Plasminogen activator urokinase interacts with the fusion protein and antagonizes the growth of Peste des petits ruminants virus.

Peste des petits ruminants is an acute and highly contagious disease caused by the Peste des petits ruminants virus (PPRV). Host proteins play a crucial role in viral replication. However, the effect of fusion (F) protein-interacting partners on PPRV infection is poorly understood. In this study, we found that the expression of goat plasminogen activator urokinase (PLAU) gradually decreased in a time- and dose-dependent manner in PPRV-infected goat alveolar macrophages (GAMs). Goat PLAU was subsequently identified using co-immunoprecipitation and confocal microscopy as an F protein binding partner. The overexpression of goat PLAU inhibited PPRV growth and replication, whereas silencing goat PLAU promoted viral growth and replication. Additionally, we confirmed that goat PLAU interacted with a virus-induced signaling adapter (VISA) to antagonize F-mediated VISA degradation, increasing the production of type I interferon. We also found that goat PLAU reduced the inhibition of PPRV replication in VISA-knockdown GAMs. Our results show that the host protein PLAU inhibits the growth and replication of PPRV by VISA-triggering RIG-I-like receptors and provides insight into the host protein that antagonizes PPRV immunosuppression.IMPORTANCEThe role of host proteins that interact with Peste des petits ruminants virus (PPRV) fusion (F) protein in PPRV replication is poorly understood. This study confirmed that goat plasminogen activator urokinase (PLAU) interacts with the PPRV F protein. We further discovered that goat PLAU inhibited PPRV replication by enhancing virus-induced signaling adapter (VISA) expression and reducing the ability of the F protein to degrade VISA. These findings offer insights into host resistance to viral invasion and suggest new strategies and directions for developing PPR vaccines.

Growth kinetics of a Vero cells adapted Bangladeshi strain of peste des petits ruminants (PPR) virus in cell culture.

Growth kinetics of a Vero cells adapted Bangladeshi strain of peste des petits ruminants virus was studied in Vero cells to determine maximum virus yield. One-step growth curve was formulated after determining virus in both supernatant (CFV) and cell lysate (CAV) at different time categories by microtitre plate titration in Vero cells and the viral presence was confirmed by real-time RT-PCR. The virus was first detected in both the supernatants and cell pellets at 12 hpi. The virus titre reached its plateau at 72 hpi. Maximum virus titre of CAV was 6.2 log10 TCID50/ml and that of CFV was 5.2 log10 TCID50/ml at 72 hpi. After that, the titer gradually declined, but maintained at 4.5 log10 TCID50/ml in case of CAV and 4.2 log10 TCID50/ml in case of CFV at 96 hpi. It was concluded that the optimum time point for harvesting Vero cell culture is 72 hpi.

Production of small ruminant morbillivirus, rift valley fever virus and lumpy skin disease virus in CelCradle™ -500A bioreactors.

BACKGROUND: Animal vaccination is an important way to stop the spread of diseases causing immense damage to livestock and economic losses and the potential transmission to humans. Therefore effective method for vaccine production using simple and inexpensive bioprocessing solutions is very essential. Conventional culture systems currently in use, tend to be uneconomic in terms of labor and time involved. Besides, they offer a limited surface area for growth of cells. In this study, the CelCradle™-500A was evaluated as an alternative to replace conventional culture systems in use such as Cell factories for the production of viral vaccines against small ruminant morbillivirus (PPR), rift valley fever virus (RVF) and lumpy skin disease virus (LSD). RESULTS: Two types of cells Vero and primary Lamb Testis cells were used to produce these viruses. The study was done in 2 phases as a) optimization of cell growth and b) virus cultivation. Vero cells could be grown to significantly higher cell densities of 3.04 × 109 using the CelCradle™-500A with a shorter doubling time as compared to 9.45 × 108 cells in Cell factories. This represents a 19 fold increase in cell numbers as compared to seeding vs only 3.7 fold in Cell factories. LT cells achieved modestly higher cell densities of 6.7 × 108 as compared to 6.3 × 108 in Cell factories. The fold change in densities for these cells was 3 fold in the CelCradle™-500A vs 2.5 fold in Cell factories. The titers in the conventional system and the bioreactor were not significantly different. However, the Cell-specific virus yield for rift valley fever virus and lumpy skin disease virus are higher (25 virions/cell for rift valley fever virus, and 21.9 virions/cell for lumpy skin disease virus versus 19.9 virions/cell for rift valley fever virus and 10 virions/cell for lumpy skin disease virus). CONCLUSIONS: This work represents a novel study for primary lamb testis cell culture in CellCradle™-500A bioreactors. In addition, on account of the high cell densities obtained and the linear scalability the titers could be further optimized using other culture process such us perfusion.

