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Comparative analysis of the attachment protein gene (H) of dolphin morbillivirus.

DMV, dolphin morbillivirus, a paramyxovirus of uncertain origin recently emerged in Mediterranean dolphins. This study presents the complete nucleotide sequence of the hemagglutinin (H) gene including the gene boundaries. The single open reading frame of the DMV H gene encodes a protein of 604 residues which exhibits overall sequence characteristics similar to the H genes of other morbilliviruses. When compared to its closest homologues, measles virus (MV) and rinderpest virus (RPV), DMV has, respectively, 44 and 46% of amino acid residues in identical positions. The primary sequence of the DMV H protein is markedly less conserved than that of the fusion protein. The comparative data at the genomic level correspond with cross-neutralization studies with different morbilliviruses. Retrospective serogical studies dating back to 1983 indicate DMV-like infections in whales of the eastern Atlantic. The presented data support and extend previous studies suggesting that this novel morbillivirus is one of the phylogenetically oldest morbilliviruses known to circulate today. The relationship of DMV and established morbilliviruses to the newly emerged candidate morbillivirus infecting horse and man is discussed.

Amino Acid Sequence↗

Ultrastructure of equine morbillivirus.

The ultrastructure of the equine morbillivirus (EMV) which was implicated in the death of one human and fourteen horses in Queensland, Australia during September 1994 and a 36 year old man from Queensland in October 1995 is described. The ultrastructure of the virus and the intracellular virus-specific structures are characteristic for the family Paramyxoviridae. Cytoplasmic nucleocapsids were observed within the infected cells monolayers, endothelial cells (lung) of infected horses and the neurons within the brain of the 36 year old Queensland man. Aggregates of smaller nucleocapsid-like structures were also observed within the brain of the same man; these did not react with sera from recovered EMV-infected horses or from a recovered EMV-infected human. Co-examination of rinderpest virus (RPV), bovine parainfluenza-3 (BPIV-3), human respiratory virus (HRSV) and Sendai virus revealed that their envelope-associated surface projections are equivalent in length to the 15 nm spikes of EMV. EMV differed from these other viruses in that the majority of virions possessed surface projections of two distinct lengths (18 and 15 nm). Further ultrastructural examinations of plaque purified EMV revealed a small percentage of EM viruses possessed a mixed array of surface projections indicating that the 'double-fringed' (DF) particles may be the result of a post-translational modification(s).

Adult↗

Round table on morbilliviruses in marine mammals.

Since 1988 morbilliviruses have been increasingly recognized and held responsible for mass mortality amongst harbour seals (Phoca vitulina) and other seal species. Virus isolations and characterization proved that morbilliviruses from seals in Northwest Europe were genetically distinct from other known members of this group including canine distemper virus (CDV), rinderpest virus, peste des petits ruminants virus and measles virus. An epidemic in Baikal seals in 1987 was apparently caused by a morbillivirus closely related to CDV so that two morbilliviruses have now been identified in two geographically distant seal populations, with only the group of isolates from Northwest Europe forming a new member of the genus morbillivirus: phocid distemper virus (PDV). Because of distemper-like disease, the Baikal seal morbillivirus was tentatively named PDV-2 in spite of its possible identity with CDV. The appearance of morbilliviruses in the Mediterranean Sea causing high mortality amongst dolphins should further increase the research activities on protection strategies for endangered species of marine mammals.

Animals↗

Editing of morbillivirus P gene transcripts in infected animals.

RNA editing in the Morbillivirus genus in vivo was investigated by applying a polymerase chain reaction-based primer extension technique to measure the edited and non-edited mRNA transcripts. In this genus of the Paramyxoviridae the P gene transcript is altered by the co-transcriptional addition of one extra G residue to produce the mRNA for the V non-structural protein. Using tissues of phocine distemper virus (PDV) infected seals, canine distemper virus (CDV) infected dogs and rinderpest virus (RPV) infected cattle, it was demonstrated that editing occurs in vivo. The P:V mRNA ratios were generally similar to those found in tissue culture infections with the same virus and a minor fraction of transcripts had 2-4 extra G residues. In one seal brain infected with PDV the ratio of P:V mRNA was reversed but no differences were found in the levels of mRNA editing in different tissues from the same animal infected with CDV or RPV. However, variation was seen between animals infected with different isolates of RPV and even between animals infected with the same isolate of RPV.

