Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

Immunoinformatics approach for predicting epitopes in HN and F proteins of Porcine rubulavirus.

Porcine rubulavirus (PRV), which belongs to the family Paramyxoviridae, causes blue eye disease in pigs, characterized by encephalitis and reproductive failure in newborn and adult pigs, respectively. There is no effective treatment against PRV and no information on the effectiveness of the available vaccines. Continuous outbreaks have occurred in Mexico since the early 1980s, which have caused serious economic losses to pig producers. Vaccination can be used to control this disease. Searching for effective antigen candidates against PRV, we first sequenced the PAC1 F protein, then we used various immunoinformatics tools to predict antigenic determinants of B-cells and T-cells against the two glycoproteins of the virus (HN and F proteins). Finally, we used AutoDock Vina to determine the binding energies. We obtained the F gene sequence of a PRV strain collected in the early 1990s in Mexico and compared its amino acid profile with previous and more recent strains, obtaining an identity similarity of 97.78 to 99.26%. For the F proteins, seven linear B-cell epitopes, six conformational B-cell epitopes and twenty-nine T-cell MHC class I epitopes were predicted. For the HN proteins, sixteen linear B-cell epitopes, seven conformational B-cell epitopes and thirty-four T-cell MHC class I epitopes were predicted. The ATRSETDYY and AAYTTTTCF epitopes of the HN protein might be important for neutralizing the viral infection. We determined the in silico binding energy between the predicted epitopes on the F and HN proteins and swine MHC-I molecules. The binding energy of these epitopes ranged from -5.8 to -7.8 kcal/mol. The present study aimed to assess the use of HN and F proteins as antigens, either as recombinant proteins or as a series of peptides that could activate different responses of the immune system. This may help identify relevant immunogens, saving time and costs in the development of new vaccines or diagnostic tools.

Alston Virus, a Novel Paramyxovirus Isolated from Bats Causes Upper Respiratory Tract Infection in Experimentally Challenged Ferrets.

Multiple viruses with zoonotic potential have been isolated from bats globally. Here we describe the isolation and characterization of a novel paramyxovirus, Alston virus (AlsPV), isolated from urine collected from an Australian pteropid bat colony in Alstonville, New South Wales. Characterization of AlsPV by whole-genome sequencing and analyzing antigenic relatedness revealed it is a rubulavirus that is closely related to parainfluenza virus 5 (PIV5). Intranasal exposure of mice to AlsPV resulted in no clinical signs of disease, although viral RNA was detected in the olfactory bulbs of two mice at 21 days post exposure. Oronasal challenge of ferrets resulted in subclinical upper respiratory tract infection, viral shedding in respiratory secretions, and detection of viral antigen in the olfactory bulb of the brain. These results imply that AlsPV may be similar to PIV5 in its ability to infect multiple mammalian host species. This isolation of a novel paramyxovirus with the potential to transmit from bats to other mammalian species reinforces the importance of continued surveillance of bats as a source of emerging viruses.

Seroprevalence of Human Parainfluenza Virus Types 1-4 Among Healthy Children Under 5 Years of Age in Korea.

Human parainfluenza viruses (HPIVs) are among the major causes of respiratory infections in children, worldwide, including in Korea. There are four types of HPIVs, each with different epidemiological characteristics. HPIV3 is the most frequently circulating HPIV type, while the epidemiology of HPIV4 remains unclear. The aim of this study was to investigate the age-stratified seropositivity rates of HPIV types 1-4 among children in Korea. These data will be useful to determine vaccine requirements. This study included 245 participants categorized into four age groups: 6-11 months, 1 year, 2 years, and 3-5 years. Hemagglutination inhibition (HAI) assay was used to measure the antibody titers in the serum samples of the subjects. Overall, a significantly higher seropositivity rate (68%) was observed for HPIV3 (p < 0.001), indicating the predominant circulation of this type. In the 3- to 5-year-old group, 97% of the participants displayed seropositivity for HPIV3, suggesting that most Korean children acquire HPIV3 infection by the age of 5 years. The seropositivity rate for HPIV3 increased with age (p < 0.001); a prompt rise was observed between the 6-11 months age group and the 1-year age group. The seropositivity rates of HPIV1, HPIV2, and HPIV4 were found to increase with age (p < 0.001), with a marked increase recorded after the age of 2 years. HPIV1, HPIV2, and HPIV4 tended to infect children later than HPIV3. Older children showed high antibody titer ranges for HPIV3 (p < 0.001), suggesting that children experience multiple HPIV3 infections. An increasing trend of HPIV4 seropositivity rates with age was observed and this was comparable to theHPIV1 and HPIV2 seropositivity rates, indicating that its incidence may have been underestimated. To reduce HPIV infection, the administration of a HPIV3 vaccine to children 1 year of age is likely to be the most effective option.

