J. Moreno-López

The structural proteins of a porcine paramyxovirus (LPMV).

The porcine paramyxovirus is a newly identified agent of a fatal disease in piglets, endemic in Mexico since 1980, where it was seen around the town of La Piedad, Michoacan, Mexico (hence LPM virus). At least six [35S]methionine-labelled proteins could be resolved by SDS-PAGE and five of them were clearly immunoprecipitated. Selective labelling of LPMV-infected cells with [3H]glucosamine revealed two bands with an Mr of about 66K and 59K, corresponding to the two viral glycoproteins, the haemagglutinin-neuraminidase protein and the fusion protein. Labelling of virus with [32P]orthophosphate disclosed one band with an Mr of 52K, corresponding to the phosphoprotein. Analysis of nucleocapsids obtained from purified virus or from a permanently infected cell line revealed one major band with an Mr of 68K, the nucleoprotein. Two other proteins were also identified, the large protein and the matrix protein, with apparent Mr of about 200K and 40K, respectively. The protein migration pattern of LPMV was compared, by SDS-PAGE, with that of Newcastle disease virus, bovine parainfluenza 3 virus and Sendai virus. Differences in the Mr of LPMV proteins and the proteins of these paramyxoviruses were observed. We propose that LPMV should be classified as a novel member of the genus Paramyxovirus.

The P gene of the porcine paramyxovirus LPMV encodes three possible polypeptides P, V and C: the P protein mRNA is edited.

The nucleotide sequence of the P gene of the porcine paramyxovirus La-Piedad-Michoacan-Mexico virus (LPMV) was analysed. Three long open reading frames (ORFs) were found in the mRNA sense. Insertion of two G residues is necessary to obtain an ORF encoding the P protein, which gives a P protein of 404 amino acids with a calculated Mr of 42475. This form of editing was demonstrated, two non-templated G residues being added in a portion of the mRNA transcripts. The LPMV V protein, which has a conserved cysteine-rich C-terminal region, is encoded by an exact copy of the P gene. The third ORF has the capacity to encode a protein of 126 amino acids, which may resemble the C proteins found in some paramyxoviruses. The ORF starts from an AUG codon down-stream of the first AUG codon of the P/V ORF.

The haemagglutinin-neuraminidase glycoprotein of the porcine paramyxovirus LPMV: comparison with other paramyxoviruses revealed the closest relationship to simian virus 5 and mumps virus

SummaryThe complete nucleotide sequence of the haemagglutinin-neuraminidase (HN) gene of the porcine paramyxovirus LPMV, was determined from cDNA derived from viral genomic RNA. The gene was 1906 nucleotides long including a putative gene end and poly A signal. One long open reading frame was found encoding a protein of 576 amino acids with a calculated molecular weight of 63,324. The protein contains four potential N-glycosylation sites and a major hydrophobic region near the N-terminal, suggesting a membrane anchor domain. Comparison of the deduced amino acid sequence of the LPMV HN protein with that of other paramyxovirus HN proteins, revealed the highest amino acid identity to simian virus 5 of 43% and mumps virus of 41%.

Analysis of the large (L) protein gene of the procine rubulavirus LPMV: identification of possible functional domains

The complete nucleotide sequence of the porcine rubulavirus LPMV (La Piedad Michoacan virus) large (L) protein gene was determined and analysed. The L mRNA was found to span 6,786 nucleotides, containing one single large open reading frame (ORF), putatively encoding a polypeptide of 2,251 amino acids. By aligning the amino acid sequence of the LPMV L-protein with L-protein of a number of viruses belonging to the order mononegavirale, a high degree of similarity between the LPMV L-protein and other rubula virus L-proteins was demonstrated, extending through almost the whole protein. Additionally we could identify several regions as being highly conserved among all studied viruses of the order mononegavirale. The significance of these regions are discussed.

The molecular biology of the porcine paramyxovirus LPMV

Protein and genomic studies of a previously uncharacterized porcine paramyxovirus (designated LPMV) confirmed that it was a member of the paramyxovirus genus. The nucleotide sequences and deduced amino acids of the complete P-gene, M-gene, F-gene and HN-gene as well as the intergenic sequences have been determined. Comparative sequence analysis of the M-gene of LPMV revealed the closest relationship of LPMV was to human mumps virus with a homology of 46% and 55% at the amino acid and nucleotide levels respectively. The P-gene of LPMV is transcribed to V protein mRNA and by editing of the gene to the P protein mRNA. The LPMV P-gene has the coding capacity for an additional protein of 126 amino acids, a C protein.

Porcine Rubulavirus Lpmv Rna Persists in the Central Nervous System of Pigs After Recovery from Acute Infection

In order to study persistence of the porcine rubulavirus LPMV, we examined tissue samples collected from pigs 53 days after experimental infection. These pigs survived the initial infection and could clinically be considered to have recovered from the infection. Two of the pigs used in this study were chemically immunosuppressed during the last 4 days before necropsy. No infectious virus or viral antigen could be detected in any tissue using standard methods for virus isolation and detection. However, the presence of viral genomic RNA and mRNA could be demonstrated in the mid brain of the convalescent pig using an optimised RT-nested PCR. Mid brain, forebrain and lung were all shown to contain LPMV RNA in the immunosuppressed convalescent pigs. In addition we examined the P-gene editing in the recovered pigs and conclude that the viral genome is transcriptionally active in these pigs. The relevance of the persistence of LPMV for maintenance and spread within and/or between pig populations is discussed.

