N6-Methyladenosine RNA Modification in Host Cells Regulates Peste des Petits Ruminants Virus Replication.

N6-methyladenosine (m6A) modification is a major RNA epigenetic regulatory mechanism. The dynamics of m6A levels in viral genomic RNA and their mRNAs have been shown to have either pro- or antiviral functions, and therefore, m6A modifications influence virus-host interactions. Currently, no reports are available on the effect of m6A modifications in the genome of Peste des petits ruminants virus (PPRV). In the present study, we took PPRV as a model for nonsegmented negative-sense single-stranded RNA viruses and elucidate the role of m6A modification on viral replication. We detected m6A-modified sites in the mRNA of the virus and host cells, as well as the PPRV RNA genome. Further, it was found that the level of m6A modification in host cells alters the viral gene expression. Knockdown of the METTL3 and FTO genes (encoding the m6A RNA modification writer and eraser proteins, respectively) results in alterations of the levels of m6A RNA modifications in the host cells. Experiments using these genetically modified clones of host cells infected with PPRV revealed that both higher and lower m6A RNA modification in the host cells negatively affect PPRV replication. We found that m6A-modified viral transcripts had better stability and translation efficiency compared to the unmodified mRNA. Altogether, from these data, we conclude that the m6A modification of RNA regulates PPRV replication. These findings contribute toward a way forward for developing novel antiviral strategies against PPRV by modulating the dynamics of host m6A RNA modification. IMPORTANCE Peste des petits ruminants virus (PPRV) causes a severe disease in sheep and goats. PPRV infection is a major problem, causing significant economic losses to small ruminant farmers in regions of endemicity. N6-methyladenosine (m6A) is an important RNA modification involved in various functions, including virus-host interactions. In the present study, we used stable clones of Vero cells, having knocked down the genes encoding proteins involved in dynamic changes of the levels of m6A modification. We also used small-molecule compounds that interfere with m6A methylation. This resulted in a platform of host cells with various degrees of m6A RNA modification. The host cells with these different microenvironments were useful for studying the effect of m6A RNA modification on the expression of viral genes and viral replication. The results pinpoint the level of m6A modifications that facilitate the maximum replication of PPRV. These findings will be useful in increasing the virus titers in cultured cells needed for the economical development of the vaccine. Furthermore, the findings have guiding significance for the development of novel antiviral strategies for limiting PPRV replication in infected animals.

Integrated analysis of long-noncoding RNA and circular RNA expression in Peste-des-Petits-Ruminants Virus (PPRV) infected marmoset B lymphocyte (B95a) cells.

Peste-des-Petits-Ruminants (PPR) or goat plague is an important viral disease of sheep and goats caused by the small ruminant morbilli virus or PPR virus (PPRV). Long non coding RNAs (lncRNA) and circular RNAs (circRNA) play a pivotal role in several biological processes including regulation of virus-host interactions. The present study explored the expression of lncRNA, circRNA and their functions in PPRV infected B-lymphocyte (B95a) cells. The results revealed a total of 4531 lncRNA and 2348 circRNA expression in both mock and PPRV infected samples. Analysis of differentially expressed (DE) RNA identified 123 DE-lncRNA and 39 DE-circRNA as significantly dysregulated. Functional analysis of cis-target genes of DE-lncRNA indicated activation of TCF dependent WNT signaling and PKN1 stimulated transcription process. Interactions (sponging) of microRNA (miRNA) revealed 344 DE-lncRNA-miRNA and 93 DE-circRNA-miRNA pairs. The competing endogenous RNA (ceRNA) network of lncRNA/circRNA-miRNA-mRNA in PPRV infected B95a cells was represented by 69 ceRNA pairs. We validated the DE-circRNA by targeted amplification and sequencing of back spliced junctions (BSJs). The present study revealed a profile of lncRNA, circRNA and their potential ceRNA network in PPRV infection. The results provide insight for better understanding of PPRV-host interactions.

