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Morbillivirus infection of marine mammals has been documented across all of the world's oceans. Whilst infection is generally demonstrated using a variety of histopathological and serological techniques, where possible, the use of molecular techniques is being used to enable accurate genetic typing of virus strains through sequence analysis. Here, we present genetic data from dolphins and pilot whales affected by morbillivirus infection in the recent outbreak in the Mediterranean Sea during a six-month period from the end of October 2006 to April 2007. To date, very few studies have looked at characterizing outbreaks of morbillivirus infections in whale species at the molecular level. Here, we provide a full sequence for the haemagglutinin (H) gene from material derived from both a dolphin and a pilot whale from the 2007 outbreak in the Mediterranean Sea and show this virus to be 100% identical across the region analysed. Furthermore, we compare partial sequence data from the nucleocapsid (N) gene of the pilot whale material with previously published data and show evidence for strong protein conservation between these different isolates. Finally, we discuss the current classification of cetacean morbilliviruses as a single species.

Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPV(KO)) can be rescued. A recombinant virus, rRPV(KO)L-RRegfpR, which grew at an indistinguishable rate and to an identical titer as rRPV(KO) in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 10(4) TCID(50) of the rRPV(KO) parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPV(KO)L-RRegfpR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.

Peste des petits ruminants (PPR) is an economically important viral disease of goats and sheep first described in west Africa in the 1940s. The virus has been circulating in parts of sub-Saharan Africa for several decades and in the Middle East and southern Asia since 1993, although the first description of the virus in India dates to 1987. To study the genetic relationship between isolates of distinct geographical origin, a selected region of the fusion (F) protein gene of the viruses was amplified using RT/PCR and the resulting DNA product sequenced for phylogenetic analysis. Viruses from 27 outbreaks in Asian and Middle Eastern countries, reported between 1993 and 2000, and two recent outbreaks from the African continent were compared with the prototype African strain. Of the four known lineages of PPR virus, lineage 1 and 2 viruses have been found exclusively in west Africa. Virus from an outbreak in Burkina Faso in 1999 fell into the lineage 1 group. Viruses of lineage 3 have been found in east Africa, where an outbreak in Ethiopia in 1996 was of this type, and also in Arabia and in southern India. However, there have been no further isolations of lineage 3 virus from India since the one reported in 1992 from Tamil Nadu. A virus of this lineage was found circulating in Yemen in 2001. In the past 8 years virus exclusively of the fourth lineage has spread across the Middle East and the Asian sub-continent, reaching east as far as Nepal and Bangladesh. This virus lineage was also reported from Kuwait in 1999. The geographical source of the new lineage 4 virus is unknown although it is most closely related to African lineage 1. The possibility that its earlier presence in northern India was masked by the circulation of Rinderpest virus, a related virus of cattle, is considered unlikely. [Copyright &y& Elsevier]

Abstract: To investigate the possible origin and spread of the dramatic re-emergent 2002 distemper epizootic observed among seals in Danish Waters, we have sequenced wild-type genes of the attachment (H) glycoproteins of viruses from both the 2002 and 1988 epizootics. Phylogenetic analysis of the H genes of phocine distemper virus (PDV) together with other morbilliviruses, suggests that the re-emergent 2002 PDV is more closely related to a putative recent ancestral PDV than the 1988 PDV isolates. Moreover, upsurges of distemper disease in land-living carnivores linked in time and locality to the 2002 seal epizootic in Danish Waters was investigated and determined to be caused by canine distemper virus, the closest relative of PDV, revealing no direct epidemiological link to the seal epizootics. [Copyright &y& Elsevier]

Abstract: Peste-des-petits ruminants virus (PPRV) causes acute febrile illness in both farmed and wild small ruminants, with associated mortality rates of 50–80%. PPRV is a member of the Morbillivirus genus within the Paramyxovirus family and although there are many full length genome sequences available for members of this family, their availability for PPRV in particular is limited. We have determined the full length sequences representing two virulent strains of PPRV, the Côte d’Ivoire 1989 (CI/89) and Nigeria 1976 (Ng76/1) strains. We present an alignment of the promoter regions of these viruses with other available PPRV promoter sequences and have identified domains in PPRV proteins believed to be critical for paramyxovirus promoter attenuation. We have also analysed the proteins of these viruses, comparing them to other available PPRV protein sequences and identified motifs that were previously recognised as being required for the function of other paramyxovirus proteins. [Copyright &y& Elsevier]

An RT-PCR/ELISA system has been developed that detects and differentiates Rinderpest virus (RPV) from the other closely related morbillivirus of ruminants, Peste des petits Ruminants virus (PPRV). In addition, using lineage specific probes, it is possible to determine whether the virus sample is wild-type or vaccine, and the likely origin of the outbreak if it is wild-type. It involves carrying out a RT-PCR with one digoxygenin (Dig)-labelled primer followed by a hybridisation step with a virus-specific, biotin-labelled, probe. The hybridisation step is carried out in an ELISA format on a streptavidin-coated plate. The DIG-labelled products are detected using a specific anti-DIG monoclonal antibody and an anti-mouse horseradish peroxidase conjugate. The hybridisation step replaces nucleotide sequencing or nested PCR for confirmation of the identity of DNA product. The assay is fast and easy to carry out and can give semi-quantitative estimates of the virus content of samples. [Copyright &y& Elsevier]