Rapid detection of CTX-M-type ESBLs and carbapenemases directly from biological samples using the BL-DetecTool.

BACKGROUND: Lateral flow immunoassays (LFIA) have shown their usefulness for detecting CTX-M- and carbapenemase-producing Enterobacterales (CPEs) in bacterial cultures. Here, we have developed and validated the BL-DetecTool to detect CTX-M enzymes and carbapenemases directly from clinical samples. METHODS: The BL-DetecTool is an LFIA that integrates an easy sample preparation device named SPID (Sampling, Processing, Incubation and Detection). It was evaluated in three University hospitals on urine, blood culture (BC) and rectal swab (RS) specimens either of clinical origin or on spiked samples. RS evaluation was done directly and after a 24 h enrichment step. RESULTS: The CTX-M BL-DetecTool was tested on 485 samples (154 BC, 150 urines, and 181 RS) and revealed a sensitivity and specificity of 97.04% (95% CI 92.59%-99.19%) and 99.43% (95% CI 97.95%-99.93%), respectively. Similarly, the Carba5 BL-DetecTool was tested on 382 samples (145 BC, 116 urines, and 121 RS) and revealed a sensitivity and specificity of 95.3% (95% CI 89.43%-98.47%) and 100% (95% CI 98.67%-100%), respectively. While with the Carba5 BL-DetecTool five false negatives were observed, mostly in RS samples, with the CTX-M BL-DetecTool, in addition to four false-negatives, two false-positives were also observed. Direct testing of RS samples revealed a sensitivity of 78% and 86% for CTX-M and carbapenemase detection, respectively. CONCLUSIONS: BL-DetecTool showed excellent biological performance, was easy-to-use, rapid, and could be implemented in any microbiology laboratory around the world, without additional equipment, no need for electricity, nor trained personnel. It offers an attractive alternative to costly molecular methods.

A novel gyrovirus in a common pheasant (Phasianus colchicus) with poult enteritis and mortality syndrome.

A novel gyrovirus was detected in an intestinal specimen of a common pheasant that died due to poult enteritis and mortality syndrome. The genome of the pheasant-associated gyrovirus (PAGyV) is 2353 nucleotides (nt) long and contains putative genes for the VP1, VP2, and VP3 proteins in an arrangement that is typical for gyroviruses. Gyrovirus-specific motifs were identified in both the coding region and the intergenic region of the PAGyV genome. The VP1 of PAGyV shares up to 67.6% pairwise nt sequence identity with reference sequences and forms a distinct branch in the phylogenetic tree. Thus, according to the recently described species demarcation criteria, PAGyV belongs to a novel species in the genus Gyrovirus, family Anelloviridae, for which we propose the name "Gyrovirus phaco 1".

Genetic characterisation of a novel reptarenavirus detected in a dead pet red-tailed boa (Boa constrictor).

Boid inclusion body disease (BIBD) is a severe and transmissible disease of snakes worldwide. Reptarenaviruses have been identified as the aetiological agents of BIBD. We determined the almost complete genome sequence of an arenavirus detected in a female red-tailed boa that had succumbed in a private collection in Hungary. We used a combination of next generation sequencing and Sanger sequencing methods. Based on the analysis of the obtained sequence data, the virus, tentatively named Coldvalley virus, seemed to belong to the Reptarenavirus genus of the Arenaviridae family. This classification was confirmed by the genome structure (bisegmented single-stranded RNA) characteristic of the genera Mammarenavirus and Reptarenavirus. The pairwise comparison of the nucleotide and amino acid sequences, as well as the topology of the maximum likelihood phylogenetic trees, suggested that the newly-characterised Coldvalley virus can be classified into the species Rotterdam reptarenavirus.

Isolation and complete genome characterization of novel reassortant orthoreovirus from common vole (Microtus arvalis).