Seasonal pattern in occurrence of rotavirus infection (RV) in diarrheic children, calves and piglets from Bareilly, India.

Rapid and reliable diagnosis for diarrhoeal disease is critically important for the differentiation of etiological agents and subsequent suitable treatment modalities. The objective of the study is to reveal the seasonal pattern in the occurrence of rotavirus in diarrheic children, calves and piglets from Bareilly, Uttar Pradesh, India. A total of 115 diarrhoeal samples were collected, out of which 51 were collected during post-monsoon/autumn (September 2018-November 2018) and 64 during the winter season (December 2018-February 2019). The samples were collected from children <5 years (n = 50), piglets <3 months (n = 35) and calves <6 months of age (n = 30). These samples were screened by ribonucleic acid-polyacrylamide gel electrophoresis (RNA-PAGE) and reverse transcriptase-polymerase chain reaction (RT-PCR) by targeting the VP6 gene of rotavirus A (RVA) and the two were compared. In RNA-PAGE 29.4% (5/17), 6.3% (1/16) and 0% (0/18) samples collected from children, calves and piglets, respectively were rotavirus positive during the autumn season while 45.5% (15/33), 21.4% (3/14) and 17.7% (3/17) samples in the winter season. In RT-PCR, 41.2% (7/17), 12.5% (2/16) and 0% (0/18) samples were rotavirus positive in the autumn season while 51.5% (17/33), 28.6% (4/14) and 29.4% (5/17) samples in winter season collected from children, calves and piglets, respectively. On statistical analysis, no significant difference between the season and number of positives in children and calves (p > 0.05) was observed, however in piglets significantly higher number of RVA positives were detected in the winter season than autumn (p < 0.01). The diagnostic test comparison of RNA-PAGE and RT-PCR showed no statistically significant difference in detecting the RVA positives (p > 0.05). Overall the percent positivity showed a seasonal pattern with higher positivity in winter as compared to autumn season.

Classical swine fever in India: current status and future perspective.

Classical swine fever (CSF) is a globally significant disease of swine caused by classical swine fever virus. The virus affects the wild boars and pigs of all age groups, leading to acute, chronic, late-onset or in-apparent course of the disease. The disease causes great economic loss to the piggery industry due to mortality, stunted growth, poor reproductive performance, and by impeding the international trade of pig and pig products. In India, CSF outbreaks are reported from most of the states wherever pig rearing is practiced and more frequently from northeast states. In spite of the highly devastating nature and frequent outbreaks, CSF remained underestimated and neglected for decades in India. The country requires rapid and sensitive diagnostic tests for an early detection of infection to limit the spread of the disease. Also, effective prophylactics are required to help in control and eradication of the disease for the development of the piggery industry. This review looks into the economic impact; epidemiology of CSF highlighting the temporal and spatial occurrence of outbreaks in the last two decades, circulation, and emergence of the virus genotypes in and around the country; and the constraints in the disease control, with the aim to update the knowledge of current status of the disease in India. The article also emphasizes the importance of the disease and the need to develop rapid specific diagnostics and effective measures to eradicate the disease.

Phylogenetic analysis of Classical swine fever virus from archival formalin fixed clinical tissues reveals vietnamese origin of the isolates.

Detection of Classical swine fever virus (CSFV) nucleic acid in archival formalin fixed tissue samples and their use for phylogenetic analysis was investigated. Ten samples were examined for the presence of CSFV nucleic acid by reverse transcriptase polymerase chain reaction (RT-PCR) amplification of 5'UTR and E2 gene. RT-PCR was found positive for 5'UTR fragment in eight samples while only one tissue samples showed amplification for E2 gene target fragment. For molecular epidemiology of the disease, 5'UTR PCR product of sample from Darbhanga (Bihar), was cloned and sequenced. The sequence was compared with the sequences available in database. The phylogenetic analysis reveals that the isolate belongs to subgroup 2.2 sharing 98.7% nucleotide identities with Vietnamese isolate (CaTh05-1, AB252170), indicating towards the possible origin of genogroup 2.2 CSFV isolates involved in the outbreak from Vietnam. From the study, it can be concluded that the tissue samples collected and stored in buffer formalin for years can be used to detect CSFV nucleic acid. Results are also suggestive of that the 5'UTR region of genome is more suitable target for RT-PCR based detection of CSFV in archival formalin fixed specimens. The study also indicates the potential of archival formalin fixed tissues for molecular epidemiology and genotyping of the CSF virus.

