact with polluted irrigation water in case of fresh produce or with contaminated harvesting water in case of bivalve molluscan shellfish. On the other hand, an infected food handler is considered as a major cause of post-harvest contamination of food products. Both transmission routes are reviewed by a summary of described NoV food borne outbreaks between 2000 and 2010. A third NoV transmission route occurs via water and the spread of NoV via river water, ground water, and surface water is reviewed. Finally, although zoonotic transmission remains hypothetical, a summary on the bovine and porcine NoV presence observed in animals is given and the presence of human infective NoV in animals is discussed.

Noroviruses and sapoviruses in pigs in Belgium

Porcine noroviruses and sapoviruses belong to the family Caliciviridae and are rarely reported in European countries. In this study, swine stools from a region representative of northern Europe were screened for these viruses by RT-PCR. Both porcine noroviruses and sapoviruses were detected, showing their circulation in this region. The porcine norovirus strains were genetically related to genotype 19 strains in the genogroup II of the genus Norovirus. The porcine sapovirus strains were genetically related to the porcine enteric calicivirus Cowden reference strain and to newly described porcine strains in the genus Sapovirus.

Molecular detection of kobuviruses and recombinant noroviruses in cattle in continental Europe

Two genotypes (Jena and Newbury2) and two intergenotype recombinant strains have been recognized in bovine noroviruses. Several studies have shown an apparent predominance of bovine infection with Newbury2-related (genotype 2) strains. Bovine stool samples were screened with two primer pairs targeting both the polymerase and the capsid genes. Among the predominant genotype 2 sequences, two were genetically related to the recombinant strain Thirsk10. The detection of sequences genetically related to Thirsk10, together with the very low rate of detection of Jena-related sequences, characterized the bovine norovirus population in Belgium, a representative region of continental Europe. Unexpectedly, bovine kobuvirus-related sequences were also amplified, extending their distribution area in Europe.

Experimental evidence of recombination in murine noroviruses.

Based on sequencing data, norovirus (NoV) recombinants have been described, but no experimental evidence of recombination in NoVs has been documented. Using the murine norovirus (MNV) model, we investigated the occurrence of genetic recombination between two co-infecting wild-type MNV isolates in RAW cells. The design of a PCR-based genotyping tool allowed accurate discrimination between the parental genomes and the detection of a viable recombinant MNV (Rec MNV) in the progeny viruses. Genetic analysis of Rec MNV identified a homologous-recombination event located at the ORF1-ORF2 overlap. Rec MNV exhibited distinct growth curves and produced smaller plaques than the wild-type MNV in RAW cells. Here, we demonstrate experimentally that MNV undergoes homologous recombination at the previously described recombination hot spot for NoVs, suggesting that the MNV model might be suitable for in vitro studies of NoV recombination. Moreover, the results show that exchange of genetic material between NoVs can generate viruses with distinct biological properties from the parental viruses.

Estimation of hepatitis E virus (HEV) pig seroprevalence using ELISA and Western blot and comparison between human and pig HEV sequences in Belgium

Zoonotic transmission of hepatitis E virus (HEV) is of special concern, particularly in high income countries were waterborne infections are less frequent than in developing countries. High HEV seroprevalences can be found in European pig populations. The aims of this study were to obtain prevalence data on HEV infection in swine in Belgium and to phylogenetically compare Belgian human HEV sequences with those obtained from swine. An ELISA screening prevalence of 73% (95% CI 68.8-77.5) was determined in Belgian pigs and a part of the results were re-evaluated by Western blot (WB). A receiver operating characteristic curve analysis was performed and scenarios varying the ELISA specificity relative to WB were analysed. The seroprevalences estimated by the different scenarios ranged between 69 and 81% and are in agreement with the high exposure of the European pig population to HEV. Pig HEV sequences were genetically compared to those detected in humans in Belgium and a predominance of genotype 3 subtype f was shown in both swine and humans. The high HEV seroprevalence in swine and the close phylogenetic relationships between pig and human HEV sequences further support the risk for zoonotic transmission of HEV between humans and pigs.

