Rinderpest virus expressing enhanced green fluorescent protein as a separate transcription unit retains pathogenicity for cattle.

A full-length DNA clone of a virulent strain of rinderpest virus was constructed with the gene for the enhanced green fluorescent protein (eGFP) inserted as a separate transcription unit between the P and M genes. Rescue of the virus from the modified clone using reverse genetics generated a virus that grew to the same levels as the virus rescued from the unmodified DNA clone in cell culture. The recombinant virus expressed eGFP to a high level and was used to follow virus replication in real-time using live-cell imaging. Cattle infected with both the recombinant wild-type virus and the recombinant eGFP expressing virus developed clinical disease similar to that of the wild-type natural virus isolate. Detection of virus in circulating peripheral blood leukocytes was equivalent to that of the animals infected with the wild-type virus. The high level of expression of soluble eGFP by this virus allowed us to detect viral replication in infected animals by confocal microscopy. Imaging vibrating microtome sections by confocal microscopy provided good preservation of tissue and cellular architecture as well as revealing the sites of replication of the virus in different tissues of infected animals.

Pathological and immunohistochemical study of experimental peste des petits ruminants virus infection in goats.

Peste des petits ruminants (PPR) is an emerging, economically important viral disease of goats and sheep in the Indian subcontinent. In the present investigation, 15 hill goats were experimentally infected with 2 ml of 10% splenic suspension of a virulent isolate of PPR virus (PPR/Izatnagar/94) that had caused heavy mortality (>75%) in goats during 1994 outbreaks in northern India. More than 86% (13 of 15) animals died between 9 and 13 days post inoculation at the height of temperature or when temperatures were declining. Necropsy findings included congestion of gastrointestinal tract (GIT), nasal sinuses, consolidation of antero-ventral lobes of lungs, engorged spleen, and occasionally oedematous lymph nodes. Histopathological examination of major organs of GIT revealed degeneration and necrosis of labial mucosa, severe mucosal and submucosal congestion, degeneration and necrosis of intestinal epithelium and lymphoid cell depletion from Peyer's patches along with presence of syncytia at times. Lungs showed broncho-interstitial changes and presence of intracytoplasmic and intranuclear eosinophilic inclusions in alveolar macrophages and syncytial cells. These changes in lungs were frequently complicated with serofibrinous pneumonia (57%, eight of 14). Lymphocytolysis and occasional syncytia formation were evident in the lymphoid tissues. Immunohistochemical (IHC) findings included presence of PPR virus antigen in the labial, intestinal, and bronchiolar epithelial cells, pneumocytes, macrophages and syncytial cells in lungs, and lymphoid (intact and necrotic) and reticular cells in lymphoid organs. The findings of the study indicated the highly virulent nature of the PPR virus isolate (PPR/Izatnagar/94), causing 100% mortality and characteristic pathological changes in the target organs such as lungs, intestines and lymphoid tissues. The results of the IHC study suggested that indirect immunoperoxidase could be an alternative method in the absence of more sophisticated methods of laboratory diagnosis of PPR virus infection in goats.

Rinderpest virus phosphoprotein gene is a major determinant of species-specific pathogenicity.

We previously demonstrated that the rinderpest virus (RPV) hemagglutinin (H) protein plays an important role in determining host range but that other viral proteins are clearly required for full RPV pathogenicity to be manifest in different species. To examine the effects of the RPV nucleocapsid (N) protein and phosphoprotein (P) genes on RPV cross-species pathogenicity, we constructed two new recombinant viruses in which the H and P or the H, N, and P genes of the cattle-derived RPV RBOK vaccine were replaced with those from the rabbit-adapted RPV-Lv strain, which is highly pathogenic in rabbits. The viruses rescued were designated recombinant RPV-lapPH (rRPV-lapPH) and rRPV-lapNPH, respectively. Rabbits inoculated with RPV-Lv become feverish and show leukopenia and a decrease in body weight gain, while clinical signs of infection are never observed in rabbits inoculated with RPV-RBOK or with rRPV-lapH. However, rabbits inoculated with either rRPV-lapPH or rRPV-lapNPH became pyrexic and showed leukopenia. Further, histopathological lesions and high virus titers were clearly observed in the lymphoid tissues from animals infected with rRPV-lapPH or rRPV-lapNPH, although they were not observed in rabbits infected with RPV-RBOK or rRPV-lapH. The clinical, virological, and histopathological signs in rabbits infected with the two new recombinant viruses did not differ significantly; therefore, the RPV P gene was considered to be a key determinant of cross-species pathogenicity.

Use of participatory epidemiology in studies of the persistence of lineage 2 rinderpest virus in East Africa.

