Human parainfluenza virus type 2 polymerase complex recognizes leader promoters of other species belonging to the genus Rubulavirus.

Leader sequence, located at the 3' terminus of paramyxovirus genomes, determines the degree of viral transcription and replication. The essential nucleotides in the leader sequence that influence viral propagation, however, have not been investigated in detail. In the present study, we show that polymerase complex of human parainfluenza virus type 2 (hPIV2) uses a luciferase-encoding hPIV2 mini-genome possessing the leader sequence from other closely related viruses as a template. Furthermore, we demonstrate that although hPIV2 polymerase complex can recognize the leader sequence of hPIV4B, mumps virus (MuV) and PIV5 as well as Newcastle disease virus (NDV), it cannot recognize measles virus, hPIV1, Sendai virus (SeV) or hPIV3. We could obtain the chimeric hPIV2 possessing the leader sequence from hPIV4B, MuV and PIV5, but not from other species, including NDV and SeV. These results reveal that although hPIV2 polymerase complex can recognize the leader sequence from rubulaviruses to achieve efficient viral infection, this does not apply to viruses belonging to other genus. A comparison of leader sequence nucleotides among paramyxoviruses highlights the importance of the conservation in the first 13 nucleotides for infectious hPIV2 growth.

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.

Full conversion of the hemagglutinin-neuraminidase specificity of the parainfluenza virus 5 fusion protein by replacement of 21 amino acids in its head region with those of the simian virus 41 fusion protein.

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.

The paramyxovirus fusion protein C-terminal region: mutagenesis indicates an indivisible protein unit.

Paramyxoviruses enter host cells by fusing the viral envelope with a host cell membrane. Fusion is mediated by the viral fusion (F) protein, and it undergoes large irreversible conformational changes to cause membrane merger. The C terminus of PIV5 F contains a membrane-proximal 7-residue external region (MPER), followed by the transmembrane (TM) domain and a 20-residue cytoplasmic tail. To study the sequence requirements of the F protein C terminus for fusion, we constructed chimeras containing the ectodomain of parainfluenza virus 5 F (PIV5 F) and either the MPER, the TM domain, or the cytoplasmic tail of the F proteins of the paramyxoviruses measles virus, mumps virus, Newcastle disease virus, human parainfluenza virus 3, and Nipah virus. The chimeras were expressed, and their ability to cause cell fusion was analyzed. The chimeric proteins were variably expressed at the cell surface. We found that chimeras containing the ectodomain of PIV5 F with the C terminus of other paramyxoviruses were unable to cause cell fusion. Fusion could be restored by decreasing the activation energy of refolding through introduction of a destabilizing mutation (S443P). Replacing individual regions, singly or doubly, in the chimeras with native PIV5 F sequences restored fusion to various degrees, but it did not have an additive effect in restoring activity. Thus, the F protein C terminus may be a specific structure that only functions with its cognate ectodomain. Alanine scanning mutagenesis of MPER indicates that it has a regulatory role in fusion since both hyperfusogenic and hypofusogenic mutations were found.

Sumoylation of the P protein at K254 plays an important role in growth of parainfluenza virus 5.

The P protein of parainfluenza virus 5 (PIV5) is an essential cofactor of the viral RNA-dependent RNA polymerase. Phosphorylation of the P protein can positively or negatively regulate viral gene expression, depending on the precise phosphorylation sites. Sumoylation, a process of adding small ubiquitin-like modifier (SUMO) to proteins posttranslationally, plays an important role in regulating protein function. In this study, we have found that the P protein of PIV5 was sumoylated with SUMO1 in both transfected and infected cells. The K254 residue of the P protein is within a consensus sumoylation motif. Mutation of the P protein at K254 to arginine (P-K254R) reduced PIV5 minigenome activity, as well as the sumoylation level of the P protein. Incorporation of K254R into a recombinant PIV5 (rPIV5-P-K254R) resulted in a virus that grew to a lower titer and had lower levels of viral RNA synthesis and protein expression than wild-type PIV5, suggesting that sumoylation of the P protein at K254 is important for PIV5 growth. Biochemical studies did not reveal any defect of P-K254R in its interactions with viral proteins NP and L or formation of homotetramers. We propose that sumoylation of the P protein at K254 regulates PIV5 gene expression through a host protein.

Co-Circulation and Excretion Dynamics of Diverse Rubula- and Related Viruses in Egyptian Rousette Bats from South Africa.