Comparative and temporal transcriptome analysis of peste des petits ruminants virus infected goat peripheral blood mononuclear cells.

Peste des petits ruminanats virus (PPRV), a morbillivirus causes an acute, highly contagious disease - peste des petits ruminants (PPR), affecting goats and sheep. Sungri/96 vaccine strain is widely used for mass vaccination programs in India against PPR and is considered the most potent vaccine providing long-term immunity. However, occurrence of outbreaks due to emerging PPR viruses may be a challenge. In this study, the temporal dynamics of immune response in goat peripheral blood mononuclear cells (PBMCs) infected with Sungri/96 vaccine virus was investigated by transcriptome analysis. Infected goat PBMCs at 48h and 120h post infection revealed 2540 and 2000 differentially expressed genes (DEGs), respectively, on comparison with respective controls. Comparison of the infected samples revealed 1416 DEGs to be altered across time points. Functional analysis of DEGs reflected enrichment of TLR signaling pathways, innate immune response, inflammatory response, positive regulation of signal transduction and cytokine production. The upregulation of innate immune genes during early phase (between 2-5 days) viz. interferon regulatory factors (IRFs), tripartite motifs (TRIM) and several interferon stimulated genes (ISGs) in infected PBMCs and interactome analysis indicated induction of broad-spectrum anti-viral state. Several Transcription factors - IRF3, FOXO3 and SP1 that govern immune regulatory pathways were identified to co-regulate the DEGs. The results from this study, highlighted the involvement of both innate and adaptive immune systems with the enrichment of complement cascade observed at 120h p.i., suggestive of a link between innate and adaptive immune response. Based on the transcriptome analysis and qRT-PCR validation, an in vitro mechanism for the induction of ISGs by IRFs in an interferon independent manner to trigger a robust immune response was predicted in PPRV infection.

Genomic analysis of host - Peste des petits ruminants vaccine viral transcriptome uncovers transcription factors modulating immune regulatory pathways.

Peste des petits ruminants (PPR), is an acute transboundary viral disease of economic importance, affecting goats and sheep. Mass vaccination programs around the world resulted in the decline of PPR outbreaks. Sungri 96 is a live attenuated vaccine, widely used in Northern India against PPR. This vaccine virus, isolated from goat works efficiently both in sheep and goat. Global gene expression changes under PPR vaccine virus infection are not yet well defined. Therefore, in this study we investigated the host-vaccine virus interactions by infecting the peripheral blood mononuclear cells isolated from goat with PPRV (Sungri 96 vaccine virus), to quantify the global changes in the transcriptomic signature by RNA-sequencing. Viral genome of Sungri 96 vaccine virus was assembled from the PPRV infected transcriptome confirming the infection and demonstrating the feasibility of building a complete non-host genome from the blood transcriptome. Comparison of infected transcriptome with control transcriptome revealed 985 differentially expressed genes. Functional analysis showed enrichment of immune regulatory pathways under PPRV infection. Key genes involved in immune system regulation, spliceosomal and apoptotic pathways were identified to be dysregulated. Network analysis revealed that the protein - protein interaction network among differentially expressed genes is significantly disrupted in infected state. Several genes encoding TFs that govern immune regulatory pathways were identified to co-regulate the differentially expressed genes. These data provide insights into the host - PPRV vaccine virus interactome for the first time. Our findings suggested dysregulation of immune regulatory pathways and genes encoding Transcription Factors (TFs) that govern these pathways in response to viral infection.