In 1994, rinderpest virus of African lineage 2 was detected in East Africa after an apparent absence of more than 30 years. In 1996, a disease search, based on participatory epidemiological techniques supplemented by serological and virological analyses, was undertaken in southern Somalia and north-eastern Kenya to collate past and current epidemiological information about rinderpest-compatible disease events, and to test the hypothesis that African lineage 2 rinderpest virus persists in populations of transhumant cattle in the Somali ethnic areas. The findings in Afmadu in Lower Juba led the search for rinderpest to the communities in the Bardera area and then on to the Kenya/Somalia border areas between Mandera and El Wak. The herders had a specific knowledge of the clinical signs of rinderpest and provided detailed and accurate descriptions of cases. They differentiated between classical acute rinderpest and a milder syndrome characterised by an ocular discharge and diarrhoea, few oral lesions, corneal opacity and occasional mortality. The studies provided evidence for the endemic occurrence of rinderpest back to at least 1981, with a periodicity of five years in the incidence of the disease. After a period of high mortality in 1992 to 1993, around Afmadu, herders reported a mild disease, with occasional increases in mortality, from other areas of Lower Juba and the Gedo Region. Reports by herders of a rinderpest-compatible disease in the El Wak area were pursued until active cases were located and rinderpest was confirmed.

Induction of apoptotic cellular death in lymphatic tissues of cattle experimentally infected with different strains of rinderpest virus.

The presence, type, and extent of cellular death in lymphatic tissues of cattle experimentally infected with rinderpest virus strains of different virulence was investigated morphologically. Cells with DNA strand breaks were identified in histological sections of palatine tonsil, spleen, and mesenteric and mandibular lymph nodes by the TUNEL (terminal desoxynucleotidyl transferase-mediated dUTP nick end labelling) assay. In addition, representative samples of lymphatic tissues were examined by transmission electron microscopy. The results indicated that cellular disassembly in lymphatic tissues was caused by both apoptosis and oncosis. Cells with DNA strand breaks were observed in follicular and parafollicular areas of lymphatic tissues and their numbers were determined. A significant correlation was found between the number of TUNEL-positive cells and viral virulence. These results suggest that, in addition to oncosis, apoptotic cellular death in lymphatic tissues contributes substantially to the pathogenesis of rinderpest.

Rinderpest and peste des petits ruminants viruses exhibit neurovirulence in mice.

Members of the morbillivirus genus, canine distemper (CDV), phocine distemper virus (PDV), and the cetacean viruses of dolphins and porpoises exhibit high levels of CNS infection in their natural hosts. CNS complications are rare for measles virus (MV) and are not associated with rinderpest virus (RPV) and peste des petits ruminants virus (PPRV) infection. However, it is possible that all morbilliviruses infect the CNS but in some hosts are rapidly cleared by the immune response. In this study, we assessed whether RPV and PPRV have the potential to be neurovirulent. We describe the outcome of infection, of selected mouse strains, with isolates of RPV, PPRV, PDV, porpoise morbillivirus (PMV), dolphin morbillivirus (DMV), and a wild-type strain of MV. In the case of RPV virus, strains with different passage histories have been examined. The results of experiments with these viruses were compared with those using neuroadapted and vaccine strains of MV, which acted as positive and negative controls respectively. Intracerebral inoculation with RPV (Saudi/81) and PPRV (Nigeria75/1) strains produced infection in Balb/C and Cd1, but not C57 suckling mice, whereas the CAM/RB rodent-adapted strain of MV infected all three strains of mice. Weanling mice were only infected by CAM/RB. Intranasal and intraperitoneal inoculation failed to produce infection with any virus strains. We have shown that, both RPV and PPRV, in common with other morbilliviruses are neurovirulent in a permissive system. Transient infection of the CNS of cattle and goats with RPV and PPRV, respectively, remains a possibility, which could provide relevant models for the initial stages of MV infection in humans.

Rinderpest epidemic in wild ruminants in Kenya 1993-97.

A severe epidemic of rinderpest, affecting mainly wild ruminants, occurred between 1993 and 1997 in East Africa. Buffalo (Syncerus caffer), eland (Taurotragus oryx) and lesser kudu (Tragelaphus imberbis) were highly susceptible. The histopathological changes, notably individual epithelial cell necrosis with syncytia formation, were consistent with an infection with an epitheliotrophic virus. Serology, the polymerase chain reaction, and virus isolation confirmed the diagnosis and provided epidemiological information. The virus was related to a strain which was prevalent in Kenya in the 1960s, of a second lineage (II), and distinct from isolations of rinderpest virus in the region since 1986. The source of the virus was presumed to be infected cattle from the Eastern region of Kenya and Somalia. The pathogenicity of the virus varied during the epidemic. The mortality in buffalo populations was estimated to be up to 80 per cent, and population data suggested that the virus had an adverse effect on a wide range of species. The virus caused only a mild disease in cattle, with minimal mortality. The results confirmed the importance of wildlife as sentinels of the disease, but although wildlife were important in the spread of the virus, they did not appear to act as reservoirs of infection.