The Egyptian rousette bat (Rousettus aegyptiacus) has previously been implicated as the natural host of a zoonotic rubulavirus; however, its association with rubulaviruses has been studied to a limited extent. Urine, spleen, and other organs collected from the R. aegyptiacus population within South Africa were tested with a hemi-nested RT-PCR assay targeting a partial polymerase gene region of viruses from the Avula- and Rubulavirus genera. Urine was collected over a 14-month period to study the temporal dynamics of viral excretion. Diverse rubulaviruses, including viruses related to human mumps and parainfluenza virus 2, were detected. Active excretion was identified during two peak periods coinciding with the host reproductive cycle. Analysis of additional organs indicated co-infection of individual bats with a number of different putative rubulaviruses, highlighting the limitations of using a single sample type when determining viral presence and diversity. Our findings suggest that R. aegyptiacus can harbor a range of Rubula- and related viruses, some of which are related to known human pathogens. The observed peaks in viral excretion represents potential periods of a higher risk of virus transmission and zoonotic disease spill-over.

Blue eye disease porcine rubulavirus (PoRv) infects pig neurons and glial cells using sialo-glycoprotein as receptor.

Pig neural cells express glycoproteins with sialylated N-linked oligosaccharide chains (SNOC) which are used by the porcine rubulavirus (PoRv) as receptors. Pig neuronal or glial cell cultures were employed to investigate (a) whether PoRv infects such cells using a molecule expressing SNOC, and (b) the role of viral envelope glycoproteins in establishing the infection. Enriched neuronal or glial cell cultures were exposed to PoRv and infection was detected immunocytochemically. Neuronal cultures prepared from neonatal pigs were treated enzymatically to eliminate sialic acid or N-linked oligosaccharide chains. Primary neural cultures were exposed to anti-HN or anti-F preincubated with PoRv to study the role of the viral glycoproteins. In enriched cultures, PoRv infected neurons and glial cells, and sialic acid expressed in N-linked oligosaccharide chains appeared to play a central role in infection. It was concluded that HN and F viral glycoproteins are required to infect neurons and glial cells.

Co-infection of classic swine H1N1 influenza virus in pigs persistently infected with porcine rubulavirus.

Porcine rubulavirus (PorPV) and swine influenza virus infection causes respiratory disease in pigs. PorPV persistent infection could facilitate the establishment of secondary infections. The aim of this study was to analyse the pathogenicity of classic swine H1N1 influenza virus (swH1N1) in growing pigs persistently infected with porcine rubulavirus. Conventional six-week-old pigs were intranasally inoculated with PorPV, swH1N1, or PorPV/swH1N1. A mock-infected group was included. The co-infection with swH1N1 was at 44 days post-infection (DPI), right after clinical signs of PorPV infection had stopped. The pigs of the co-infection group presented an increase of clinical signs compared to the simple infection groups. In all infected groups, the most recurrent lung lesion was hyperplasia of the bronchiolar-associated lymphoid tissue and interstitial pneumonia. By means of immunohistochemical evaluation it was possible to demonstrate the presence of the two viral agents infecting simultaneously the bronchiolar epithelium. Viral excretion of PorPV in nasal and oral fluid was recorded at 28 and 52 DPI, respectively. PorPV persisted in several samples from respiratory tissues (RT), secondary lymphoid organs (SLO), and bronchoalveolar lavage fluid (BALF). For swH1N1, the viral excretion in nasal fluids was significantly higher in single-infected swH1N1 pigs than in the co-infected group. However, the co-infection group exhibited an increase in the presence of swH1N1 in RT, SLO, and BALF at two days after co-infection. In conclusion, the results obtained confirm an increase in the clinical signs of infection, and PorPV was observed to impact the spread of swH1N1 in analysed tissues in the early stage of co-infection, although viral shedding was not enhanced. In the present study, the interaction of swH1N1 infection is demonstrated in pigs persistently infected with PorPV.

Accessory genes of the paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, as a focus of active investigation by reverse genetics.

The Paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, comprises several genera each containing important human and animal pathogens. They possess in common six basal genes essential for viral replication and, in addition, a subset of accessory genes that are largely unique to each genus. These accessory genes are either encoded in one or more alternative overlapping frames of a basal gene, which are accessed transcriptionally or translationally, or inserted before or between the basal genes as one or more extra genes. However, the question of how the individual accessory genes contribute to actual viral replication and pathogenesis remained unanswered. It was not even established whether they are dispensable or indispensable for the viral life cycle. The plasmid-based reverse genetics of the full-length viral genome has now come into wide use to demonstrate that most, if not all, of these putative accessory genes can be disrupted without destroying viral infectivity, conclusively defining them as indeed dispensable accessory genes. Studies on the phenotypes of the resulting gene knockout viruses have revealed that the individual accessory genes greatly contribute specifically and additively to the overall viral fitness both in vitro and in vivo.