Tetherin antagonism by V proteins is a common trait among the genus Rubulavirus.

Tetherin (BST-2/CD317/HM1.24) is an anti-viral factor that restricts the budding of several enveloped viruses. Most of these viruses have evolved to encode tetherin antagonists. Our previous study demonstrated that the growth of human parainfluenza virus type 2 (hPIV-2), a member of the genus Rubulavirus in the family Paramyxoviridae, was inhibited by tetherin, and its V protein decreases the amount of cell surface tetherin by the interaction. In the present study, we investigated whether tetherin inhibits the growth of other rubulaviruses including PIV-5, mumps virus (MuV), simian virus 41, and hPIV-4, and whether their V proteins act as tetherin antagonists. Plaque assay demonstrated that the growth of PIV-5 and MuV was inhibited by tetherin. Flow cytometry and immunoblot analyses revealed that the infection of PIV-5 and MuV caused reduction of cell surface tetherin without affecting total amount of tetherin. Immunoprecipitation analysis showed that all V proteins of rubulaviruses tested bound to tetherin. These results suggest that tetherin antagonism by V proteins is common among the genus Rubulavirus.

Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus.

Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease.

La Piedad Michoacán Mexico Virus V protein antagonizes type I interferon response by binding STAT2 protein and preventing STATs nuclear translocation.

La Piedad Michoacán Mexico Virus (LPMV) is a member of the Rubulavirus genus within the Paramyxoviridae family. LPMV is the etiologic agent of "blue eye disease", causing a significant disease burden in swine in Mexico with long-term implications for the agricultural industry. This virus mainly affects piglets and is characterized by meningoencephalitis and respiratory distress. It also affects adult pigs, causing reduced fertility and abortions in females, and orchitis and epididymitis in males. Viruses of the Paramyxoviridae family evade the innate immune response by targeting components of the interferon (IFN) signaling pathway. The V protein, expressed by most paramyxoviruses, is a well-characterized IFN signaling antagonist. Until now, there were no reports on the role of the LPMV-V protein in inhibiting the IFN response. In this study we demonstrate that LPMV-V protein antagonizes type I but not type II IFN signaling by binding STAT2, a component of the type I IFN cascade. Our results indicate that the last 18 amino acids of LPMV-V protein are required for binding to STAT2 in human and swine cells. While LPMV-V protein does not affect the protein levels of STAT1 or STAT2, it does prevent the IFN-induced phosphorylation and nuclear translocation of STAT1 and STAT2 thereby inhibiting cellular responses to IFN α/ß.

Serological survey of veterinarians to assess the zoonotic potential of three emerging swine diseases in Mexico.

We conducted an immunological assay of blood samples taken from 85 swine-specialist veterinarians attending the Congress of the Mexican Association of Swine Specialist Veterinarians in Mexico in 2011. Serum samples were assayed for Porcine rubulavirus (PorPV), Encephalomyocarditis virus (EMCV) and Leptospira spp. antibodies. Using a hemagglutination inhibition test, we registered 2.3% and 27% seropositivity for PorPV and EMCV, respectively. Using viral neutralization tests, we registered 5.8% and 47% seropositivity for PorPV and EMCV, respectively. For Leptospira spp., we registered a seropositivity of 38.8%. The variables (sex, age, years of exposure, number of visited farms, biosecurity level and region) showed no significant effect (P > 0.05) on the seropositivity for EMCV, PorPV and Leptospira spp. except for number of visited farms on HI seropositivity for EMCV (P < 0.05; odds ratio: 1.38). The data obtained provide information on the epidemiology of emerging diseases with zoonotic potential in occupational risk groups.

Efficacy of parainfluenza virus 5 mutants expressing hemagglutinin from H5N1 influenza A virus in mice.

Parainfluenza virus 5 (PIV5) is a promising viral vector for vaccine development. PIV5 is safe, stable, efficacious, cost-effective to produce and, most interestingly, it overcomes preexisting antivector immunity. We have recently reported that PIV5 expressing the hemagglutinin (HA) from highly pathogenic avian influenza (HPAI) virus H5N1 (PIV5-H5) provides sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. It is thought that induction of apoptosis can lead to enhanced antigen presentation. Previously, we have shown that deleting the SH gene and the conserved C terminus of the V gene in PIV5 results in mutant viruses (PIV5ΔSH and PIV5VΔC) that enhance induction of apoptosis. In this study, we inserted the HA gene of H5N1 into PIV5ΔSH (PIV5ΔSH-H5) or PIV5VΔC (PIV5VΔC-H5) and compared their efficacies as vaccine candidates to PIV5-H5. We have found that PIV5ΔSH-H5 induced the highest levels of anti-HA antibodies, the strongest T cell responses, and the best protection against an H5N1 lethal challenge in mice. These results suggest that PIV5ΔSH is a better vaccine vector than wild-type PIV5.

