Reimar Johne

Uniformity of Rotavirus Strain Nomenclature Proposed by the Rotavirus Classification Working Group (RCWG)

In April 2008, a nucleotide-sequence-based, complete genome classification system was developed for group A rotaviruses (RVs). This system assigns a specific genotype to each of the 11 genome segments of a particular RV strain according to established nucleotide percent cutoff values. Using this approach, the genome of individual RV strains are given the complete descriptor of Gx-P[x]-Ix-Rx-Cx-Mx-Ax-Nx-Tx-Ex-Hx. The Rotavirus Classification Working Group (RCWG) was formed by scientists in the field to maintain, evaluate and develop the RV genotype classification system, in particular to aid in the designation of new genotypes. Since its conception, the group has ratified 51 new genotypes: as of April 2011, new genotypes for VP7 (G20-G27), VP4 (P[28]-P[35]), VP6 (I12-I16), VP1 (R5-R9), VP2 (C6-C9), VP3 (M7-M8), NSP1 (A15-A16), NSP2 (N6-N9), NSP3 (T8-T12), NSP4 (E12-E14) and NSP5/6 (H7-H11) have been defined for RV strains recovered from humans, cows, pigs, horses, mice, South American camelids (guanaco), chickens, turkeys, pheasants, bats and a sugar glider. With increasing numbers of complete RV genome sequences becoming available, a standardized RV strain nomenclature system is needed, and the RCWG proposes that individual RV strains are named as follows: RV group/species of origin/country of identification/common name/year of identification/G- and P-type. In collaboration with the National Center for Biotechnology Information (NCBI), the RCWG is also working on developing a RV-specific resource for the deposition of nucleotide sequences. This resource will provide useful information regarding RV strains, including, but not limited to, the individual gene genotypes and epidemiological and clinical information. Together, the proposed nomenclature system and the NCBI RV resource will offer highly useful tools for investigators to search for, retrieve, and analyze the ever-growing volume of RV genomic data.

PCR inhibitors – occurrence, properties and removal

The polymerase chain reaction (PCR) is increasingly used as the standard method for detection and characterization of microorganisms and genetic markers in a variety of sample types. However, the method is prone to inhibiting substances, which may be present in the analysed sample and which may affect the sensitivity of the assay or even lead to false‐negative results. The PCR inhibitors represent a diverse group of substances with different properties and mechanisms of action. Some of them are predominantly found in specific types of samples thus necessitating matrix‐specific protocols for preparation of nucleic acids before PCR. A variety of protocols have been developed to remove the PCR inhibitors. This review focuses on the general properties of PCR inhibitors and their occurrence in specific matrices. Strategies for their removal from the sample and for quality control by assessing their influence on the individual PCR test are presented and discussed.

VP6-sequence-based cutoff values as a criterion for rotavirus species demarcation

Indirect immunofluorescence techniques targeting the rotavirus (RV) protein VP6 are used to differentiate RV species. The ICTV recognizes RV species A to E and two tentative species, F and G. A potential new RV species, ADRV-N, has been described. Phylogenetic trees and pairwise identity frequency graphs were constructed with more than 400 available VP6 sequences and seven newly determined VP6 sequences of RVD strains. All RV species were separated into distinct phylogenetic clusters. An amino acid sequence cutoff value of 53% firmly permitted differentiation of RV species, and ADRV-N was tentatively assigned to a novel RV species H (RVH).

Detection of a novel hepatitis E-like virus in faeces of wild rats using a nested broad-spectrum RT-PCR

Hepatitis E is a rare human disease in developed countries. It is caused by hepatitis E virus (HEV), which is probably transmitted zoonotically to humans from domestic pigs and wild boars. Multiple reports on the detection of HEV-specific antibodies in rats have suggested the presence of an HEV-related agent; however, infectious virus or a viral genome has not been demonstrated so far. Here, a nested broad-spectrum RT-PCR protocol was developed capable of detecting different HEV types including those derived from wild boar and chicken. Screening of 30 faecal samples from wild Norway rats (Rattus norvegicus) from Hamburg (Germany) resulted in the detection of two sequences with similarities to human, mammalian and avian HEV. Virus particles with a morphology reminiscent of HEV were demonstrated by immunoelectron microscopy in one of these samples and the virus was tentatively designated rat HEV. Genome fragments with sizes of 4019 and 1545 nt were amplified from two samples. Sequence comparison with human and avian strains revealed only 59.9 and 49.9 % sequence identity, respectively. Similarly, the deduced amino acid sequence for the complete capsid protein had 56.2 and 42.9 % identity with human and avian strains, respectively. Inoculation of the samples onto three different permanent rat liver cell lines did not result in detectable virus replication as assayed by RT-PCR with cells of the fifth virus passage. Further investigations are necessary to clarify the zoonotic potential of rat HEV and to assess its suitability to serve in a laboratory rat animal model for human hepatitis E.

