Zoonotic Infection With Pigeon Paramyxovirus Type 1 Linked to Fatal Pneumonia.

The characteristics and risk factors of pigeon paramyxovirus type 1 (PPMV-1) infection in humans are poorly known. We performed virological, pathological, and epidemiological analyses of a Dutch case, and compared the results with those of a US case. Both infections occurred in transplant patients under immunosuppressive therapy and caused fatal respiratory failure. Both virus isolates clustered with PPMV-1, which has pigeons and doves as reservoir. Experimentally inoculated pigeons became infected and transmitted the virus to naive pigeons. Both patients were likely infected by contact with infected pigeons or doves. Given the large populations of feral pigeons with PPMV-1 infection in cities, increasing urbanization, and a higher proportion of immunocompromised individuals, the risk of severe human PPMV-1 infections may increase. We recommend testing for avian paramyxovirus type 1, including PPMV-1, in respiratory disease cases where common respiratory pathogens cannot be identified.

Case of seasonal reassortant A(H1N2) influenza virus infection, the Netherlands, March 2018.

A seasonal reassortant A(H1N2) influenza virus harbouring genome segments from seasonal influenza viruses A(H1N1)pdm09 (HA and NS) and A(H3N2) (PB2, PB1, PA, NP, NA and M) was identified in March 2018 in a 19-months-old patient with influenza-like illness (ILI) who presented to a general practitioner participating in the routine sentinel surveillance of ILI in the Netherlands. The patient recovered fully. Further epidemiological and virological investigation did not reveal additional cases.

Metagenomic sequencing for virus identification in a public-health setting.

The use of metagenomics for virus discovery in clinical samples has opened new opportunities for understanding the aetiology of unexplained illness. This study explores the potential of this sequence-independent approach in a public-health setting, by systematic analysis of samples cultured from patients with unexplained illness through a combination of PCR-based assays and viral metagenomics. In total, 1834 cell-culture isolates were collected between 1994 and 2007 through the Enterovirus Surveillance programme in the Netherlands. During the 13 year period, seven samples that exhibited reproducible cytopathogenic effects in cell culture tested negative in standard PCR assays for a range of viruses. In order to fill the diagnostic gap, viral metagenomics was applied to these culture supernatants, resulting in the rapid identification of viruses in all of the samples. The unexplained samples contained BK polyomavirus, herpes simplex virus, Newcastle disease virus and the recently discovered Saffold viruses (SAFV) (which dominated the unexplained samples; n=4). The full genomic sequences of four SAFV genotype 3 (SAFV-3) viruses, which share 88-93 % nucleotide identity with known SAFV-3 viruses, are reported. Further screening for SAFV in additional cultured, unidentified clinical isolates from 2008 and 2009 resulted in identification of another SAFV-positive sample. Although the pathogenicity of the identified viruses has not been established, this study demonstrates that viral metagenomics is a powerful tool that can be integrated into public-health monitoring efforts to investigate unidentified viruses in cell cultures from clinical isolates where standard PCR assays fail to detect viruses.

Epidemiology and genotype analysis of emerging sapovirus-associated infections across Europe.

Sapoviruses (SaVs) belong to the Caliciviridae family and can cause gastroenteritis in humans and swine. Despite extensive testing, human sapoviruses have been found only in sporadic cases and in one mixed outbreak in children between 1994 and 2007 in the Netherlands. Here we describe a change in sapovirus epidemiology in the Netherlands resulting in sapovirus outbreaks and infections in adults. From November 2007 to January 2009, 478 outbreaks of acute gastroenteritis were reported to the National Institute for Public Health and the Environment in the Netherlands as a part of ongoing surveillance. Sapoviruses were found to be the most likely cause of 19 outbreaks (4%). During the same 2-year period, sapovirus infections were reported in Sweden, Slovenia, and Hungary. In the Netherlands, further characterization of outbreak strains showed that 12 (63%) sapovirus outbreaks were caused by genotype I.2 viruses. Most patients were adults older than 60 years (range, 1 to 100 years). Phylogenetic analysis using all presently available SaV sequences showed high homology between genotype I.2 strains detected in different geographical regions (Sweden, Slovenia, Taiwan, Japan, and Russia) since 2007. These first reported outbreaks of sapovirus infections in adults in the Netherlands were remarkable. Detection of identical genotypes in many samples might suggest that these viruses have the same origin, and since the infection is spreading fast, the prevalence of sapovirus infection may be increasing. The incidence of sapovirus infections in these countries suggests that a substantial part of Europe is affected by this virus.