Animals↗

Rapid isolation of monoclonal hybridoma cultures by a 'fusion-cloning' method: the requirement for aminopterin.

Hybridomas were generated by fusing the Balb/c SP2/0 myeloma-like cell line with either: (i) splenocytes from Balb/c mice immunized with foot-and-mouth disease virus (FMDV), rinderpest virus (RPV), peste des petits ruminants virus (PPRV), African swine fever virus (ASFV) or pig thymocytes; or (ii) lymph node cells from cattle immunized with FMDV. If the fusion mixtures were plated in cloning medium of methyl cellulose and HAT medium, small hybridoma colonies developed which rarely survived. Fusion mixtures were then plated in liquid HT medium on to 3T3/A31 feeder layers in 75 cm2 flasks, incubated at 37 degrees C for 24 h before adding aminopterin, and incubated for a further 2 to 4 days before cloning in methyl cellulose/HT medium. Without the aminopterin in the cloning medium, colonies of hybridomas, which could be cultured, developed from the majority of fusions. These colonies were isolated in HT medium over feeder layers and given two subcultures in HAT medium as a precaution against any reversion to aminopterin sensitivity during the cloning. No evidence of such reversions were seen, and recloning results suggested that the initial cloning was highly efficient at generating monoclonal cultures.

Aminopterin↗

Characterization of monoclonal antibodies directed against the canine distemper virus nucleocapsid protein.

We have established four monoclonal antibodies (MAbs) against the nucleocapsid protein (NP) of canine distemper virus (CDV). A competitive binding assay has revealed that the MAbs are directed against two antigenic domains. An immunofluorescence assay using a series of deletion clones of the NP and an immunoprecipitation assay using the NP have revealed that two of the MAbs recognize the C-terminal region of the NP while the other two recognize the tertiary structure of the N-terminal domain. These MAbs reacted with all eight strains of CDV used in this study, but showed different reactivities against measles virus and rinderpest virus.

Animals↗

Antibody seroprevalences against peste des petits ruminants (PPR) virus in camels, cattle, goats and sheep in Ethiopia.

A questionnaire-survey data indicated that 26% of 276 farmers reported the presence of respiratory disease in their herds in 2001. The incidence was perceived as "high" in small ruminants and camels, but as "low" in cattle. Simultaneously, 2815 serum samples from camels (n=628), cattle (n=910), goats (n=442) and sheep (n=835) were tested. The peste des petits ruminants (PPR) antibody seroprevalence was 3% in camels, 9% in cattle, 9% in goats and 13% in sheep. The highest locality-specific seroprevalences were: camels 10%, cattle 16%, goats 22% and sheep 23%. The animals had not been vaccinated against rinderpest or PPR. Antibody seroprevalences detected in camels, cattle, goats and sheep confirmed natural transmission of PPR virus under field conditions.

Animals↗

Booster immunization of antigen primed mice with anti-idiotypic T cells generates antigen-specific memory T cell response.

Immunological memory is characterized by quick and enhanced immune response after re-exposure to the same antigen. In the present work, we have shown that anti-idiotypic T cells are generated in mice after immunization with idiotypic T cell clone or polyclonal T cell specific for nucleocapsid protein of Rinderpest virus. Further, we have shown that N specific idiotypic T cell receptors from apoptotic idiotypic T cells are processed by bone marrow derived dendritic cells and presented to cognate anti-idiotypic T cells. Evidence has been provided for the existence of antigen specific T cell idiotypic network in the body. Boosting with antigen specific anti-idiotypic T cells generates memory response in the antigen-primed mice.

Adoptive Transfer↗

Morbilliviral infection in a pygmy sperm whale (Kogia breviceps) from Taiwanese waters.