Molecular characterization of peste-des-petits ruminants virus (PPRV) isolated from an outbreak in the Indo-Bangladesh border of Tripura state of North-East India.

Peste-des-petits- ruminants (PPR) is a highly contagious and devastating disease of goats and sheep. Although India is endemic for PPR, Tripura, a state in North East India has never been reported confirmed PPR outbreaks. Recently, an outbreak of PPR occurred in non-descript goats at the Sabroom town of Tripura state in North-East India in June, 2013. The causative agent, PPR virus (PPRV) was confirmed by sandwich ELISA, virus isolation and N gene based RT-PCR and sequencing. The sequence and phylogenetic analysis confirmed the involvement of lineage IV PPR virus in the outbreak. The outbreak viruses from Tripura state were clustered mainly with circulating viruses from Bangladesh, India, China, Pakistan, Tajikistan, Dubai and Kurdistan. However, the nucleotide sequence homology ranged from 99.2 to 99.6% with the PPR strains circulating in Bangladesh during 2011 and 2012 whereas 95.5-98% homology has been observed with the viruses from India and other countries. These findings suggest the transboundary circulation of PPR virus between India and Bangladesh border, which warrant immediate vaccination across the international border to create an immune belt.

Antidiabetic activity and molecular docking of fructooligosaccharides produced by Aureobasidium pullulans in poloxamer-407-induced T2DM rats.

This study evaluated the beneficial effects of fructooligosaccharide (FOS) intake from Aureobasidium pullulans using poloxamer-407 (PX-407) induced type 2 diabetes mellitus (T2DM) in rat. Administration of FOS enhanced enzymatic activities of catalase and glutathione reductase in a dose-dependent manner. Significant reduction in fasting plasma triacylglycerol and very low-density lipoprotein level coupled with slight increase in fasting plasma insulin level was observed. Significant decrease in severe glucosuria, proteinuria, blood creatinine, urea and advanced glycation end products was also observed. Supplementation of FOS increased glucagon like peptide-1 content as well as Bifidobacteria and Lactobacilli populations in the caecum. Molecular docking by Gold and Glide software revealed that three sugar types present in the FOS (1-kestose, nystose, and 1-ß-fructofuranosyl nystose) are potent dipeptidyl peptidase-IV inhibitors as well as peroxisome proliferator-activated receptor-gamma agonists. This work indicates that FOS can be positioned as a nutraceutical product, beneficial in diabetes-associated metabolic abnormalities.

Antihyperglycemic activity with DPP-IV inhibition of alkaloids from seed extract of Castanospermum australe: Investigation by experimental validation and molecular docking.

The antidiabetic actions of Castanospermum australe Cunn., seed (CAS) extract were evaluated in Poloxamer-407 (PX-407) induced T2DM rats. The CAS extract (100 and 150 mg/kg body weight) was administered orally once a day for 5 weeks after the animals were confirmed diabetic. A significant increase in blood glucose, HbA1c and serum insulin levels were observed in T2DM rats in comparison to citrate control rats. Treatment with CAS extract in T2DM rats reduced the elevated levels of blood glucose, HbA1c and insulin with significant (p≤0.001) improvement in OGT. The CAS extract treatment also increased (p≤0.001) the K(ITT) and prevented increase in HOMA-R level in T2DM rats. The DPP-IV inhibitory potential of CAS extract showed IC50 value of 13.96 µg/ml whilst the standard Diprotin A displayed the IC50 value of 1.543 µg/ml. Molecular docking of the three reported alkaloids from the seeds of C. australe showed comparable DPP-IV inhibition with berberine. Our data suggest that CAS extract (150 mg/kg body weight) normalizes hyperglycemia in T2DM rats with strong DPP-IV inhibitory potential. The molecular docking showed that among the three alkaloids of seed extract 7-Deoxy-6-epi-castanospermine is a potent DPP-IV inhibitor similar to berberine.