Cattle plague in Shangri-La: observations on a severe outbreak of rinderpest in northern Pakistan 1994-1995.

Between April 1994 and November 1995 the most severe epidemic of rinderpest reported in the world for over a decade affected domestic livestock in the Northern Areas of Pakistan. As many as 40,000 cattle and yaks died, more by some estimates, and mortality rates may have exceeded 80 per cent in these species in several villages. This report describes some of the clinicopathological and epidemiological features peculiar to the outbreak, including laboratory-confirmed rinderpest in a goat, and the difficulties encountered before the disease was eradicated. It also describes the human costs and emphasises the need to accelerate the global eradication of this most eradicable disease.

A review of three pathology-based techniques for retrospective diagnosis of rinderpest, with comparison to virus isolation.

Base of tongue, eyelid, and retropharyngeal lymph node were collected from three animals experimentally infected with rinderpest and utilised in a study comparing virus isolation with histopathology, immunohistochemistry, and in situ hybridisation to determine the usefulness of the latter three techniques as retrospective diagnostic aids for this disease. Virus isolation was positive for all nine samples. Histopathology was suggestive in all the tissues and definitive in some. Immunohistochemistry and in situ hybridisation highlighted the presence of rinderpest antigen of rinderpest nucleic acid in all of the sections. However, in situ hybridisation was more specific than immunohistochemistry.

Immunohistochemical detection of rinderpest virus: effects of autolysis and period of fixation.

Samples of eyelid, tongue, soft palate and palatine tonsil were collected from calves infected experimentally with rinderpest virus. The tissues were fixed in 10 per cent neutral buffered formalin immediately, 24 or 48 hours post mortem. Then, after three days, 10 days, 28 days or three months in formalin, they were processed into paraffin blocks and examined immunohistochemically for rinderpest viral antigen. The tonsil was the best of the four tissues in providing a consistently positive immunohistochemical signal for the presence of virus, despite autolytic changes and/or prolonged fixation.

Pathomorphological and immunohistological findings in cattle experimentally infected with rinderpest virus isolates of different pathogenicity.

Experimental infection of nine cattle with seven rinderpest virus strains of different pathogenicity resulted in significant variations of clinical signs, morphological lesions and distribution of viral antigen in tissues. The severity of clinical disease was correlated with the extent of tissue alterations and the amount of immunohistologically detectable viral antigen. Both mild and virulent strains of rinderpest share essentially the same tissue tropisms in vivo, i.e. epithelio- and lympho-tropism. However, rinderpest virus isolates of higher pathogenicity showed a more rapid and wider distribution with more extensive lesions than milder strains, which probably accounts for the higher mortality.

Immunohistochemical studies of lymphoid tissues of rabbits infected with rinderpest virus.

The pathogenesis of infection with the L-strain of rinderpest virus (RPV) in rabbits was investigated. Of several lymphoid tissues examined, those associated with the gut showed the most marked virus growth. The virus titres were maximal 4 days after inoculation but had declined at day 6. The distribution of viral antigen was examined immunohistochemically with the recently established anti-rabbit CD5 monoclonal antibody (MoAb), which is a pan-T-cell marker, and the anti-RPV-nucleoprotein MoAb. The virus antigen was localized in the CD5+ area at the initial stage of infection but spread to all areas of the lymphoid tissues at the later stages. By flow cytometric analysis with both rabbit CD5 and CD4 MoAbs, a decrease of the CD4+ and CD5+ subpopulations was observed in the spleen and mesenteric lymph nodes.

Distribution of antigen in cattle infected with rinderpest virus.