Characterization of defective interfering (DI) particles of Pestedes petitsruminants vaccine virus Sungri/96 strain-implications in vaccine upscaling.

The present investigation deals with the characterization of defective interfering (DI) particles of Peste-des-petits ruminants (PPR) vaccine Sungri/96 strain generated as a result of high MOI in Vero cells. During the serial 10 passages, infectivity titres drastically reduced from 6.5 to 2.25 log10TCID50/ml at high MOI. Further, attenuation of CPE with high MOI indicated generation of DI particles that resulted in no/slow progression of CPE during the late passages. Monoclonal antibody based cell ELISA indicated normal protein (N & H) packaging in samples with DI activity. At genomic level, inconsistency in amplicon intensity of H gene was observed in RT-PCR, indicating a possible defect of H gene. Further analysis of copy number of PPRV by RT-qPCR indicated intermittent fluctuations of viral genomic RNA copies. The significant decline of viral RNA copies with MOI 3 (314 copies) compared to low MOI (512804 copies), proved that higher DI multiplicities cause more interference with the replication process of the standard virus. Therefore, MOI is critical for manufacturing of vaccines. These investigations will help in upscaling of PPR vaccines in view of ongoing National and Global PPR control and eradication programme.

Characterization of ovine Nectin-4, a novel peste des petits ruminants virus receptor.

Small ruminants infected with peste des petits ruminants virus exhibit lesions typical of epithelial infection and necrosis. However, the only established host receptor for this virus is the immune cell marker signaling lymphocyte activation molecule (SLAM). We have confirmed that the ovine Nectin-4 protein, when overexpressed in epithelial cells, permits efficient replication of PPRV. Furthermore, this gene was predominantly expressed in epithelial tissues and encoded by multiple haplotypes in sheep breeds from around the world.

Rescue of eGFP-expressing small ruminant morbillivirus for identifying susceptibilities of eight mammalian cell lines to its infection.

Small ruminant morbillivirus (SRMV), formerly called peste-des-petits-ruminants virus (PPRV), is classified into the genus Morbillivirus in the family Paramyxoviridae. If genetically modified using reverse genetics, the SRMV would be a useful vector to express foreign proteins in vitro and in vivo. In this study, a recombinant SRMV was rescued by reverse genetics for efficiently expressing an enhanced green fluorescent protein (eGFP) in vitro. Based on green fluorescence-tracked characteristics of the recombinant SRMV, eight mammalian cell lines (BHK-21, F81, MDBK, RK13, MDCK, PK15, Vero and GT) were selected for identifying their susceptibilities to SRMV infection. The result showed that all cell lines could be infected with the recombinant SRMV but at different efficiencies. The Vero and PK15 cell lines showed the highest and lowest susceptibilities to its infection, respectively, if merely comparing the proportions of green fluorescence-emitting cells among eight cell monolayers.

Development of reverse genetics system for small ruminant morbillivirus: Rescuing recombinant virus to express Echinococcus granulosus EG95 antigen.

Peste des petits ruminants and cystic hydatidosis may be simultaneously endemic in a given area. Their pathogens are small ruminant morbillivirus (SRMV) and Echinococcus granulosus (E. granulosus), respectively. The SRMV, formerly called peste-des-petits-ruminants virus (PPRV), is classified into the genus Morbillivirus in the family Paramyxoviridae. This virus is an ideal vaccine vector to deliver immunogenic proteins. In this study, a reverse genetics system was developed to rescue a recombinant SRMV (Nigeria 75/1 strain) expressing E. granulosus EG95 antigen in vitro. The recombinant SRMV, albeit replicating more slowly than its parental virus, could effectively express the EG95 antigen in cells by analyses of Western blot, indirect immunofluorescence and mass spectrometry. An EG95 subunit vaccine has been widely used for prevention of cystic hydatidosis in some areas of China. The EG95-expressing SRMV, if proven to induce effective immune responses against both diseases in a future animal experiment, would become a potential candidate of bivalent vaccine.

Effect of siRNA mediated suppression of signaling lymphocyte activation molecule on replication of peste des petits ruminants virus in vitro.

