Annette Mankertz

PCV-2 genotype definition and nomenclature

SIR, — To date, at least three different phylogenetic groups of porcine circo-virus type 2 (pcv-2) have been recognised ([Gagnon and others 2007][1], [Dupont and others 2008][2], [Grau-Roma and others 2008][3], [Timmusk and others 2008][4]). However, the mandate of the International Committee on

Identification of a protein essential for replication of porcine circovirus

The largest open reading frame of porcine circovirus (ORF 4) encodes a protein of 312 amino acids. The predicted gene product of ORF 4 shows similarities to Rep proteins of other plant circoviruses and geminiviruses. Three motifs have been identified that are characteristic for proteins involved in rolling circle replication and the consensus sequence for a putative dNTP-binding box (GKS) has been found. In this paper, experimental evidence is presented which indicates that ORF 4 encodes the replication protein of porcine circovirus. After cloning of the ORF 4 gene product, it was supplied in trans in a transient replication assay. The ORF 4 gene product promoted the replication of plasmid pOP11, which carries the origin of DNA replication of porcine circovirus. Since pOP11 itself is unable to replicate in virus-free porcine kidney cells, the ORF 4 gene product must be essential for replication of porcine circovirus.

Characterisation of PCV-2 isolates from Spain, Germany and France

The new isolated circovirus variant PCV-2 is discussed to be the etiological agent of a new emerging swine disease with a variable morbidity and high lethality, postweaning multisystemic wasting syndrome (PMWS). PMWS has been diagnosed in North America and West Europe. Clinical signs include dyspnea, loss of weight, lymph node enlargement and lymphocyte depletion in lymphoid tissues. This report describes the characterisation of PCV-2 isolates from animals affected with PMWS from Germany, Spain and France. We could demonstrate the presence of circovirus by electron microscope, in situ hybridisation and PCR. PCR revealed incidence of PCV-2 in many tissues of one infected animal with the exception of heart and nervous system. The phylogenetic analysis of all PCV-2 isolates yet published in the database, showed relationship of isolates from Spain, Germany and France, with three sequences from Canada determined recently and two isolates from Taiwan, while other North American sequences display a separate cluster. PCR screening of randomly collected organ samples from German pigs not affected with PMWS, revealed a rate of infection with PCV-1 of 5% and with PCV-2 of 26.8%, while blood samples showed a lower incidence.

Global Distribution of Measles Genotypes and Measles Molecular Epidemiology

A critical component of laboratory surveillance for measles is the genetic characterization of circulating wild-type viruses. The World Health Organization (WHO) Measles and Rubella Laboratory Network (LabNet), provides for standardized testing in 183 countries and supports genetic characterization of currently circulating strains of measles viruses. The goal of this report is to describe the lessons learned from nearly 20 years of virologic surveillance for measles, to describe the global databases for measles sequences, and to provide regional updates about measles genotypes detected by recent surveillance activities. Virologic surveillance for measles is now well established in all of the WHO regions, and most countries have conducted at least some baseline surveillance. The WHO Global Genotype Database contains >7000 genotype reports, and the Measles Nucleotide Surveillance (MeaNS) contains >4000 entries. This sequence information has proven to be extremely useful for tracking global transmission patterns and for documenting the interruption of transmission in some countries. The future challenges will be to develop quality control programs for molecular methods and to continue to expand virologic surveillance activities in all regions.

Porcine circoviruses—Small but powerful

When porcine circovirus type 1 (PCV1) was isolated more than 40 years ago as a non-pathogenic contaminant of a porcine kidney cell line, enthusiasm and curiosity kept within reasonable limits. Virologists became more interested, when a second variant was isolated and termed PCV2, because PCV2 is linked to postweaning multisystemic wasting disease (PMWS), a new emerging multifactorial disease in swine. Both PCV1 and PCV2 are small and rather simply organized and express only few proteins. Therefore, it was expected that the factor(s) triggering PMWS should be easily identified, but more than one decade of PCV research has not yet singled out a molecule inducing the disease onset. Unravelling the molecular features of PCV and the channels through which the virus interacts with its host are key to manage, prevent and treat PMWS and other PCV-associated diseases. Since we have learned many aspects of the molecular biology of PCV in the last years, it is time for a résumé!