Analysis of the Fusion Protein Gene of the Porcine Rubulavirus LPMV: Comparative Analysis of Paramyxovirus F Proteins

Complementary DNA clones representing the fusion (F) protein gene of the porcine rubulavirus LPMV were isolated and sequenced. The F gene was found to be 1,845 nucleotides long containing one long open reading frame capable of encoding a protein of 541 amino acids. The cleavage motif for F0 into F1 and F2 is His-Arg-Lys-Lys-Arg. A sequence comparison and a phylogenetic analysis was performed in order to identify possible functional domains of paramyxovirus fusion proteins and also to classify the porcine rubulavirus. The F gene of LPMV is most closely related to the human mumps virus and simian virus type 5 F genes, and is therefore classified into the rubulavirus genus. A coding region for a small hydrophobic protein was however not found between the F and hemagglutinin-neuraminidase (HN) genes as previously found in both SV5 and mumps.

Full-length genome sequence and genetic relationship of two paramyxoviruses isolated from bat and pigs in the Americas

SummaryMapuera virus (MPRV) was isolated from a fruit bat in Brazil in 1979, but its host range and disease-causing potential are unknown. Porcine rubulavirus (PoRV) was identified as the aetiological agent of disease outbreaks in pigs in Mexico during early 1980s, but the origin of PoRV remains elusive. In this study, the completed genome sequence of MPRV was determined, and the complete genome sequence of PoRV was assembled from previously published protein-coding genes and the non-coding genome regions determined from this study. Comparison of sequence and genome organization indicated that PoRV is more closely related to MPRV than to any other members of the genus Rubulavirus. In the P gene coding region of both viruses, there is an ORF located at the 5′ end of the P gene overlapping with the P protein coding region, similar to the C protein ORF present in most viruses of the subfamily Paramyxovirinae, but absent in other known rubulaviruses. Based on these findings, we hypothesise that PoRV may also originate from bats, and spillover events from bats to pigs, either directly or via an intermediate host, were responsible for the sporadic disease outbreaks observed in Mexico.

Organization and expression of the transforming region from the European elk papillomavirus (EEPV).

The nucleotide sequence of the early (transforming) region from the European elk papillomavirus (EEPV) double-stranded DNA has been determined together with flanking regions. The established sequence, which is 5732 bp long, shows that the genome of EEPV is closely related to the previously sequenced bovine papillomavirus type 1 (BPV-1) and deer papillomavirus (DPV) genomes. Seven open reading frames (ORFs), designated E1-E7, were identified in similar positions as in the BPV-1 genome. The E1 and E5 regions were best conserved. The strong homology between the E5 ORFs of EEPV, BPV-1 and DPV is interesting in the light of the recent proposal that these ORFs encode a major transforming function (Schiller et al., 1986; DiMaio et al., 1986). A set of mRNAs, comprising six size classes, were identified in EEPV-transformed cells. At least two different promoters appear to control EEPV transcription in transformed cells.

Experimental porcine rubulavirus (La Piedad-Michoacan virus) infection in pregnant gilts.

Porcine rubulavirus (La Piedad-Michoacan virus) (PoRV-LPMV) is a member of the Paramyxoviridae family that causes encephalitis in young piglets and infertility in adult sows and boars. Infertility in sows naturally infected by PoRV-LPMV is characterized by an increased number of returns to oestrus, stillbirths and mummified fetuses. In this study, nine seronegative gilts were inoculated intranasally with the PAC-3 strain of PoRV-LPMV at week 6 or 10 of gestation. These animals were then killed at weeks 8 or 15 of gestation (seven gilts) or after natural parturition (two gilts). Four control gilts were mock-infected at gestation week 6 or 10 and killed between 2 and 4 weeks later. Gross lesions of focal congestion and haemorrhage were seen in the placenta and endometrium of one gilt infected at gestation week 6 and one infected at gestation week 10. PoRV-LPMV was isolated, at 2-6 weeks post-inoculation (pi), from lung, tonsils, ovary, placenta, uterus and lymph nodes of three of the gilts infected at gestation week 6 and at 2-3 weeks pi from lung, tonsil and ovary of two gilts infected at gestation week 10. Many of the fetuses of eight infected gilts were smaller than normal and had dermal ecchymoses. Dehydrated or mummified fetuses were present in six of the infected gilts but not in any control animal. PoRV-LPMV was isolated from brain, lung and liver of fetuses from two gilts infected at gestation week 6, and from two infected at gestation week 10. These results indicate that, after experimental infection, PoRV can replicate in tissues of seronegative pregnant gilts, cross the placenta, and cause fetal death and mummification.