Signaling lymphocyte activation molecule (SLAM) expression was inhibited in B95a cell line using siRNA and the effect of SLAM inhibition on peste des petits ruminants virus (PPRV) replication and infectivity titre was studied. SLAM suppression was assessed using real-time PCR and flow cytometry to confirm suppression at the m-RNA and protein levels, respectively. Three chemically synthesized siRNAs were transfected individually using oligofectamine into B95a cell line. This resulted in SLAM suppression from 48 to 454-folds, in comparison to the untransfected B95a cell line. When the SLAM suppressed B95a cell line was infected with PPRV, replication was reduced by 12-143-folds and virus titre was reduced from log10 1.09 to 2.28. siRNA 3 showed the most potent inhibition of SLAM expression both at m-RNA and protein levels. This also caused the maximum reduction of virus replication and virus titre. A 100-fold reduction in PPRV titres was seen in anti-SLAM antibody neutralized B95a cell line. This further confirms that SLAM is one of the (co) receptors for PPRV. However, the presence of other putative virus receptor(s) is/are not ruled out.

Early detection of viral excretion from experimentally infected goats with peste-des-petits ruminants virus.

We observed 15 goats for 9 days after subcutaneous infection with 10(3) TCID(50) with isolates of peste-des-petits ruminants virus from Africa and India and five concurrent, uninfected control goats. Typical clinical signs of the infection were present in all 15 infected goats by day 8 and in most by day 6 and some signs were present by day 4. However, 6 out of 15 goats already have detectable virus shedding by day 3 and four more were shedding by day 4 and every goat had virus shedding for at least 1 day before the recognition of clinical signs. This experiment indicates that incubatory carriers therefore might play a role in the transmission of PPRV among small ruminants.

Complexities in Isolation and Purification of Multiple Viruses from Mixed Viral Infections: Viral Interference, Persistence and Exclusion.

Successful purification of multiple viruses from mixed infections remains a challenge. In this study, we investigated peste des petits ruminants virus (PPRV) and foot-and-mouth disease virus (FMDV) mixed infection in goats. Rather than in a single cell type, cytopathic effect (CPE) of the virus was observed in cocultured Vero/BHK-21 cells at 6th blind passage (BP). PPRV, but not FMDV could be purified from the virus mixture by plaque assay. Viral RNA (mixture) transfection in BHK-21 cells produced FMDV but not PPRV virions, a strategy which we have successfully employed for the first time to eliminate the negative-stranded RNA virus from the virus mixture. FMDV phenotypes, such as replication competent but noncytolytic, cytolytic but defective in plaque formation and, cytolytic but defective in both plaque formation and standard FMDV genome were observed respectively, at passage level BP8, BP15 and BP19 and hence complicated virus isolation in the cell culture system. Mixed infection was not found to induce any significant antigenic and genetic diversity in both PPRV and FMDV. Further, we for the first time demonstrated the viral interference between PPRV and FMDV. Prior transfection of PPRV RNA, but not Newcastle disease virus (NDV) and rotavirus RNA resulted in reduced FMDV replication in BHK-21 cells suggesting that the PPRV RNA-induced interference was specifically directed against FMDV. On long-term coinfection of some acute pathogenic viruses (all possible combinations of PPRV, FMDV, NDV and buffalopox virus) in Vero cells, in most cases, one of the coinfecting viruses was excluded at passage level 5 suggesting that the long-term coinfection may modify viral persistence. To the best of our knowledge, this is the first documented evidence describing a natural mixed infection of FMDV and PPRV. The study not only provides simple and reliable methodologies for isolation and purification of two epidemiologically and economically important groups of viruses, but could also help in establishing better guidelines for trading animals that could transmit further infections and epidemics in disease free nations.

VeroNectin-4 is a highly sensitive cell line that can be used for the isolation and titration of Peste des Petits Ruminants virus.

Peste des Petits Ruminants virus (PPRV) is a member of the Morbillivirus subgroup of the family Paramyxoviridae, and is one of the most contagious diseases of small ruminants throughout Africa and the rest of the world. Different cell lines have previously been used to isolate PPRV but with limited success. Thus, to improve the isolation of Morbilliviruses, human, canine, and goat homologues of the lymphocyte receptor signaling lymphocyte activation molecule (SLAM) have been introduced into cells that can support virus replication. However, the amino acid sequence of SLAM varies between species, and often requires adaptation of a particular virus to different versions of the receptor. The protein sequence of Nectin-4 is highly conserved between different mammals, which eliminate the need for receptor adaptation by the virus. Cell lines expressing Nectin-4 have previously been used to propagate measles and canine distemper viruses. In this study, we compared infections in Vero cells expressing canine SLAM (VeroDogSLAM) to those in Vero cells expressing Nectin-4 (VeroNectin-4), following inoculations with wild-type strains of PPRV. Virus isolation using VeroNectin-4 cells was successful with 23% of swabbed samples obtained from live infected animals, and was 89% effective using post-mortem tissues of infected sheep. By contrast, only 4.5% efficiency was observed from swab samples and 67% efficiency was obtained in virus isolation from post-mortem tissues using VeroDogSLAM cells. The average incubation period for virus recovery from post-mortem tissues was 3.4 days using VeroNectin-4 cells, compared with 5.5 days when using VeroDogSLAM cells. The virus titers of PPRV obtained from VeroNectin-4 cells were also higher than those derived from VeroDogSLAM cells. A comparison of the growth kinetics for PPRV in the two cell lines confirmed the superiority of VeroNectin-4 cells for PPR diagnostic purposes and vaccine virus titration.