Cloning and sequencing of columbid circovirus (CoCV), a new circovirus from pigeons

Summary. The complete nucleotide sequence of columbid circovirus (CoCV) isolated from pigeons is described. CoCV was amplified using a consensus primer PCR approach directed against conserved sequences within the rep genes of vertebrate circoviruses. The genome of CoCV is circular and 2037 nt in size. It displays 55% homology to the genome of psittacine beak and feather disease virus and is more distantly related (< 40% homology) to porcine circovirus type 1 and 2. Two major open reading frames were identified, encoding the replicase and the putative capsid protein of CoCV. A region similar to the origin of replication of other circoviruses was found: it encompasses a stem-loop structure with the nonamer 5′-TAGTATTAC, conserved in circo-, nano- and geminiviruses. Phylogenetic analyses suggest classification of CoCV as member of the genus Circovirus of the virus family Circoviridae.

Serum antibodies to porcine circovirus type 1 and type 2 in pigs with and without PMWS

PORCINE circovirus (PCV) is a small, single-stranded, circular, covalently-closed DNA virus, which was first detected as a contaminant in the pig kidney cell line PK-15 (ATCC CCL-33) (Tischer and others 1974). PK-15-derived PCV is considered apathogenic (Tischer and others 1986, Allan and others 1995), and serological studies for the detection of PCV antibodies in pig sera from Germany, Canada, New Zealand, Great Britain, Northern Ireland and the USA have shown a widespread presence of PCV antibodies in fattening and adult pigs, ranging from 25 to 98 per cent of the investigated sera. Therefore, it has been suggested that PCV infection is ubiquitous throughout the world (Allan 1996). Since 1991 in Canada (Clark 1997) and during the past two years in several other countries, including the USA, France, Spain, the UK, Denmark, Italy and Germany, a new disease called postweaning multisystemic wasting syndrome (PMWS) has been identified. PMWS affects nursery and fattening pigs, and i s histologically characterised by lymphocyte depletion with histiocytic infiltrates within lymphoid organs, and bronchointerstitial pneumonia (Clark 1997, Segalés and others 1997). An unequivocal aetiological relationship between PCV and PMWS has not been demonstrated; however, pigs affected with PMWS are regularly infected with a novel strain of PCV. This new strain of PCV has a nucleotide sequence similarity of 75 per cent when compared with PCV derived from PK-15 cells (Hamel and others 1998, Meehan and others 1998); therefore, the nomenclature PCV type 1 (PCV-1) and PCV type 2 (PCV-2) has been suggested for the PK-15and PMWS-derived PCV, respectively (Allan and others 1999). Antigenic differences between PCV-1 and PCV-2 do exist, as some monoclonal antibodies raised against PCV-1 are not able to recognise PCV-2, and monoclonal antibodies raised against PCV-2 cannot recognise PCV-1 in infected cell monolayers by immunofluorescence (Allan and others 1999). In addition, hyperimmune rabbit serum raised by immunisation with PCV-2 showed a high specific titre against PCV-2 but only a low titre against PCV-1 in cell monolayers (Allan and others 1998). The aim of this study was to compare serum antibody titres against PCV-1 and PCV-2 in necropsied pigs, with and without PMWS, using an immunoperoxidase monolayer assay (IPMA) technique. A total of 90 oneto five-month-old pigs from 37 different Spanish farms were submitted to the laboratory of the Veterinary School of Barcelona for pathological examination between May 1997 (when PMWS was recognised in Spain) and July 1998. Pigs were bled at the jugular vein and a 10 ml blood sample was collected in a Vacutainer (Venoject; Terumo Europe). The sample was allowed to clot, centrifuged and then the serum was frozen at –80°C until required for testing. The pigs were necropsied and tissue samples (including the lungs, lymphoid organs, livers and kidneys) were fixed in 10 per cent buffered formalin, embedded in paraffin wax and routinely processed for histopathology. Pigs were classified as PCV-2Short Communications