Epidemiological study of bovine norovirus infection by RT-PCR and A VLP-based Antibody ELISA

Abstract Noroviruses, belonging to the family Caliciviridae, have been identified in human beings and in several animal species including cattle. The distribution of bovine norovirus infections was investigated by both RT-PCR to detect norovirus genomes and a virus-like particles-based ELISA to detect genotype 2 bovine norovirus antibodies. During a 1-year systematic study, a virus prevalence of 7.5% (CI 95%: [3.7; 13.4%]) (10 out of 133 samples) was found in stool samples from diarrhoeic calves screened by RT-PCR. Nucleotide sequencing performed on the polymerase region classified all the norovirus amplicons in the bovine norovirus genotype 2. Rather surprisingly, some rotavirus sequences were also detected. On the basis of the polymerase region, genotype 1 bovine norovirus was not identified. Other enteropathogens were found in all samples. By ELISA, a genotype 2 seroprevalence of 93.2% (CI 95%: [90.4; 95.3%]) was found from calves and adult cattle. Antibody levels against genotype 2 bovine noroviruses rose in the first 6 months of life and were maintained in adults. Together the results of virus prevalence and seroprevalence studies suggest that bovine norovirus infection occurs early in life and that re-infection with serologically related bovine noroviruses strains could occur in adult cattle as reported for rotaviruses. The antibody rise against genotype 2 bovine noroviruses in the adult cattle also suggests a short lived and/or strain specific immunity as already shown in human noroviruses. Genotype 2 bovine noroviruses are endemic in the region investigated.

Molecular Detection and Genotyping of Noroviruses

Noroviruses (NoVs) are a major cause of gastroenteritis worldwide in humans and animals and are known as very infectious viral agents. They are spread through feces and vomit via several transmission routes involving person-to-person contact, food, and water. Investigation of these transmission routes requires sensitive methods for detection of NoVs. As NoVs cannot be cultivated to date, detection of these viruses relies on the use of molecular methods such as (real-time) reverse transcriptase polymerase chain reaction (RT-PCR). Regardless of the matrix, detection of NoVs generally requires three subsequent steps: a virus extraction step, RNA purification, and molecular detection of the purified RNA, occasionally followed by molecular genotyping. The current review mainly focused on the molecular detection and genotyping of NoVs. The most conserved region in the genome of human infective NoVs is the ORF1/ORF2 junction and has been used as a preferred target region for molecular detection of NoVs by methods such as (real-time) RT-PCR, NASBA, and LAMP. In case of animal NoVs, broad range molecular assays have most frequently been applied for molecular detection. Regarding genotyping of NoVs, five regions situated in the polymerase and capsid genes have been used for conventional RT-PCR amplification and sequencing. As the expected levels of NoVs on food and in water are very low and inhibition of molecular methods can occur in these matrices, quality control including adequate positive and negative controls is an essential part of NoV detection. Although the development of molecular methods for NoV detection has certainly aided in the understanding of NoV transmission, it has also led to new problems such as the question whether low levels of human NoV detected on fresh produce and shellfish could pose a threat to public health.

Isolation and characterization of bovine parainfluenza virus type 3 from water buffaloes (Bubalus bubalis) in Argentina

BackgroundParainfluenza virus type 3 (PIV3) was isolated from dairy buffaloes (Bubalus bubalis) naturally affected with respiratory and reproductive clinical conditions.ResultsExamination of nasal and vaginal swabs collected from 12 diseased buffaloes led to the isolation of three paramyxovirus isolates from two animals. Antigenic, morphological and biological characteristics of these three isolates were essentially similar to those of members of the Paramyxoviridae family. Antigenic analysis by direct immunofluorescence and cross neutralization test placed these isolates together with bovine parainfluenza virus type 3 (BPIV3). Nucleotide and amino acid phylogenetic analysis of partial matrix gene sequences of the buffalo isolates and six field BPIV3 isolates from bovines in Argentina were studied. Buffalo isolates were similar to genotype B (BPIV3b) while the six BPIV3 isolates were similar to genotypes A (BPIV3a) and C (BPIV3c).ConclusionsThis is the first characterization of BPIV3 in water buffalo.According to the samples analyzed, in Argentina, the genotype B was found in buffalo and the genotypes A and C were found in cattle.

Hepatitis E Virus and Related Viruses in Animals.

&NA; Hepatitis E is an acute human liver disease in healthy individuals which may eventually become chronic. It is caused by the hepatitis E virus (HEV) and can have a zoonotic origin. Nearly 57,000 people die yearly from hepatitis E‐related conditions. The disease is endemic in both developing and developed countries with distinct epidemiologic profiles. In developing countries, the disease is associated with inadequate water treatment, while in developed countries, transmission is associated with animal contact and the ingestion of raw or uncooked meat, especially liver. All human HEV are grouped into at least four genotypes, while HEV or HEV‐related viruses have been identified in an increasing number of domestic and wild animal species. Despite a high genetic diversity, only one single HEV serotype has been described to date for HEV genotypes 1–4. The discovery of new HEV or HEV‐related viruses leads to a continuing increase in the number of genotypes. In addition, the genome organization of all these viruses is variable with overlapping open reading frames (ORF) and differences in the location of ORF3. In spite of the role of some domestic and wild animals as reservoir, the origin of HEV and HEV‐related viruses in humans and animals is still unclear. This review discusses aspects of the detection, molecular virology, zoonotic transmission and origin of HEV and HEV‐related viruses in the context of ‘One Health’ and establishes a link between the previous and the new taxonomy of this growing virus family.

