Emergence of porcine circovirus 2g (PCV2g) and evidence for recombination between genotypes 2g, 2b and 2d among field isolates from non-vaccinated pigs in Mizoram, India.

The molecular genetics of fourteen Porcine Circovirus 2 (PCV2) isolates from non-vaccinated pigs that died of porcine circovirus associated disease (PCVAD) between 2012 and 2019 in the Mizoram state of North East India, was studied. The PCVAD in these pigs, that had shown characteristic clinical signs and lesions associated with post-weaning multi-systemic wasting syndrome and reproductive failure was confirmed with detection of PCV2 DNA in the tissue samples. Complete viral genomes of these fourteen field isolates were sequenced following in house developed overlapping PCR. The multiple sequence alignment of viral capsid proteins or the open reading frame 2 (ORF2) sequences showed highly conserved residues known for antibody recognition and genotype specificity, however, variations were noticed in the amino acid residues previously known as important for in vitro replication of PCV2. The phylogenetic analyses based on the complete genome sequences enabled identification of genotype PCV2g (9/14, 64.29%) for the first time in India along with genotypes PCV2d (3/14, 21.43%) and PCV2b (2/14, 14.29%). Further, recombination analyses showed evidence for recombination between the genotypes 2b, 2g and 2d. This is the first report on the prevalence of genotype PCV2g and natural inter-genotypic (2g-2b, 2g-2d and 2d-2g) recombinants in India. The findings indicate a non-vaccine driven, natural genotypic shift and signify the need for routine PCV2 surveillance and genotyping. Our analyses also provide a solid ground for future studies to understand the consequences of multiple PCV2 genotypes within a pig population with respect to vaccination, diagnostics and emergence of new genotypes.

The combination of FLT3 and DNA methyltransferase inhibition is synergistically cytotoxic to FLT3/ITD acute myeloid leukemia cells.

Effective treatment regimens for elderly acute myeloid leukemia (AML) patients harboring internal tandem duplication mutations in the FMS-like tyrosine kinase-3 (FLT3) gene (FLT3/ITD) are lacking and represent a significant unmet need. Recent data on the effects of FLT3 tyrosine kinase inhibitors on FLT3/ITD(+) AML showed promising clinical activity, including in elderly patients. DNA methyltransferase (DNMT) inhibitors such as decitabine (5-aza-2-deoxycytidine, DEC) and 5-azacitidine (AZA) demonstrated clinical benefit in AML, are well tolerated and are associated with minimal increases in FLT3 ligand, which can represent a potential resistance mechanism to FLT3 inhibitors. In addition, both FLT3 and DNMT inhibition are associated with the induction of terminal differentiation of myeloid blasts. Consequently, there is a strong theoretical rationale for combining FLT3 and DNMT inhibition for FLT3/ITD(+) AML. We therefore sought to study the anti-leukemic effects of DEC, AZA and FLT3 inhibitors, either as single agents or in combination, on AML cell lines and primary cells derived from newly diagnosed and relapsed AML patients. Our studies indicate that combined treatment using FLT3 inhibition and hypomethylation confers synergistic anti-leukemic effects, including apoptosis, growth inhibition and differentiation. The simultaneous administration of AZA and FLT3 inhibition appears to be the most efficacious combination in this regard. These drugs may provide a novel therapeutic approach for FLT3/ITD(+) AML, in particular for older patients.

Indian porcine reproductive and respiratory syndrome virus bears discontinuous deletion of 30 amino acids in nonstructural protein 2.

Since its first outbreak in 2013, porcine reproductive and respiratory syndrome (PRRS) has established as an enzootic disease in pig population of Mizoram state, India. Our previous studies based on phylogenetic analysis of ORF5 and ORF7 gene sequences revealed close relationship of Indian PRRSV with the highly pathogenic variant of PRRSV (HP-PRRSV) of Chinese origin. Despite the control measures, second major outbreak of the disease was recorded in Aizawl district of Mizoram in 2015. The objective of the present study was to examine the origin of PRRSV isolates of 2015 outbreak, identification of deleted region in Nsp2 gene and determination of any genetic variation between 2013 and 2015 isolates of PRRSV. The outbreak was confirmed by the detection of PRRSV-specific antibodies in 57 out of 92 serum samples (61.96 %) and also by RT-PCR in 42 out of 42 necropsy samples (100 %). Nucleotide sequence analysis of Nsp2 coding region of Indian isolates and comparison with reference sequences revealed 90 nucleotides discontinuous deletion further establishes the closeness of Indian PRRSV to Chinese HP-PRRSV. Further, sequence and phylogenetic analysis of ORF5, ORF7 and Nsp2 genes of Indian PRRSV from both 2013 and 2015 revealed that the outbreaks were caused by two different strains of HP-PRRSV closely associated with the Chinese 10 HEB-3 isolate and 07QN isolates of Vietnam origin respectively. The present study confirms that the Indian PRRSV is a highly pathogenic variant of PRRSV and this study serves as the basis for developing practical and effective control measures against this disease.