A novel mammalian orthoreovirus (MRV) strain was isolated from the lung tissue of a common vole (Microtus arvalis) with Tula hantavirus infection. Seven segments (L1-L3, M2-M3, S2, and S4) of the Hungarian MRV isolate MORV/47Ma/06 revealed a high similarity with an MRV strain detected in bank vole (Myodes glareolus) in Germany. The M1 and S3 segment of the Hungarian isolate showed the closest relationship with the sequence of a Slovenian human and a French murine isolate, respectively. The highest nucleotide and amino acid identity values were above 90 and 95% in all of the comparisons to the reference sequences in GenBank, except for the S1 with a maximum of 69.6% nucleotide and 75.4% amino acid identity. As wild rodents are among the main sources of zoonotic infections, the reservoir role of these animals and zoonotic potential of rodent origin MRVs need to be further investigated.

Detection and sequencing of West Nile virus RNA from human urine and serum samples during the 2014 seasonal period.

West Nile virus, a widely distributed mosquito-borne flavivirus, is responsible for numerous animal and human infections in Europe, Africa and the Americas. In Hungary, the average number of human infections falls between 10 and 20 cases each year. The severity of clinically manifesting infections varies widely from the milder form of West Nile fever to West Nile neuroinvasive disease (WNND). In routine laboratory diagnosis of human West Nile virus infections, serological methods are mainly applied due to the limited duration of viremia. However, recent studies suggest that detection of West Nile virus RNA in urine samples may be useful as a molecular diagnostic test for these infections. The Hungarian National Reference Laboratory for Viral Zoonoses serologically confirmed eleven acute human infections during the 2014 seasonal period. In three patients with neurological symptoms, viral RNA was detected from both urine and serum specimens, albeit for a longer period and in higher copy numbers with urine. Phylogenetic analysis of the NS3 genomic region of three strains and the complete genome of one selected strain demonstrated that all three patients had lineage-2 West Nile virus infections. Our findings reaffirm the utility of viral RNA detection in urine as a molecular diagnostic procedure for diagnosis of West Nile virus infections.

Taxonomy of the order Mononegavirales: update 2016.

In 2016, the order Mononegavirales was emended through the addition of two new families (Mymonaviridae and Sunviridae), the elevation of the paramyxoviral subfamily Pneumovirinae to family status (Pneumoviridae), the addition of five free-floating genera (Anphevirus, Arlivirus, Chengtivirus, Crustavirus, and Wastrivirus), and several other changes at the genus and species levels. This article presents the updated taxonomy of the order Mononegavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).

Candidate new rotavirus species in sheltered dogs, Hungary.

We identified unusual rotavirus strains in fecal specimens from sheltered dogs in Hungary by viral metagenomics. The novel rotavirus species displayed limited genome sequence homology to representatives of the 8 rotavirus species, A-H, and qualifies as a candidate new rotavirus species that we tentatively named Rotavirus I.

Sequencing and phylogenetic analysis identifies candidate members of a new picornavirus genus in terrestrial tortoise species.

Near-complete genome sequences of seven picornavirus (PV) strains isolated from different terrestrial tortoise species were determined and characterized. The genome organization of the strains proved to be similar and displayed a typical PV layout, and the polyprotein-encoding regions showed low similarity to those of other PVs. The predicted regions of the tortoise PV genomes were related to the corresponding genome parts of viruses belonging to distinct genera, implying modular evolution of these novel viruses. Our results suggest that these tortoise PVs belong to a prototype species in a separate proposed genus in the family Picornaviridae, tentatively called Topivirus (Tortoise picornavirus).

Coding-complete sequencing classifies parrot bornavirus 5 into a novel virus species.