Bluetongue in captive yaks.

To the Editor: In August 2006, several northern European countries including Belgium reported cases of bluetongue (BT) (1). This noncontagious, arthropod-borne animal disease is caused by Bluetongue virus (BTV), genus Orbivirus, family Reoviridae. The genome of BTV consists of 10 segments of double-stranded RNA; 24 serotypes have been reported (2). Serotype 8 (BTV-8) was implicated in the emergence in Belgium (3). All ruminant species are thought to be susceptible to BT (2). We report laboratory-confirmed clinical cases of BT in yaks (Bos grunniens grunniens). Yaks living in captivity in a Belgian animal park showed clinical signs of BT. A clinical examination performed on 1 yak showed loss of weight associated with a progressive weakness linked to anorexia, ulcerative and necrotic lesions on the muzzle with some crusts and mucopurulent nasal discharge, and udder erythema with papules and crusts. The tongue was severely swollen and cyanotic and protruded from the mouth (Figure). The animal was reluctant to move and was recumbent (possibly as a consequence of podal lesions linked to BT); it died 7 days after examination. Necropsies were performed on carcasses of this and another yak. The main lesions found were severe diffuse congestion of the lungs with edema and emphysema, acute hemorrhagic enteritis restricted to the ileum and jejunum, and petechial hemorrhages on the abomasums. No lesions characteristic of coronitis were noted. Figure A captive yak infected with bluetongue virus. Tongue is swollen, cyanotic, and protruding from the mouth. Samples of spleen and bone marrow were taken and prepared according to the method of Parsonson and McColl (4). A real-time reverse transcription quantitative–PCR (RT-qPCR) targeting BTV segment 5 (RT-qPCR_S5) was used to detect BTV RNA in tissues samples. Each test was performed in parallel with a RT-qPCR that amplifies β-actin mRNA as an internal control (RT-qPCR_ACT). Both assays were conducted according to Toussaint and others (5), with slight modifications. Briefly, total RNA was purified from 25 mg of tissue by Trizol extraction (Invitrogen, Carlsbad, CA, USA) and denatured by heating for 3 min at 95°C with 10% dimethylsulfoxide (Sigma-Aldrich, St. Louis, MO, USA). Reverse transcription reactions were conducted by using the Taqman reverse transcription reagents according to the manufacturer instructions (Applied Biosystems, Foster City, CA, USA). RT-qPCR reactions consisted of 1× concentrated Taqman fast universal PCR master mix (Applied Biosystems), 375 nM (β actin) or 500 nM (BTV) of each primer, 250 nM Taqman probe, and 5 μL cDNA. Cycling conditions were as follows: 1 cycle at 95°C for 20 s, followed by 45 cycles of 1 s at 95°C, and 20 s at 60°C. The specificity of the RT-qPCR used had been previously tested against prototype strains of genetically related viruses (9 strains of epizootic hemorrhagic disease virus and 9 strains of African horse sickness virus) (5). The RT-qPCR tests confirmed BTV viremia. The yak species in its natural biotope is usually rarely exposed to competent Culicoides vectors. Antibodies against BTV have been found in many wild ruminants (6), and our results extend the number of ruminant species susceptible to BTV. In the northern European BT outbreak, lesions in cattle and sheep were mainly localized to the regions of the muzzle, mouth, and eye; clinical signs were not always obvious (7,8). As in cattle and sheep, clinical signs in yaks were observed on the muzzle, in the periocular region, and around and inside the mouth. These signs clearly reflected viral-induced endothelial damage triggering disseminated intravascular coagulation and a hemorrhagic diathesis commonly described in sheep and cattle (2). In our case, lesions depicted pronounced microvascular damage. According to the severity of the lesions and rates of illness and death observed, the yak, like sheep, appears to be highly susceptible to BTV. In the epidemiology of BT in African countries, cattle and wild ruminant species such as antelopes play a role as asymptomatic reservoir hosts of the virus (2). Some wild ruminant species in captivity could also play this role in European countries affected by the recent BT outbreak. These cases could be of particular concern for all parks and zoos that gather numerous wild ruminants. Illness, reproductive failure, and deaths usually reported with BT (9) could generate substantial losses on these premises. Moreover, the source of BTV-8 in the northern European outbreak remains unclear, and the role of wild ruminant species has to be taken into account. In the future, European authorities should consider vaccination to prevent the spread of the disease in European member states (10). All premises with wild ruminants need to be involved in BT control and prophylaxis.