Morbilliviral infection was diagnosed in an adult male pygmy sperm whale (Kogia breviceps) from southwestern Taiwan on the basis of pathological findings, immunohistochemical staining, and reverse transcription-polymerase chain reaction. The whale was found alive stranded on the beach and died after 5 days of medical care. It was thin and had dozens of nematode in the first stomach. The lungs were dark red and heavy. Histopathological examination revealed diffuse, moderate bronchointerstitial pneumonia. Intranuclear and intracytoplasmic inclusions with occasional syncytial cell formation were noted in the lungs, lymph nodes, and spleen. The RNA extracted from lung tissue was subjected to morbilliviral gene amplification. After priming with specific oligonucleotides, the cDNA covering the phosphoprotein (P) gene was copied and then amplified by PCR. The gene fragment amplified from the lung tissue was sequenced. Phylogenetic analysis of partial P gene revealed 97.6% sequence identity to the dolphin morbillivirus and 90.2% similarity to the pilot whale morbillivirus. Morbilliviral antigens were detected in the lungs, lymph nodes, and spleen by immunohistochemistry using polyclonal antibody against rinderpest virus. This is the first report of morbilliviral infection with genetic evidence in a pygmy sperm whale from the Western Pacific Ocean around Taiwan.

Animals↗

Morbillivirus nucleoprotein possesses a novel nuclear localization signal and a CRM1-independent nuclear export signal.

Morbilliviruses, which belong to the Mononegavirales, replicate its RNA genome in the cytoplasm of the host cell. However, they also form characteristic intranuclear inclusion bodies, consisting of nucleoprotein (N), in infected cells. To analyze the mechanisms of nucleocytoplasmic transport of N protein, we characterized the nuclear localization (NLS) and nuclear export (NES) signals of canine distemper virus (CDV) N protein by deletion mutation and alanine substitution of the protein. The NLS has a novel leucine/isoleucine-rich motif (TGILISIL) at positions 70-77, whereas the NES is composed of a leucine-rich motif (LLRSLTLF) at positions 4-11. The NLS and NES of the N proteins of other morbilliviruses, that is, measles virus (MV) and rinderpest virus (RPV), were also analyzed. The NLS of CDV-N protein is conserved at the same position in MV-N protein, whereas the NES of MV-N protein is located in the C-terminal region. The NES of RPV-N protein is also located at the same position as CDV-N protein, whereas the NLS motif is present not only at the same locus as CDV-N protein but also at other sites. Interestingly, the nuclear export of all these N proteins appears to proceed via a CRM1-independent pathway.

Amino Acid Sequence↗

The application of molecular biology to the study of veterinary infectious diseases.

The practical impact that the techniques of molecular biology is having on the study of infectious diseases of veterinary importance is discussed. The areas highlighted include the development of novel diagnostic assays such as the polymerase chain reaction, the use of recombinant antigen in ELISA tests, and the potential of genetically engineered vaccines to alter radically the disease status of animals in developed and developing countries. Novel vaccine strategies such as the use of rationally attenuated viruses as has been successfully employed with pseudorabies virus, and the use of viruses such as vaccinia as vaccine vectors to carry the genes encoding potent immunogens from a range of pathogens (such as rabies and rinderpest) are described. Contributions to the study of epidemiology, mechanisms of pathogenesis and immune responses are also discussed.

Animals↗

Human contact with cattle, dogs and monkeys and genetic diversity of measles viruses.

There is a very close contact among humans, cattle, dogs and monkeys in developing countries. That could lead to genetic alteration in morbillivirus isolates of measles, rinderpest, canine distemper. Emergence of novel morbillivirus strains and failures of prophylactic viral vaccines should be monitored by characterisation of genetic profiles of isolates in different species. Incorporation of additional epitopes in existing prophylactic vaccines should minimise vaccine failures attributable to such novel strains.

Animals↗

Idiotype and anti-idiotype specific T cell responses on transplantation with hybridomas reactive to viral hemagglutinin and human tumor antigen.

B cell hybridomas expressing class I and II MHC molecules and producing antibodies directed against hemagglutinin protein of Rinderpest virus and human Mucin-1 have been used as surrogate B cells to study T cell responses against the antigens. The observed CTL and lymphoproliferative response indicates that anti-idiotypic B cells termed Jerne cells stimulate both T helper and T cytotoxic cells by virtue of their ability to present recycled or regurgitated peptido-mimics of antigen to T helper cells through class II MHC and de novo synthesized peptido-mimics of antigens to CTLs. Thus, T cell memory response can be perpetuated by anti-idiotypic Jerne B cells and these findings lend support to the earlier proposed relay hypothesis for perpetuation of immunological memory (IM).

Animals↗

The hemagglutinin-neuraminidase protein of peste des petits ruminants virus is biologically active when transiently expressed in mammalian cells.