In this study, we determined the sequence of the coding region of an avian bornavirus detected in a blue-and-yellow macaw (Ara ararauna) with pathological/histopathological changes characteristic of proventricular dilatation disease. The genomic organization of the macaw bornavirus is similar to that of other bornaviruses, and its nucleotide sequence is nearly identical to the available partial parrot bornavirus 5 (PaBV-5) sequences. Phylogenetic analysis showed that these strains formed a monophyletic group distinct from other mammalian and avian bornaviruses and in calculations performed with matrix protein coding sequences, the PaBV-5 and PaBV-6 genotypes formed a common cluster, suggesting that according to the recently accepted classification system for bornaviruses, these two genotypes may belong to a new species, provisionally named Psittaciform 2 bornavirus.

Complete genome analysis identifies Tvärminne avian virus as a candidate new species within the genus Orthoreovirus.

Orthoreoviruses have been associated with a variety of diseases in domesticated poultry and wild-living birds. In 2002, a reovirus strain named Tvärminne avian virus (TVAV), was identified in Finland in a crow showing neurological disorders. The objective of this study was the molecular characterization of this novel reovirus strain. Genome sequencing was performed by combining semiconductor sequencing and traditional capillary sequencing. Sequence and phylogenetic analyses showed that TVAV shares low nucleotide sequence identity with other reoviruses (range for each gene, 31-72 %) including strains belonging to the species Avian orthoreovirus. The most closely related reovirus strain was an isolate identified in Steller sea lion. Our data indicate that TVAV is a divergent reovirus of avian origin that may be the first representative of a distinct virus species within the genus Orthoreovirus.

The complete genome sequence of a European goose reovirus strain.

The complete genomic sequence of a Hungarian goose orthoreovirus strain (D20/99) is reported in this study. The genome of D20/99 is 22,969 bp in length (range, 3958 bp for L1 to 1124 bp for S4) and encodes 11 putative proteins. Pairwise sequence comparisons and phylogenetic analyses indicated that D20/99 shares genetic signatures with some contemporary Chinese duck and goose reovirus strains, except for the µA, µNS and σA protein coding genes, which represented independent genetic lineages. This study implies a greater genetic diversity among waterfowl-origin orthoreoviruses than hitherto recognized.

Molecular detection and characterization of human gyroviruses identified in the ferret fecal virome.

The recently described novel gyroviruses may infect chickens and/or humans; however, their pathogenic potential is unknown. In our metagenomic investigation, we detected many of the novel gyroviruses in the fecal viromes of ferrets with lymph node and organ enlargement. The complete genomic sequences of selected gyrovirus strains showed 90.7-99.4 % similarity to homologous reference gyrovirus strains. This study did not demonstrate an association between gyrovirus shedding from ferrets and the observed background disease; however, it provides evidence for genetic diversity among gyroviruses and raises the possibility that pet ferrets may transmit gyroviruses to heterologous hosts, e.g., humans.

Whole-genome sequencing of a green bush viper reovirus reveals a shared evolutionary history between reptilian and unusual mammalian orthoreoviruses.

In this study, we sequenced the whole genome of a reovirus isolated from a green bush viper (Atheris squamigera). The bush viper reovirus shared several features with other orthoreoviruses, including its genome organization. In phylogenetic analysis, this strain was monophyletic with Broome virus and baboon orthoreovirus, indicating that these viruses might have originated from a common ancestor. These new molecular data supplement previous information based mainly on biological properties of reptilian reoviruses, confirming their taxonomic position and broadening our knowledge of the evolution of members of the genus Orthoreovirus.

Classification and characterization of a laboratory chicken rotavirus strain carrying G7P[35] neutralization antigens on the genotype 4 backbone gene configuration.

The laboratory rotavirus strain, BRS/115, has been used for more than two decades to monitor rotaviruses in specific pathogen free flocks of laying hens. However, the virus strain has not been characterized in detail. Therefore we aimed at the description of molecular features of BRS115 by using random primed reverse transcription-PCR of the genomic RNA followed by massive parallel sequencing using the semiconductor sequencing technology. Over 64,000 trimmed reads mapped to reference sequences obtained from GenBank. The strain classified into the species Rotavirus A and genotyped G7-P[35]-I4-R4-C4-M4-A16-N4-T4-E11-H4 according to guidelines of the Rotavirus Classification Working Group. Phylogenetic analysis identified shared features with chicken, turkey and pigeon origin rotaviruses. This study demonstrates the robustness of next generation sequencing in the characterization of reference virus materials used in specialized laboratories.