Genetic and antigenic changes in porcine rubulavirus.

Blue eye disease, caused by a porcine rubulavirus (PoRV), is an emergent viral swine disease that has been endemic in Mexico since 1980. Atypical outbreaks were detected in 1990 and 2003. Growing and adult pigs presented neurological signs, mild neurological signs were observed in piglets, and severe reproductive problems were observed in adults. Amino acid sequence comparisons and phylogenetic analysis of the hemagglutinin-neuraminidase (HN) protein revealed genetically different lineages. We used cross-neutralization assays, with homologous and heterologous antisera, to determine the antigenic relatedness values for the PoRV isolates. We found antigenic changes among several strains and identified a highly divergent one, making up a new serogroup. It seems that genetically and antigenically different PoRV strains are circulating simultaneously in the swine population in the geographical region studied. The cross neutralization studies suggest that the HN is not the only antigenic determinant participating in the antigenic changes among the different PoRV strains.

Identification of antigenic variants of the porcine rubulavirus in sera of field swine and their seroprevalence.

We sampled sera from 1013 non-vaccinated swine from four states in Mexico, Guanajuato, Jalisco, Michoacán and the Estado de Mexico, to analyse anti-porcine rubulavirus antibody titres against three different porcine rubulavirus isolates (PAC-4/1993, PAC-6/2001, and PAC-9/2003) using a hemagglutination inhibition assay. The results revealed that there were antigenic differences among the isolates assessed. In particular, the estimated correlation between the PAC-4/1993 and PAC-6/2001 (0.50) isolates and between the PAC-4/1993 and PAC-9/2003 isolates (0.56) displayed a moderate positive correlation. In contrast, there was a strong positive correlation between the PAC-6/2001 and PAC-9/2003 isolates (0.73). We also found that in the state of Guanajuato, PAC-4/1993 was the isolate that was most frequently identified; in Jalisco, the isolate was PAC-6/2001; and in Michoacán, the isolate was PAC-9/2003. By contrast, in the Estado de Mexico, all three isolates appeared to circulate with a low seroprevalence. In general, the analysed sera from the four states displayed a porcine rubulavirus serological prevalence ranging from 9% to 23.7%. These data indicate that there is not complete antibody cross-antigenicity among the three isolates, and the antigenic variations in the antibody response found in this study implies that the use of a monovalent vaccine would not generate complete protection against the different antigenic subtypes.

Mumps outbreaks in vaccinated populations: are available mumps vaccines effective enough to prevent outbreaks?

Increased reports of mumps in vaccinated populations prompted a review of the performance of mumps vaccines. The effectiveness of prior vaccination with 1 dose of vaccine ranged from 72.8% to 91% for the Jeryl Lynn strain, from 54.4% to 93% for the Urabe strain, and from 0% to 33% for the Rubini strain. Vaccine effectiveness after 2 doses of mumps vaccine was reported in 3 outbreaks and ranged from 91% to 94.6%. There was evidence of waning immunity, which is a likely factor in mumps outbreaks, aggravated by possible antigenic differences between the vaccine strain and outbreak strains. Inadequate vaccine coverage or use of the Rubini vaccine strain accounted for the majority of outbreaks reviewed; however, some outbreaks could not be prevented, despite high vaccination coverage with 2 doses of the Jeryl Lynn vaccine strain. Our findings indicate the need for more-effective mumps vaccines and/or for review of current vaccination policies to prevent future outbreaks.

Identification of potential B cell epitope determinants by computer techniques, in hemagglutinin-neuraminidase from the porcine rubulavirus La Piedad Michoacan.