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.

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.

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.

Persistence of porcine rubulavirus in experimentally infected boars.

Porcine rubulavirus is the etiological agent of blue eye disease in pigs. In boars, this virus causes orchitis and epididymitis and reduces seminal quality. The objective of this study was to determine the persistence of porcine rubulavirus in experimentally infected boars. Nine 12-month-old boars were infected with 5 ml of the PAC-3 strain of porcine rubulavirus at 1 × 10(5) TCID(50)/ml and held for 142 days post infection (DPI) to evaluate humoral immune response. The virus was isolated in cell cultures and detected by RT-PCR. Infection with porcine rubulavirus produced clinical signs beginning at 5 DPI. Necropsy results showed that 3 boars had lesions in the testicles and epididymes. Histological analysis showed the characteristic lesions in all infected boars. Porcine rubulavirus antibodies were detected in the second week post infection and increased significantly (P<0.05) over time. Isolation of the virus from semen was achieved between 5 DPI and 48 DPI and from the testicles and epididymes between 64 DPI and 142 DPI. Viral RNA was detected in the serum between 2 DPI and 64 DPI and in the semen until 142 DPI. These results confirm that the RNA of the porcine rubulavirus persists in the semen and that this virus remains in the reproductive tract for prolonged periods of infection. Semen of persistently infected boars, therefore, represents an important source of the virus and a risk factor for the spread of blue eye disease in swine populations.

The V protein of Tioman virus is incapable of blocking type I interferon signaling in human cells.

The capacity of a virus to cross species barriers is determined by the development of bona fide interactions with cellular components of new hosts, and in particular its ability to block IFN-α/ß antiviral signaling. Tioman virus (TioV), a close relative of mumps virus (MuV), has been isolated in giant fruit bats in Southeast Asia. Nipah and Hendra viruses, which are present in the same bat colonies, are highly pathogenic in human. Despite serological evidences of close contacts between TioV and human populations, whether TioV is associated to some human pathology remains undetermined. Here we show that in contrast to the V protein of MuV, the V protein of TioV (TioV-V) hardly interacts with human STAT2, does not degrade STAT1, and cannot block IFN-α/ß signaling in human cells. In contrast, TioV-V properly binds to human STAT3 and MDA5, and thus interferes with IL-6 signaling and IFN-ß promoter induction in human cells. Because STAT2 binding was previously identified as a host restriction factor for some Paramyxoviridae, we established STAT2 sequence from giant fruit bats, and binding to TioV-V was tested. Surprisingly, TioV-V interaction with STAT2 from giant fruit bats is also extremely weak and barely detectable. Altogether, our observations question the capacity of TioV to appropriately control IFN-α/ß signaling in both human and giant fruit bats that are considered as its natural host.

Comparison of differing cytopathic effects in human airway epithelium of parainfluenza virus 5 (W3A), parainfluenza virus type 3, and respiratory syncytial virus.

Parainfluenza virus 5 (PIV5) infects a wide range of animals including dogs, pigs, cats, and humans; however, its association with disease in humans remains controversial. In contrast to parainfluenza virus 3 (PIV3) or respiratory syncytial virus (RSV), PIV5 is remarkably non-cytopathic in monolayer cultures of immortalized epithelial cells. To compare the cytopathology produced by these viruses in a relevant human tissue, we infected an in vitro model of human ciliated airway epithelium and measured outcomes of cytopathology. PIV5, PIV3 and, RSV all infected ciliated cells, and PIV5 and PIV3 infection was dependent on sialic acid residues. Only PIV5-infected cells formed syncytia. PIV5 infection resulted in a more rapid loss of infected cells by shedding of infected cells into the lumen. These studies revealed striking differences in cytopathology of PIV5 versus PIV3 or RSV and indicate the extent of cytopathology determined in cell-lines does not predict events in differentiated airway cells.

Failure to activate the IFN-ß promoter by a paramyxovirus lacking an interferon antagonist.