Porcine reproductive and respiratory syndrome virus (PRRSV) from the first outbreak of India shows close relationship with the highly pathogenic variant of China.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is an economically important viral disease of pigs worldwide. India has reported the first outbreak of PRRS in the pig population of Mizoram state to the Office International des Epizooties on the 26 June 2013. HYPOTHESIS/OBJECTIVES: The aim of the present study was to determine the genotype and origin of porcine reproductive and respiratory syndrome virus (PRRSV) from the first outbreak in the pig population of Mizoram state, India, in 2013. ANIMALS AND METHODS: A total of 880 affected pigs from the outbreak were clinically examined, 51 animals were necropsied and tested by reverse transcription polymerase chain reaction (RT-PCR) to detect PRRSV and 148 serum samples were tested to detect PRRSV-specific antibodies. The full open reading frame 5 (ORF5) gene sequences from 12 and ORF7 gene sequences from three clinical cases were sequenced and analysed for genomic characterization, respectively. RESULTS: The outbreak was confirmed by the detection of PRRSV-specific antibodies in 109 out of 148 serum samples (74%) and also by RT-PCR in 46 out of 51 necropsy samples (90%). Notably, ORF5 and ORF7 genes of Indian strain shares the same nucleotide positions i.e. 13,698-14,300 and 14,799-15,170, respectively, with the highly pathogenic (HP) strain of China and were grouped together in a phylogenetic tree. CONCLUSIONS AND CLINICAL IMPORTANCE: Sequence and phylogenetic analysis of ORF5 and ORF7 confirmed that the Indian strain has a close link with the HP-PRRSV of China. The current study forms an essential step for better understanding of the epidemiology as well as the movement and spread of the disease in India.

Pathology and molecular diagnosis of classical swine fever in Mizoram.

AIM: Clinical histopathological and molecular diagnosis of classical swine fever disease in pigs of Mizoram. MATERIALS AND METHODS: Totally, 31 clinically suspected pigs from 6 districts of Mizoram were examined, and clinical symptoms were recorded. Detailed post mortem examination of all the 31 dead animals was conducted, and gross changes were recorded. Tissue samples were collected for histopathological examination and molecular diagnosis. The collected tissues (tonsil, lymph nodes, spleen) were also processed for RNA extraction. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the specific gene fragments of classical swine fever virus (CSFV). RESULTS: Clinical examination of all the 31 suspected pigs revealed typical clinical signs of CSF. All the animals also showed typical gross and microscopic lesions of CSF. RT-PCR on tissue samples amplified the 421bp, 449bp and 735bp region of 5´NCR, non-structural protein 5B and E(rns) gene regions of CSFV, respectively. Nested PCR for internal region of E2 gene also amplified the expected product of 271bp using PCR product of whole E2 region as template DNA. CONCLUSION: CSF is highly endemic disease in Mizoram. The viral strains circulating in this region are highly virulent. The disease can be diagnosed specifically using RT-PCR.

Emergence of 2.1. subgenotype of classical swine fever virus in pig population of India in 2011.

BACKGROUND: Limited studies are available on molecular epidemiology of classical swine fever virus (CSFV) in India and are restricted to domestic pigs. These studies show the presence of 1.1. genotype. HYPOTHESIS/OBJECTIVES: The aim of the present study was to subgenotype four CSFV isolates, two each from the outbreaks of CSF in wild (Sus scrofa) and domestic pigs of Mizoram state, India, in 2011. ANIMALS AND METHODS: CSFV isolates were subjected to nucleotide sequencing in E2 and NS5B genomic regions. Phylogenetic analysis of the isolates in both genomic regions was carried out with 39 Indian isolates (4 isolates from the present study of Mizoram state and 35 isolates from the other states of India) and 57 reference sequences retrieved from the GenBank database. Two of the 39 isolates from India were collected from wild boar and were subgenotyped as 2.1. Out of 37 isolates from domestic pigs, only two were subgenotyped as 2.1. RESULTS: The analysis revealed the emergence of 2.1. subgenotype of CSFV in both wild and domestic pigs in India. CONCLUSIONS AND CLINICAL IMPORTANCE: The isolates from domestic pigs of Mizoram state (CSF/MZ/KOL/73 and CSF/MZ/AIZ/115) were grouped in genotype 1 and subgenotype 1.1., thus confirming that the source of CSF outbreaks in domesticated pigs in Mizoram was not from wild pigs. The current study forms an essential step for better understanding of the epidemiology of 2.1 subgroup as well as the movement and spread of the disease in India.