Metagenomic Identification of Novel Eukaryotic Viruses with Small DNA Genomes in Pheasants.

A panel of intestinal samples collected from common pheasants (Phasianus colchicus) between 2008 and 2017 was used for metagenomic investigation using an unbiased enrichment protocol and different bioinformatic pipelines. The number of sequence reads in the metagenomic analysis ranged from 1,419,265 to 17,507,704 with a viral sequence read rate ranging from 0.01% to 59%. When considering the sequence reads of eukaryotic viruses, RNA and DNA viruses were identified in the samples, including but not limited to coronaviruses, reoviruses, parvoviruses, and CRESS DNA viruses (i.e., circular Rep-encoding single-stranded DNA viruses). Partial or nearly complete genome sequences were reconstructed of at least three different parvoviruses (dependoparvovirus, aveparvovirus and chaphamaparvovirus), as well as gyroviruses and diverse CRESS DNA viruses. Generating information of virus diversity will serve as a basis for developing specific diagnostic tools and for structured epidemiological investigations, useful to assess the impact of these novel viruses on animal health.

One year survey of human rotavirus strains suggests the emergence of genotype G12 in Cameroon.

In this study the emergence of rotavirus A genotype G12 in children <5 years of age is reported from Cameroon during 2010/2011. A total of 135 human stool samples were P and G genotyped by reverse transcriptase PCR. Six different rotavirus VP7 genotypes were detected, including G1, G2, G3, G8, G9, and G12 in combinations with P[4], P[6] and P[8] VP4 genotypes. Genotype G12 predominated in combination with P[8] (54.1%) and P[6] (10.4%) genotypes followed by G1P[6] (8.2%), G3P[6] (6.7%), G2P[4] (5.9%), G8P[6] (3.7%), G2P[6] (0.7%), G3P[8] (0.7%), and G9P[8] (0.7%). Genotype P[6] strains in combination with various G-types represented a substantial proportion (N=44, 32.6%) of the genotyped strains. Partially typed strains included G12P[NT] (2.2%); G3P[NT] (0.7%); G(NT)P[6] (1.5%); and G(NT)P[8] (0.7%). Mixed infections were found in five specimens (3.7%) in several combinations including G1+ G12P[6], G2+ G3P[6] + P[8], G3+ G8P[6], G3 + G12P[6] + P[8], and G12P[6] +P[8]. The approximately 10% relative frequency of G12P[6] strains detected in this study suggests that this strain is emerging in Cameroon and should be monitored carefully as rotavirus vaccine is implemented in this country, as it shares neither G- nor P-type specificity with strains in the RotaTeq® and Rotarix® vaccines. These findings are consistent with other recent reports of the global spread and increasing epidemiologic importance of G12 and P[6] strains.

Detection and characterization of a divergent avian reovirus strain from a broiler chicken with central nervous system disease.

Avian orthoreoviruses have been associated with a variety of diseases in chickens, including tenosynovitis, runting-stunting syndrome, hepatitis, myocarditis, osteoporosis, respiratory diseases, and central nervous system disease. The primary objective of our study was the molecular characterization of an avian reovirus strain, T1781, which was isolated from a broiler chicken with a central nervous system disorder in Hungary during 2012. The complete genome sequence was determined using a traditional sequencing method after cell culture adaptation of the strain. Sequence and phylogenetic analyses showed that T1781 shared only moderate nucleic acid sequence identity in several genes to previously analyzed reovirus strains from chickens, and each gene formed separate branches in the corresponding phylogenetic trees. The maximum nucleotide sequence identities of strain T1781 genes to reference avian reovirus strains ranged from 79 % to 90 %. Collectively, our analyses indicated that T1781 is a divergent chicken reovirus strain. The genetic background of this and other avian reoviruses associated with various disease manifestations needs further investigation.