Cloning and sequencing of Duck circovirus (DuCV)

Summary. The genome of Duck circovirus (DuCV) is circular and 1996 nts in size. Two major open reading frames were identified, encoding the replicase (V1) and the capsid protein (C1). A stem-loop structure comprising the nonamer 5′-TATTATTAC, conserved in all circo-, nano- and geminiviruses, was found. Unique to DuCV, the region between the 3′-ends of the rep and cap gene contains four repeats of a 44-bp sequence. Phylogenetic analysis shows close relation of DuCV with Goose circovirus and suggests classification of DuCV as a new member of the genus Circovirus of the virus family Circoviridae.

Further efforts needed to achieve measles elimination in Germany: results of an outbreak investigation

OBJECTIVE To determine morbidity and costs related to a large measles outbreak in Germany and to identify ways to improve the countrys national measles elimination strategy. METHODS We investigated a large outbreak of measles in the federal state of North Rhine-Westphalia (NRW) that occurred in 2006 after 2 years of low measles incidence (< 1 case per 100,000). WHOs clinical case definition was used, and surveillance data from 2006 and 2001 were compared. All cases notified in Duisburg, the most severely affected city, were contacted and interviewed or sent a questionnaire. Health-care provider costs were calculated using information on complications, hospitalization and physician consultations. FINDINGS In NRW, 1749 cases were notified over a 48-week period. Compared with 2001, the distribution of cases shifted to older age groups (especially the 10-14 year group). Most cases (n = 614) occurred in Duisburg. Of these, 81% were interviewed; 15% were hospitalized and two died. Of the 464 for whom information was available, 80% were reported as unvaccinated. Common reasons for non-vaccination were parents either forgetting (36%) or rejecting (28%) vaccination. The average cost per measles case was estimated at 373 euros. CONCLUSION An accumulation of non-immune individuals led to this outbreak. The shift in age distribution has implications for the effectiveness of measles control and the elimination strategy in place. Immediate nationwide school-based catch-up vaccination campaigns targeting older age groups are needed to close critical immunity gaps. Otherwise, the elimination of measles in Germany and thus in Europe by 2010 will not be feasible.

New Reporter Gene-Based Replication Assay Reveals Exchangeability of Replication Factors of Porcine Circovirus Types 1 and 2

ABSTRACT Two types of porcine circovirus (PCV), which differ in their pathogenicity, are known. PCV type 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome in swine, while PCV1 has not yet been linked to a disease. Corroborating earlier observations in PCV1, transcript mapping revealed that the rep gene of PCV2 encodes two products, the full-length protein Rep and the spliced version Rep′ and that the simultaneous expression of Rep and Rep′ proteins is essential for initiation of replication of PCV2. The interchangeability of the replication factors of PCV1 and PCV2 was examined. The rep gene products of PCV2 were not only able to bind the PCV2 origin but also the origin of PCV1 and vice versa. To investigate the competence of the Rep/Rep′ proteins to initiate replication at the heterologous origin, a new replication assay was developed. It measures the expression of a luc reporter gene present on a plasmid carrying the origin of the investigated replicon. Replication is initiated by expression of the appendant replicase from a second plasmid and results in replication of the origin plasmid coupled with an increase in the Luc activity. Using this method to compare replication of PCV1 and PCV2 in cell culture, it was shown that the Rep/Rep′ protein of PCV2 initiated replication at the origin of PCV1, as did the reciprocal combination. Our results indicate that the cis- and trans-acting replication factors of the two viruses are functionally exchangeable.