It is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (IFN) induction are produced during virus replication and, to limit the activation of the IFN response by these PAMPs, viruses encode antagonists of IFN induction. Here we have studied the induction of IFN by parainfluenza virus type 5 (PIV5) at the single-cell level, using a cell line expressing GFP under the control of the IFN-ß promoter. We demonstrate that a recombinant PIV5 (termed PIV5-VΔC) that lacks a functional V protein (the viral IFN antagonist) does not activate the IFN-ß promoter in the majority of infected cells. We conclude that viral PAMPs capable of activating the IFN induction cascade are not produced or exposed during the normal replication cycle of PIV5, and suggest instead that defective viruses are primarily responsible for inducing IFN during PIV5 infection in this system.

Synthesis and biological evaluation of analogues of AKT (protein kinase B) inhibitor-IV.

Inhibitors of the PI3-kinase/AKT (protein kinase B) pathway are under investigation as anticancer and antiviral agents. The benzimidazole derivative AKT inhibitor-IV (ChemBridge 5233705) affects this pathway and exhibits potent anticancer and antiviral activity. To probe its biological activity, we synthesized AKT inhibitor-IV and 21 analogues using a novel six-step route based on ZrCl(4)-catalyzed cyclization of 1,2-arylenediamines with α,ß-unsaturated aldehydes. We examined effects on viability of HeLa carcinoma cells, viability of normal human cells (NHBE), replication of recombinant parainfluenza virus 5 (PIV5) in HeLa cells, and replication of the intracellular bacterium Mycobacterium fortuitum in HeLa cells. Replacement of the benzimidazole N-ethyl substitutent of AKT inhibitor-IV with N-hexyl and N-dodecyl groups enhanced antiviral activity and cytotoxicity against the cancer cell line, but these compounds showed substantially lower toxicity (from 6-fold to >20-fold) against NHBE cells and no effect on M. fortuitum, suggesting inhibition of one or more host protein(s) required for proliferation of cancer cells and PIV5. The key structural elements identified here may facilitate identification of targets of this highly biologically active scaffold.

Viral morphogenesis and morphological changes in human neuronal cells following Tioman and Menangle virus infection.

Tioman virus (TioPV) and Menangle virus (MenPV) are two antigenically and genetically related paramyxoviruses (genus: Rubulavirus, family: Paramyxoviridae) isolated from Peninsular Malaysia (2001) and Australia (1997), respectively. Both viruses are potential zoonotic agents. In the present study, the infectivity, growth kinetics, morphology and morphogenesis of these two paramyxoviruses in a human neuronal cell (SK-N-SH) line were investigated. Sub-confluent SK-N-SH cells were infected with TioPV and MenPV at similar multiplicity of infection. These cells were examined by conventional and immunoelectron microscopy, and virus titres in the supernatants were assayed. Syncytia were observed for both infections in SK-N-SH cells and were more pronounced during the early stages of TioPV infection. The TioPV titre increased consistently (10(1)) every 12 h after infection. In MenPV-infected cells, cellular material was frequently observed within budding virions, and microfilaments and microtubules were abundant. Viral budding was common, and extracellular MenPVs tended to be more pleomorphic compared to TioPVs, which appeared to be more spherical in appearance. The MenPV cytoplasmic viral inclusion appeared to be comparatively smaller, loose and interspersed with randomly scattered circle-like particles, whereas huge tubule-like cytoplasmic inclusions were observed in TioPV-infected cells. Both viruses also displayed different cellular pathology in the SK-N-SH cells. The intracellular ultrastructural characteristics of these two viruses in infected neuronal cells may allow them to be differentiated by electron microscopy.

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.

Multiple factors including subgenomic RNAs and reduced viral protein expression are associated with a persistent infection by porcine rubulavirus (LPMV).

The synthesis of virus specific RNA and the expression of viral proteins in PK-15 cells persistently infected with the porcine rubulavirus LPMV have been studied at two different cell-passages following establishment of persistency (passages 25 and 65). Protein analysis of persistently infected cells and the virus particles released from these failed to demonstrate the presence of the polymerase (L) protein. A decrease in the amount of the phospho- (P) protein was also noted. The genome and mRNAs, both mono- and bicistronic, could readily be identified in the persistently infected cells with the exception of the L mRNA. By analysis of transcription gradients generated using the NIH Image analysis software, as well as analysis of the editing frequency, it was concluded that the changes in viral protein levels in persistently infected cells could be associated with a reduction in the amount of L mRNA and a shift in editing of the P gene. In addition, several large subgenomic RNAs of both the internally deleted and copy-back type were found in the persistently infected cells. The relevance of these findings to the persistent state is discussed.