A simplified objective method for quantification of peste des petits ruminants virus or neutralizing antibody.

A simplified and standardized assay based on haemagglutination of infected culture supernatants was developed to detect peste des petits ruminants (PPR) virus growth in vero cells and to quantify PPR neutralizing antibody. Virus titres estimated by visual reading of cytopathic effects as a criterion were compared with those estimated by haemagglutination of infected supernatants and no statistically significant differences were seen between them. During quantification of PPR antibodies, the titres based on haemagglutination of supernatants on day 5 post-infection had a high sensitivity, specificity and agreement in qualititative comparison with those determined by visual examination of cytopathic effects. In quantitative comparison, the correlation coefficient between serum neutralization titres estimated by visual examination of cytopathic effects or haemagglutination of supernatants was 0.96, when haemagglutination was done on day 5 post-infection. The virus and serum neutralization titres can thus be estimated objectively using the haemagglutination of supernatants as criterion to measure endpoints. The haemagglutination-inhibition titres also correlated well with serum neutralization titres with a coefficient of 0.78. Thus the haemagglutination-inhibition test appears to be a suitable alternative to the serum neutralization test for quantification of PPR neutralizing antibody.

Improved technique for transient expression and negative strand virus rescue using fowlpox T7 recombinant virus in mammalian cells.

The suitability of recombinant T7 polymerase produced using either the highly attenuated MVA strain of vaccinia (MVA-T7) or fowlpox virus (FP-T7) for transient expression and negative strand virus rescue was compared in two mammalian cell lines (MDBK and Vero) and in primary cells of bovine, ovine and caprine origin. Such primary cells are more permissive for the growth of wild type strains of morbilliviruses, such as Rinderpest virus and Peste des petits ruminants virus. MVA-T7 was found to be highly cytopathic in the primary cells, multiplying rapidly and killing the cells within 3-5 days of infection, even when very low multiplicities of infection (MOI) were used. In contrast, FP-T7, which appeared to express similar amounts of T7 polymerase, was found to be non-cytopathic in a variety of primary and established cell lines of mammalian origin and was suitable for use in virus rescue experiments. MDBK cells and primary cells, unlike Vero cells, could not be efficiently transfected and so were unsuitable for virus rescue. Optimal conditions for rinderpest virus rescue in Vero cells were established using FP-T7 in place of MVA-T7. This system will be suitable for rescuing other viruses which grow in Vero cells.

Assessment of the cytotoxic and anti-viral potential of aqueous extracts from different parts of Acacia nilotica (Linn) Delile against Peste des petits ruminants virus.

Peste des petits ruminants virus (PPRV); a negative sense single stranded RNA enveloped virus that causes Peste des petits ruminants (PPR), is dramatically affecting the health status of ruminants all around the world resulting in extensive economical losses in livestock sector. Acacia nilotica (Linn) Delile; a tannin-rich medicinal plant distributed throughout subcontinent, is traditionally used as food for ruminants and possesses anti-viral potential against different RNA viruses. In the current study, aqueous extracts from the bark, leaves and pods of A. nilotica (Linn) Delile indica were evaluated for their cytotoxicity and anti-viral activities against PPRV by adopting MTT colorimetric assay and anti-viral assay using Vero cell line. Aqueous extract from the leaves presented significantly better (P<0.05) anti-PPRV activities in comparison to pods extract. On the contrary, bark extract did not show any anti-viral activity. The data presented in the study could pave a way toward the discovery of novel anti-viral chemicals in the plants against PPRV and other viral diseases.

Isolation, identification and characterization of a Peste des Petits Ruminants virus from an outbreak in Nanakpur, India.