Hemagglutinin-neuraminidase (HN) from porcine rubulavirus La Piedad Michoacan (RvpLPM) is one of the most antigenic proteins known, and is responsible for virus-host cell interaction. We analyzed the amino acid sequence of HN, using computer-assisted techniques to identify B cell epitopes. From a pool of 18 possible antigenic peptides, we evaluated the antigenicity of the 2 peptides with the highest scores and the 1 with lowest score. Antibodies from RvpLPM-infected pigs recognized the synthesized HN-A, HN-B, and HN-R peptides (optical density [OD]: 0.33 +/- 0.02 for HN-A, 0.20 +/- 0.02 for HN-B, and 0.07 +/- 0.01 for HN-R); bovine serum albumin-coupled HN-A and HN-B induced rabbit anti-RvpLPM antibodies (OD: 0.39 +/- 0.01 for HN-A and 0.35 +/- 0.02 for HN-B). Loop 5 from the outer membrane protein, OmpC, from Salmonella typhi was replaced with HN-B; this protein was then expressed in Escherichia coli UH302. BALB/c mice were challenged intraperitoneally or orogastrically with the fusion protein expressed in E. coli and murine antibodies obtained from both types of administration inhibited virus-hemagglutinating activity, as did the antibodies from RvpLPM-infected swine. These results suggest that HN-A and HN-B are peptides involved in RvpLPM cell carbohydrate recognition, and could therefore be considered potential targets for vaccine and diagnostic procedures development.

Mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading STATs.

Mapuera virus (MPRV) is a paramyxovirus that was originally isolated from bats, but its host range remains unknown. It was classified as a member of the genus Rubulavirus on the basis of structural and genetic features. Like other rubulaviruses it encodes a V protein (MPRV/V) that functions as an interferon (IFN) antagonist. Here we show that MPRV/V differs from the IFN antagonists of other rubulaviruses in that it does not induce the proteasomal degradation of STAT proteins, key factors in the IFN signalling cascade. Rather, MPRV/V prevents the nuclear translocation of STATs in response to IFN stimulation and inhibits the formation of the transcription factor complex ISGF3. We also show that MPRV/V blocks IFN signalling in cells from diverse mammalian species and discuss the IFN response as a barrier to cross-species infections.

Interferon-induced inhibition of parainfluenza virus type 5; the roles of MxA, PKR and oligo A synthetase/RNase L.

We have previously reported that the addition of interferon (IFN) to the culture medium of Vero cells (which cannot produce IFN) that were infected with the CPI- strain of parainfluenza virus 5 (PIV5, formally known as SV5), that fails to block IFN signaling, rapidly induces alterations in the relative levels of virus mRNA and protein synthesis. In addition, IFN treatment also caused a rapid redistribution of virus proteins and enhanced the formation of cytoplasmic viral inclusion bodies. The most studied IFN-induced genes with known anti-viral activity are MxA, PKR and the Oligo A synthetase/RNase L system. We therefore examined the effects of these proteins on the replication cycle of PIV5. These studies revealed that while these proteins had some anti-viral activity against PIV5 they were not primarily responsible for the very rapid alteration in virus protein synthesis observed following IFN treatment, nor for the IFN-induced formation of virus inclusion bodies, in CPI- infected cells.

Declining population immunity to mumps among Israeli military recruits.

Population-based seroepidemologic data on mumps have not been available in Israel since 1987, and the effects of mass immigration from the Commonwealth of Independent States during the 1990s have not been investigated. We conducted a seroprevalence study of mumps antibodies among 353 Israeli military recruits aged 18-19, based on a representative sample of sera collected in 1999. The overall seroprevalence rate was 83.3%, which was significantly lower than that measured in 1987 (94.1%, P<0.001). Foreign-born subjects had substantially lower seroporevalence rates than their native Israeli counterparts (68.5% versus 86.1%, P<0.001). Recent seroprevalence levels are below those required for herd immunity, and most likely contributed to an outbreak of mumps observed among young adults in Israel in 2005. Immigration appears to be a contributing factor to the decrease in population seroprevalence over time.

[A virological description of serous meningitis in children immunized with vaccine against epidemic parotitis]. / Virusologicheskaia kharakteristika seroznykh meningitov u detei, immunizirovannykh vaktsinoi protiv épidemicheskogo parotita.

The morbidity structure was analyzed in children vaccinated against epidemic parotitis in 1993-2002. Eight children (4 with serous meningitis and 4 with lesions of the salivary glands) underwent virologic and immunologic examinations. The molecular typing of the SH-gene fragment of the parotitis virus showed the process in 7 cases to be provoked by the vaccination strain. Presumedly, progressing vaccine-associated meningitis inhibits antibody formation. The total incidence of vaccine-associated meningitis was shown, according to Saint Petersburg data, to be not high, which testifies to a low reactogenicity of the Russian vaccine strain.

Prevalence of rabies and LPM paramyxovirus antibody in non-hematophagous bats captured in the Central Pacific coast of Mexico.