Taxonomical developments in the family Polyomaviridae

The Polyomaviridae Study Group of the International Committee on Taxonomy of Viruses (ICTV) has recommended several taxonomical revisions, as follows: The family Polyomaviridae, which is currently constituted as a single genus (Polyomavirus), will be comprised of three genera: two containing mammalian viruses and one containing avian viruses. The two mammalian genera will be designated Orthopolyomavirus and Wukipolyomavirus, and the avian genus will be named Avipolyomavirus. These genera will be created by the redistribution of species from the current single genus (Polyomavirus) and by the inclusion of several new species. In addition, the names of several species will be changed to reflect current usage.

Novel Hepatitis E Virus Genotype in Norway Rats, Germany

Human hepatitis E virus infections may be caused by zoonotic transmission of virus genotypes 3 and 4. To determine whether rodents are a reservoir, we analyzed the complete nucleotide sequence of a hepatitis E–like virus from 2 Norway rats in Germany. The sequence suggests a separate genotype for this hepatotropic virus.

Rolling-circle amplification of viral DNA genomes using phi29 polymerase.

Techniques for the single-step amplification of whole genomes have been developed into powerful tools for phylogenetic analyses, epidemiological studies and studies on genome organization. Recently, the bacteriophage phi29 DNA polymerase has been used for the efficient amplification of circular DNA viral genomes without the need of specific primers by a rolling-circle amplification (RCA) mechanism. Various protocols have been applied for detection of novel viruses, for differentiation between circular and linear forms of viral genomes and for generation of infectious genomic clones directly from specimens. Here, we summarize the broad application of the RCA technique to DNA viruses infecting humans, animals and plants.

Proposed reference sequences for hepatitis E virus subtypes.

The nomenclature of hepatitis E virus (HEV) subtypes is inconsistent and makes comparison of different studies problematic. We have provided a table of proposed complete genome reference sequences for each subtype. The criteria for subtype assignment vary between different genotypes and methodologies, and so a conservative pragmatic approach has been favoured. Updates to this table will be posted on the International Committee on Taxonomy of Viruses website (http://talk.ictvonline.org/r.ashx?C). The use of common reference sequences will facilitate communication between researchers and help clarify the epidemiology of this important human pathogen. This subtyping procedure might be adopted for other taxa of the genus Orthohepevirus.

Evidence of interspecies transmission and reassortment among avian group A rotaviruses.

Avian rotaviruses are broadly distributed among birds, but only scarcely characterized on the molecular level. The VP4-, VP6-, VP7- and NSP5-encoding sequences of eight group A rotaviruses from chickens and turkeys determined here indicate a low degree of sequence similarity with mammalian rotaviruses. An NSP6-encoding region was missing in all chicken isolates except for isolate Ch2. Four novel genotypes (P[30], P[31], G22 and H8) were assigned by the Rotavirus Classification Working Group. Generally, chicken and turkey isolates clustered into separate branches of phylogenetic trees. However, chicken isolate Ch2 consistently clustered together with turkey isolates. Chicken isolate 06V0661G1 has a VP4-encoding sequence of unknown origin, but possesses VP6, VP7 and NSP5 genotypes typical for chicken isolates. These results might indicate interspecies transmission and reassortment among avian group A rotaviruses under field conditions. PCR protocols enabling amplification of avian and mammalian group A rotaviruses were developed for use in further epidemiological studies.

Detection of hepatitis E virus in wild boars of rural and urban regions in Germany and whole genome characterization of an endemic strain

BackgroundHepatitis E is an increasingly diagnosed human disease in Central Europe. Besides domestic pigs, in which hepatitis E virus (HEV) infection is highly prevalent, wild boars have been identified as a possible source of human infection. In order to assess the distribution of HEV in the wild boar population of Germany, we tested liver samples originating from different geographical regions for the presence of the HEV genome and compared the detected sequences to animal and human HEV strains.ResultsA total of 148 wild boar liver samples were tested using real-time RT-PCR resulting in an average HEV detection rate of 14.9% (95% CI 9.6–21.6). HEV was detected in all age classes and all geographical regions. However, the prevalence of HEV infection was significantly higher in rural as compared to urban regions (p < 0.001). Sequencing of the PCR products indicated a high degree of heterogenicity of the detected viruses within genotype 3 and a grouping according to their geographical origin. The whole genome sequence of an HEV isolate (wbGER27) detected in many wild boars in the federal state of Brandenburg was determined. It belongs to genotype 3i and shows 97.9% nucleotide sequence identity to a partial sequence derived from a human hepatitis E patient from Germany.ConclusionThe results indicate that wild boars have to be considered as a reservoir for HEV in Germany and that a risk of HEV transmission to humans is present in rural as well as urban regions.