Integrated circoviral rep-like sequences in the genome of cyprinid fish.

Recently a new group of circoviruses have been detected in tissues of Barbel fish and European catfish in Hungary. In our study circovirus genomes were screened in eight additional fish species for the detection and characterization of circoviruses. Two species of these bore circoviral sequences based on conventional PCR assay targeting the replication-associated protein coding gene fragments. Interestingly, the methods successfully used before failed to amplify other parts of the circular viral genome, suggesting the presence of partial, integrated genetic elements in the genome of the host. The successfully sequenced fragments of the Indian rohu (Labeo rohita) encoded mutations which may cause frameshifts or termination in the coding region described previously in other vertebrates. Phylogenetic analyses presumed that integration of the viral genetic elements might have progressed concurrently or following the diversification of cyprinid fish. Further studies on the nature of whole circovirus genomes and integrated elements may help to understand their potential role and evolution in different fish species.

Genetic diversity among reptilian orthoreoviruses isolated from pet snakes and lizards.

Reovirus infections in reptiles are frequently detected and associated with various clinical diseases; yet, our knowledge about their genetic diversity and evolutionary relationships remains limited. In this study, we characterize at the genomic level five reptile origin orthoreovirus strains isolated from exotic snakes and lizards in Hungary and Germany. The genomic organization of the study strains was similar to that of the representative strains of reptile origin reoviruses belonging to species Reptilian orthoreovirus and Testudine orthoreovirus. Additionally, all five study strains clustered with the bush viper origin reference Reptilian orthoreovirus strain, 47/02. The nucleotide sequence divergence among strains fell from 56.64 to 99.36%. Based on genome segment constellations two well separated groups were observed, which may represent two genetic lineages of reptilian orthoreoviruses we tentatively referred here as genogroups, classifying two squamata origin strains with available whole genome sequences into genogroup I (GGI) and four strains into genogroup II (GGII). The representative GGI and GGII Reptilian orthoreovirus strains are characterized by moderate-to-high nucleotide and amino acid similarities within genogroups (range, 69.45 to 99.36% and 74.64 to 100.00%), whereas lower nucleotide and amino acid similarities (range, 56.64 to 77.24% and 54.53 to 93.85%) and different structures of the bicistronic S1 segment were found between genogroups. Further studies are needed to explore the genomic diversity among reptilian reoviruses of squamata origin; this would be critical to establish a robust classification system for these viruses and to see if interaction among members of distinct lineages may result in viable progenies with novel genetic features.

Marked Genotype Diversity among Reoviruses Isolated from Chicken in Selected East-Central European Countries.

The concern that the vaccines currently used against Avian orthoreovirus (ARV) infections are less efficient in the field justifies the need for the close monitoring of circulating ARV strains. In this study, we collected necropsy samples from various chicken breeds and tested for ARV by virus isolation, RT-PCR assay and sequence analysis. ARVs were isolated from birds showing runting-stunting syndrome, uneven growth, lameness or increased mortality, with relative detection rates of 38%, 35%, 6% and 25%, respectively. Partial σC gene sequences were determined for nearly 90% of ARV isolates. The isolates could be classified into one of the major genetic clusters. Interestingly, cluster 2 and cluster 5 were isolated from vaccinated broiler breeders, while clusters 1 to 4 were isolated from unvaccinated broilers. The isolates shared less than 75% amino acid identities with the vaccine strains (range, 44.3-74.6%). This study reaffirms the global distribution of the major genetic clusters of ARVs in chicken. The diversity of ARV strains isolated from unvaccinated broilers was greater than those detected from vaccinated animals, however, the relative importance of passive and active immunity on the selection of novel strains in different chicken breeds needs to be better understood.