Novel approach for detection of enteric viruses to enable syndrome surveillance of acute viral gastroenteritis.

Acute gastroenteritis is one of the most common diseases worldwide, with viruses, particularly noroviruses, being the leading cause in developed countries. In The Netherlands, systematic surveillance of gastroenteritis outbreaks of suspected viral etiology was established by the National Institute for Public Health and the Environment in 1994. Since 2002, the total number of outbreaks reported has been increasing, and with that comes the need for sensitive assays that can be performed quickly. In addition, the diagnostic demand changed so that now the proportion of samples from hospitals is higher and there is a need for patient-based test results. In order to target the diagnosis of acute gastroenteritis, we reviewed our data on outbreaks of gastroenteritis and the prevalence of individual viruses to provide a priority list of viruses for which samples should be evaluated. Random primers were used to replace the separate specific primers for each virus used in the reverse transcription steps. The individual PCR assays were replaced by multiplex PCR assays. We employed a two-step method in which in the first step we screened for the most common causes of viral gastroenteritis, noroviruses of genogroup II and rotaviruses of group A, with equine arteritis virus used as the internal control. Subsequently, in the second step, two parallel PCR assays were developed for the detection of noroviruses of genogroup I and equine arteritis virus in one run and adenoviruses, sapoviruses, and astroviruses in the other run. The specificities of the assays were calculated to be 92.5% for the assay for noroviruses of genogroup I and 100% for the assays for all other viruses, the detection limits were equal for all viruses, and the turnaround time was reduced to 1 day compared to the at least 3 days required for the methods used previously. This approach allows the targeted, rapid, and cost-effective elucidation of the causes of acute gastroenteritis outbreaks.

A new generic real-time reverse transcription polymerase chain reaction assay for vesiviruses; vesiviruses were not detected in human samples.

Different viruses belonging to the genus Vesivirus infect a broad range of animals, and cause gastroenteritis, vesicular skin lesions, hemorrhagic disease, respiratory diseases and other conditions. A recent report on Vesivirus viremia, as detected by PCR, in samples from patients with hepatitis of unknown etiology in the USA suggested a zoonotic potential for vesiviruses. These results have not been confirmed by another laboratory. In order to do so, a generic PCR assay on the RNA polymerase region was developed, and validated with RNA from 69 different Vesivirus species. Except SMSV serotype-8, all species tested were detected, including the ones that were suggested to be involved in zoonotic transmission in the USA (SMSV serotype-5). The generic Vesivirus assay was used on RNA extracted from serum samples from patients with hepatitis, stool samples from patients with gastroenteritis, throat-swab specimens of patients with rash illnesses, throat-swab and nose-swabs of patients with acute respiratory diseases, and cell cultures with cytopathologic effect from enterovirus surveillance in which no pathogen was found. None were found positive. In this study a generic Vesivirus assay was developed and it was concluded that vesiviruses are an unlikely cause of common illnesses in humans in the Netherlands.

Establishment of a genotyping scheme for Coxiella burnetii.

Coxiella burnetii is the causative agent of Q fever. The bacterium is highly infectious and is classified as a category B biological weapon. The tools of molecular biology are of utmost importance in a rapid and unambiguous identification of C. burnetii in naturally occurring Q fever outbreaks, or in cases of a deliberate release of the infectious agent. In this work, development of a multiple locus variable number tandem repeats (VNTR) analysis (MLVA) for the characterization of C. burnetii is described. Sixteen C. burnetii isolates and five passage history/laboratory variants were characterized. The VNTR markers revealed many polymorphisms resulting in nine unique MLVA types that cluster into five different clusters. This proves that the MLVA system is highly discriminatory. The selected VNTR markers were stable. The MLVA method developed in this report is a promising tool for the characterization of C. burnetii isolates and their epidemiological study.