Five Holstein heifers (approximately 8 months of age and weighing 225-275 kg) were inoculated subcutaneously with 1,000 TCID50 of rinderpest virus, virulent Kabete O strain. They become clinically ill 2 to 5 days post-inoculation, with fever (40 C to 41.5 C), conjunctivitis, and diarrhea. All were euthanatized when moribund at 6 days postinoculation. The following tissues were collected in formalin, embedded in paraffin, and subsequently subjected to histopathologic and immunohistochemical examination: tongue, buccal mucosa, soft palate, esophagus, rumen, abomasum, duodenum, jejunum with and without Peyer's patch, ileum, cecum, proximal colonic lymphoid patch, spiral colon, eyelid, gall bladder, spleen, tonsil, trachea, lungs, and numerous lymph nodes. Immunohistochemical examination was accomplished using a primary rabbit anti-rinderpest antibody, and either a peroxidase-diaminobenzidine or alkaline phosphatase-Vector Red detection substrate system. In the lymph nodes, spleen, and tonsil, depletion of lymphocytes from all areas was extensive, with antigen most prominent in persisting reticular cells throughout the tissues. In the intestine, necrotizing and ulcerative changes in the mucosa were extensive and widespread. Damage was most severe in areas overlying lymphoid patches. In both small and large intestine, antigen was distributed predominantly in epithelial cells, histiocytic cells in the lamina propria, and in remaining reticular cells of lymphoid patches. In oral mucosa, there were multiple ulcerations and numerous multinucleate syncytial cells, both containing and without antigen. Lungs and trachea had subtle yet consistent necrosis of epithelial cells, with antigen often distributed in a circumferential manner in epithelium of bronchioles.

Comparison of the pathogenicity of rinderpest virus in different strains of rabbits.

Pathogenicity of the lapinized Nakamura-III (L) strain of rinderpest virus (RPV) was examined in four strains of rabbits consisting of two inbred strains (NW-NIBS and DUY-NIBS) and two outbred strains maintained in closed colony (NW-NIBS and JW-NIBS) using a marmoset lymphoblastoid cell line, B95a cell-passaged virus and tissue homogenates of virus-infected rabbits. The cell culture virus was found to maintain virulence for rabbits of both closed colony and inbred NW-NIBS strain similar to the homogenate virus. Among the strains investigated, inbred NW-NIBS strain showed the highest susceptibility to RPV. Thus experimental model in an inbred rabbit using cell culture virus became useful.

Viral antigen distribution in organs of cattle experimentally infected with rinderpest virus.

The distribution of viral antigen in various organs of four approximately 10-month-old castrated male Friesian cattle experimentally infected with a highly virulent strain of rinderpest virus was studied. A monoclonal antibody with genus-specific reactivity for morbilliviruses was applied in an indirect immunoperoxidase method performed on formalin-fixed, paraffin-embedded tissue sections. Rinderpest viral antigen was located mainly in the cytoplasm of the epithelial cells of the digestive, respiratory, and urinary tracts, as well as in the cells of endocrine glands (adrenal, thyroid) and exocrine glands (salivary glands, sebaceous glands, exocrine pancreas). Furthermore, different types of cells in lymphatic organs contained rinderpest viral antigen. In contrast to the documented results of studies carried out with other morbilliviruses, tissues of the central nervous system did not contain viral antigen. Various types of epithelial and lymphoreticular cells are the main targets of a virulent strain of rinderpest virus in vivo.

An immunohistochemical study of the pneumonia caused by peste des petits ruminants virus.

Ten goats were inoculated with peste des petits ruminants virus, a paramyxovirus closely related to rinderpest virus. All goats developed severe clinical disease, 8/10 having coughing or dyspnea as prominent clinical signs. In addition, all of the goats had stomatitis and diarrhea. Histopathologic and immunohistochemical studies were done only on the respiratory tracts. Pathologic changes ranged from mild multifocal bronchiolitis and bronchitis to severe bronchointerstitial pneumonia. Lesions were more severe in anteroventral than caudal lobes. The histologic nature of the viral process in the goat lungs had many features in common with the processes of pneumonia in dogs, due to canine distemper, or pneumonia in human beings, due to measles virus. Immunohistochemical staining of formalin-fixed, paraffin-embedded respiratory tract tissue was performed using an indirect system with rabbit anti-rinderpest virus serum, biotinylated anti-rabbit antibody, streptavidin-alkaline phosphatase, and nitroblue tetrazolium chromogen. Staining was sensitive, highlighting the presence of viral antigen in both lung and trachea of all goats. Viral antigen was found in both cytoplasm and nucleus of tracheal, bronchial, and bronchiolar epithelial cells, type II pneumocytes, syncytial cells, and alveolar macrophages. In general, the amount of staining correlated directly with the severity of the inflammatory process.

Evidence of identification of peste des petits ruminants from goats in Egypt.

Peste des petits ruminants virus was isolated from young goats in an outbreak of the disease for the first time in Egypt. Affected goats showed symptoms simulating rinderpest, disease in cattle. The mortality rate was about 30%, and morbidity reached 90%. History, clinical symptoms, postmortem lesions, and diagnosis were discussed.