A Peste des Petits Ruminants virus (PPRV) was isolated from an outbreak that occurred in sheep and goats in Nanakpur village of Mathura District in Uttar Pradesh (India). Based on hemagglutination of chicken red blood cells (rbcs), cytopathic effect similar to that caused by the Morbilliviruses in Vero cells, and amplification and sequence analysis of the viral nucleoprotein (N) gene, the identity of the virus was confirmed as PPRV and named PPRV/C. hircus-tc/India/2012/Nanakpur1 (in short PPRV/Nkp1/2012). However, based on its poor neutralization with monoclonal antibodies, escape detection by commercial ELISA, and unsuccessful amplification of the hemagglutinin (H) and the fusion (F) genes by several pairs of published PCR primers it was concluded that the PPRV/Nkp1/2012 may not be closely related to lineage type IV PPR viruses believed to be present in the Indian subcontinent. A plaque assay for titration of the PPRV was developed for the first time. The virus was plaque purified and its growth characteristics were studied in the African green monkey kidney (Vero) cells and baby hamster kidney (BHK-21) cells. In a one-step growth curve analysis it was concluded that the duration of the PPRV life cycle is 6-8h, an uncharacterized part of PPRV replication. These findings provide information for devising control strategies against PPR in India by choosing a homologous candidate vaccine prototype.

Potential of adenovirus and baculovirus vectors for the delivery of shRNA against morbilliviruses.

Morbilliviruses are important pathogens of humans, ruminants, carnivores and marine mammals. Although good vaccines inducing long-term immunity are available, recurrent outbreaks of measles, canine distemper and peste des petits ruminants (PPR) are observed. In control strategies, antivirals thus could be useful to confine virus spread and application of interfering RNAs is a promising approach, provided they can be delivered efficiently into the host cells. We have constructed recombinant adenovirus and baculovirus vectors expressing short hairpin RNAs (shRNAs) against the PPR virus (PPRV) and compared them in vitro. It was found that both recombinant viruses inhibited PPRV replication with the baculovirus vector, which inhibited generation of infectious progeny by more than 2 log10 and the nucleoprotein expression of PPRV by 73%, being the more efficient. The results show that baculoviral shRNA-expressing vectors have the potential for therapeutic use against morbillivirus infections.

A vero cell derived combined vaccine against sheep pox and Peste des Petits ruminants for sheep.

The combined sheep pox and Peste des Petits ruminants (PPR) vaccine was prepared in lyophilized form containing recommended doses of both vaccine viruses. Safety and immunogenicity of this combined vaccine was evaluated in sheep. Sheep immunized subcutaneously with 1ml of live attenuated vaccine consisting of 10(3)TCID(50) each of sheep pox virus (SPV) Romanian Fanar (RF) strain and Peste des Petits ruminants virus (PPRV-Sungri/96 strain) were monitored for clinical and serological responses for a period of four weeks post immunization (pi) and two week post challenge (pc). Specific antibodies directed to sheep pox virus could be demonstrated by indirect ELISA and serum neutralization test (SNT). Competitive ELISA and SNT were used for demonstration of antibodies to PPR virus. All the immunized animals resisted challenge with virulent SPV or PPRV on day 30pi, while control animals developed characteristic signs of disease. Specific virus could be detected in the unvaccinated control animals after challenge but not from any of the immunized sheep. Combined vaccine was found to be safe and potent as evident from sero conversion as well as challenge studies in sheep. This indicates that component vaccines did not interfere each other and can be used in target population for economic vaccination strategies.

Scalable culture systems using different cell lines for the production of Peste des Petits ruminants vaccine.

Peste des Petits ruminants (PPR) is considered as one of the major constraints to the productivity of small ruminants in Africa and Asian countries. Currently PPR control is done by vaccination with an attenuated PPR strain (Nigeria 75/1) produced in monolayers of Vero cells grown in roller bottles or static flasks. This work focuses on the production of a PPR vaccine strain using stirred conditions as an advanced option for process scale-up. Non-porous microcarriers (Cytodex-1) were used to support Vero cell growth in suspension cultures. The use of Ex-Cell medium could improve cell specific productivities obtained with standard serum containing medium, independently of the type of system used, i.e. static as well as suspension stirred cultures. As an alternative, several cell lines adapted to grow as single cells in suspension (CHO-K1, BHK-21A and 293) and another anchorage-dependent (MRC-5) were evaluated in their capacity to produce a PPR vaccine. BHK-21A and 293 cells grown as single-cell suspension in serum free medium were both suited to produce PPR vaccine with productivities similar to Vero cells, namely 10(6)TCID(50)/mL. However, for the 293 cells, these results were only obtained 2-3 days later. CHO-K1 and MRC-5 cells have shown not to be suitable to adequately produce this virus. These results provide further insights into the feasibility of applying microcarrier cell culture technology to produce PPR vaccine in Vero cells as well as in the alternative use of single-cell suspension cultures of BHK-21A, significantly simplifying the existing production process.