Development and validation of a microarray for the confirmation and typing of norovirus RT-PCR products.

Noroviruses are implicated in many worldwide institutional, food and waterborne outbreaks each year. Genetic typing of isolates is valuable for monitoring outbreak spread as well as variation in circulating strains. Microarrays have the potential to provide rapid genotype information for norovirus samples. The NoroChip v3.0 provides an oligonucleotide hybridization platform to screen for over 600 potential interactions in each experiment. The NoroChip v3.0 was developed at Health Canada and validated in seven international partner laboratories. Each laboratory validated the NoroChip v3.0 using norovirus amplicons routinely characterized in their testing protocols. Fragments from the capsid region (region C) and a 2.4 kb amplicon spanning polymerase and capsid sequences (region AD) were validated in six of the partner laboratories and provided correct genogroup typing information (GI or GII) when hybridized to the NoroChip v3.0. Results indicate that the current limiting factor for implementing the NoroChip v3.0 as a strain typing tool is the difficulty obtaining a long, specific amplicon from all circulating norovirus strains. Data obtained with the longer region AD amplicon provided the best discrimination between norovirus strains.

Etiological role of viruses in outbreaks of acute gastroenteritis in The Netherlands from 1994 through 2005.

Acute gastroenteritis is one of the most common diseases worldwide. In developed countries, viruses, particularly noroviruses, are recognized as the leading cause. In The Netherlands, the surveillance of gastroenteritis outbreaks with suspected viral etiologies (as determined by Kaplan criteria) was established by the National Institute for Public Health and the Environment in 1994. This paper presents an overview of viral gastroenteritis outbreaks reported from 1994 through 2005. A minimum epidemiological data set consisting of the associated setting(s), the probable transmission mode, the date of the first illness and the date of sampling, the number of persons affected, and the number of hospitalizations was requested for each reported outbreak. Stool samples were tested for the presence of norovirus, sapovirus, rotavirus, astrovirus, adenovirus, and Aichi virus by electron microscopy, enzyme-linked immunosorbent assay, and/or reverse transcription-PCR. A total of 6,707 stool samples from 941 gastroenteritis outbreaks were investigated. Noroviruses were detected as the causative agent in 735 (78.1%) of the outbreaks, and rotaviruses, adenoviruses, and astroviruses were found to be responsible for 46 (4.9%), 9 (1.0%), and 5 (0.5%) outbreaks, respectively. Among the gastroenteritis outbreaks in which a mode of transmission was identified, most outbreaks (38.1%) were associated with person-to-person transmission, and the majority (54.9%) of the outbreaks investigated were reported by residential institutions. Since 2002, the total number of outbreaks reported and the number of unexplained outbreaks have increased. Furthermore, the number of rotavirus-associated outbreaks has increased, especially in nursing homes. Despite thorough testing, 115 (12.2%) outbreaks suspected of having viral etiologies remain unexplained. Increases in numbers of reported outbreaks may indicate undefined changes in the criteria for reporting or the emergence of new pathogens.

Rickettsia prowazekii and real-time polymerase chain reaction.

Rickettsia prowazekii is the causative agent of epidemic typhus and a potential bioterrorism agent. Sensitive and specific rapid assays are needed to complement existing methods of detecting this organism. We developed a real-time quantitative polymerase chain reaction assay by using a species-specific probe targeting the gltA gene. This assay, which was rapid, specific for R. prowazekii only, and sensitive (cutoff detection of 1 to 5 copies per sample), detected and directly identified R. prowazekii in blood of 12 experimentally infected mice sampled at day 3 and 6 postinfection or in naturally or experimentally infected lice. Because our assay is highly standardized and easily adaptable, it could improve epidemic typhus surveillance in public health programs, especially for countries with underdiagnosed or unrecognized human cases.