To investigate if non-hematophagous bats play a role in outbreaks of rabies and blue eye disease (LPMV), we studied the seroprevalence against both agents in several species of non-hematophagous bats on the sub-tropical Pacific coast of the state of Colima, Mexico. The survey covered a predominantly agricultural area (disturbed), and an area dominated by semideciduous dry forest (undisturbed). A total of 151 non-hematophagous bats of 16 species were captured from the two areas. Fifty-six (37%) had antirabic antibodies (Ab) while 87 (58%) did not and 8 samples (5%) had to be discarded because of hemolysis. A much lower (P<0.05) prevalence of antirabic Ab was found in bats caught in disturbed areas (22.7%) compared with those from undisturbed areas (51.9%). The presence of antirabic Ab was not related to sex, genera or feeding habits. The higher prevalence found in bats in the undisturbed area may be the result of more frequent interspecies encounters. Of the 108 sera analyzed for antibodies against LPMV, only one was positive (a male Rhogeessa parvula major, captured in the undisturbed area). This suggests that bats in the surveyed localities do not play a role in the epidemiology of LPMV.

Apoptosis in lymph nodes and changes in lymphocyte subpopulations in peripheral blood of pigs infected with porcine rubulavirus.

In a first experiment, five pigs were inoculated intranasally with porcine rubulavirus (PoRV) at 5 days of age and killed 7 days post-infection (pi). In a second experiment, four pigs were infected with the same virus at 17 days of age and killed at 9 or 15 days pi. Control piglets in each experiment received uninfected cell culture supernate. All PoRV-infected pigs developed respiratory and nervous signs, and histological lesions of non-suppurative encephalitis and interstitial pneumonia. All control pigs remained clinically normal and did not have histological lesions. Significantly increased numbers of apoptotic cells were detected by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) in tonsil and lymph nodes of the pigs infected at 7 days of age and killed at 7 days pi. Significantly increased percentages of CD2(+) and CD8(+) T lymphocytes were also found in peripheral blood of these animals at this time, while the percentages of CD4(+) and MHC class II lymphocytes were significantly reduced. Significantly increased numbers of apoptotic cells were detected in lymphoid tissues of the pigs infected at 17 days of age and killed at 9 days pi. The percentages of CD2(+), CD8(+) and MHC class II lymphocytes in peripheral blood were also significantly increased at this time; the percentage of MHC class II lymphocytes remained elevated at 15 days pi. These results indicate that induction of apoptosis is an important mechanism in the pathogenesis of PoRV infection in young pigs, and that this virus induces changes in lymphocyte subpopulations in peripheral blood.

Uptake of porcine rubulavirus (LPMV) by PK-15 cells.

BACKGROUND: The porcine virus denominated La Piedad Michoacan Virus (LPMV) is a member of the family Paramyxoviridae and is the cause of a disease in pigs present only in Mexico. The disease is characterized by meningoencephalitis and respiratory distress in young pigs, epididymitis and orchitis in boars, and reproductive failure and abortion in sows. METHODS: The cytopathology, morphology, and distribution of the hemagglutination neuraminidase (HN) and nucleoprotein (NP) proteins of LPMV were investigated following inoculation into PK-15 cells. The cytopathic effect was characterized by cytoplasmic vacuolation and the formation of syncytia and cytoplasmic inclusion bodies. RESULTS: In immunofluorescence assays using a monoclonal antibody (MAb) against the HN protein at 5-60 min post-infection (early infection), a diffuse immunofluorescence was observed near the cell membrane and adjacent to the nuclear membrane. At 24 h post-infection (late infection), a dust-like immunofluorescence was observed throughout the cytoplasm. LPMV-infected cells incubated with the MAb against the NP protein showed punctate cytoplasmic fluorescence during the early stages of infection. At the late infection stage, these fluorescent particles became larger and were seen predominantly in the cytoplasm of syncytia. This pattern was also apparent by immunohistochemical labeling and immunogold electron microscopy. The latter technique revealed that HN protein was diffusely distributed throughout the cytoplasm. When using the MAb against the NP protein, nucleocapsid organization was the most prominent feature and resulted in the formation of cytoplasmic inclusion bodies visible by light and electron microscopy. Immunogold labeling of purified nucleocapsids was shown by electron microscopy. Virus particles and nucleocapsids were morphologically similar to members of the Paramyxoviridae family. CONCLUSIONS: The morphologic characteristics of the virions and the distribution patterns of the HN and NP proteins in PK-15 infected cells indicate that the mechanisms of LPMV replication are generally similar to those of the members of the Paramyxoviridae family.