The genes coding for the surface glycoproteins hemagglutinin-neuraminidase (HN) of the peste des petits ruminants virus (PPRV) and hemagglutinin (H) of rinderpest virus (RPV) were cloned in a cytomagalovirus promoter driven expression vector and expressed transiently in mammalian cells. The protein expression was apparent 24 h after transfection and the expressed proteins were detected at the cell surface. The transiently expressed PPRV HN protein was found to be biologically active in possessing hemadsorption and neuraminidase activities. On the other hand, RPV H protein exhibited neuraminidase activity but was deficient in hemadsorption activity. The substrate specificity of the neuraminidase activity of these two proteins differed distinctly. The presence of neuraminidase activity in both PPRV HN and RPV H proteins is unusual among members of the morbillivirus genus.

Animals↗

Recent epidemiology of peste des petits ruminants virus (PPRV).

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.

Africa↗

Morbillivirus infections, with special emphasis on morbilliviruses of carnivores.

Morbilliviruses infections cause significant mortality in human beings and animals. Measles virus is responsible for up to two million childhood deaths annually in the developing world, while rinderpest and peste des petits ruminants cause severe epizootics in domestic and wild ruminants in areas of the world where they remain endemic. Canine distemper virus (CDV) is a cause of fatal disease in many species of carnivores. Distemper is controlled by vaccination in domestic dogs and farmed mink, but it may be impossible to eradicate the virus because of its global distribution and wide variety of susceptible host species, which includes both freshwater and marine seals. Research is currently under way to develop new recombinant vaccines, since the currently available live attenuated vaccines for CDV are not safe for use for all species and many valuable zoo animals need to be protected from CDV. New morbilliviruses with potentially disastrous ecological consequences for marine mammals have been discovered in the past decade; phocid distemper virus (PDV) in seals and the cetacean morbillivirus (CMV) has been found in dolphins, whales and porpoises. Reverse transcription, coupled with the polymerase chain reaction (RT/PCR) and nucleic acid sequencing, has been used to characterise the morbilliviruses and has given insights into the evolution of this virus genus.

Animals↗

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus.

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.

Journal Article↗

Monitoring trypanosomiasis in space and time.

The paper examines the possible contributions to be made by Geographic Information Systems (GIS) to studies on human and animal trypanosomiasis in Africa. The epidemiological characteristics of trypanosomiasis are reviewed in the light of the formula for the basic reproductive rate or number of vector-borne diseases. The paper then describes how important biological characteristics of the vectors of trypanosomiasis in West Africa may be monitored using data from the NOAA series of meteorological satellites. This will lead to an understanding of the spatial distribution of both vectors and disease. An alternative, statistical approach to understanding the spatial distribution of tsetse, based on linear discriminant analysis, is illustrated with the example of Glossina morsitans in Zimbabwe, Kenya and Tanzania. In the case of Zimbabwe, a single climatic variable, the maximum of the mean monthly temperature, correctly predicts the pre-rinderpest distribution of tsetse over 82% of the country; additional climatic and vegetation variables do not improve considerably on this figure. In the cases of Kenya and Tanzania, however, another variable, the maximum of the mean monthly Normalized Difference Vegetation Index, is the single most important variable, giving correct predictions over 69% of the area; the other climatic and vegetation variables improve this to 82% overall. Such statistical analyses can guide field work towards the correct biological interpretation of the distributional limits of vectors and may also be used to make predictions about the impact of global change on vector ranges. Examples are given of the areas of Zimbabwe which would become climatically suitable for tsetse given mean temperature increases of 1, 2 and 3 degrees Centigrade. Five possible causes for sleeping sickness outbreaks are given, illustrated by the analysis of field data or from the output of mathematical models. One cause is abiotic (variation in rainfall), three are biotic (variation in vectorial potential, host immunity, or parasite virulence) and one is historical (the impact of explorers, colonizers and dictators). The implications for disease monitoring, in order to anticipate sleeping sickness outbreaks, are briefly discussed. It is concluded that present data are inadequate to distinguish between these hypotheses. The idea that sleeping sickness outbreaks are periodic (i.e. cyclical) is only barely supported by hard data. Hence it is even difficult to conclude whether the major cause of sleeping sickness outbreaks is biotic (which, in model situations, tends to produce cyclical epidemics) or abiotic.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