Reverse Genetics and Its Usage in the Development of Vaccine Against Poultry Diseases.

Vaccines are the most effective and economic way of combating poultry viruses. However, the use of traditional live-attenuated poultry vaccines has problems such as antigenic differences with the currently circulating strains of viruses and the risk of reversion to virulence. In veterinary medicine, reverse genetics is applied to solve these problems by developing genotype-matched vaccines, better attenuated and effective live vaccines, broad-spectrum vaccine vectors, bivalent vaccines, and genetically tagged recombinant vaccines that facilitate the serological differentiation of vaccinated animals from infected animals. In this chapter, we discuss reverse genetics as a tool for the development of recombinant vaccines against economically devastating poultry viruses.

Inhibition of immunosuppressive indoleamine 2,3-dioxygenase by targeting the heme and apo-form.

Inhibition of immunomodulating enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is considered one of the potential approaches in the fight against cancer and other diseases. Comprehensive biophysical and cellular studies have shown that quinine derivatives effectively inhibit the activity of IDO1. Mechanistic studies revealed that the potent quinine derivatives compete with heme for binding to apo-IDO1 and perturb its reversible binding propensity to apo-IDO1 via the formation of a heme-inhibitor complex. This IDO1 inhibitory pathway could provide new avenues to immunotherapy-based drug discovery strategies.

The Natural History, Pathobiology, and Clinical Manifestations of SARS-CoV-2 Infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2, is a positive-sense single-stranded RNA virus with epithelial cell and respiratory system proclivity. Like its predecessor, SARS-CoV, COVID-19 can lead to life-threatening disease. Due to wide geographic impact affecting an extremely high proportion of the world population it was defined by the World Health Organization as a global public health pandemic. The infection is known to readily spread from person-to-person. This occurs through liquid droplets by cough, sneeze, hand-to-mouth-to-eye contact and through contaminated hard surfaces. Close human proximity accelerates SARS-CoV-2 spread. COVID-19 is a systemic disease that can move beyond the lungs by blood-based dissemination to affect multiple organs. These organs include the kidney, liver, muscles, nervous system, and spleen. The primary cause of SARS-CoV-2 mortality is acute respiratory distress syndrome initiated by epithelial infection and alveolar macrophage activation in the lungs. The early cell-based portal for viral entry is through the angiotensin-converting enzyme 2 receptor. Viral origins are zoonotic with genomic linkages to the bat coronaviruses but without an identifiable intermediate animal reservoir. There are currently few therapeutic options, and while many are being tested, although none are effective in curtailing the death rates. There is no available vaccine yet. Intense global efforts have targeted research into a better understanding of the epidemiology, molecular biology, pharmacology, and pathobiology of SARS-CoV-2. These fields of study will provide the insights directed to curtailing this disease outbreak with intense international impact. Graphical Abstract.

Structure analysis of the nucleoprotein of Newcastle disease virus: An insight towards its multimeric form in solution.

Newcastle disease virus (NDV) has been explored to a great extent to understand the biology of negative-sense RNA viruses. Nucleoprotein (N) is the most abundant protein in the virus particles, and its primary function is to encapsidate the virus genome for its transcription, replication, and packaging. Here, we report the structural investigations of the N protein of NDV (NDV-N) in solution. The N gene of NDV was cloned and expressed in E. coli as a soluble protein of ~53 kDa in size. The FE-TEM imaging of the purified NDV-N displayed a nearly spherical shape with a diameter of 28 nm and the DLS analysis of the purified NDV-N displayed a monodispersed nature, with averaged hydrodynamic radius, 26.5 nm. The conformational behavior of the NDV-N in solution was studied by SAXS analysis, which suggested two ring structures of NDV-N formed by thirteen monomeric units each. Each ring interacts with RNA molecules and forms a large molecule with a size of ~1450 kDa and are stacked on each other in a spiral arrangement. More profound knowledge of the N protein structure will help us in deciphering the control of viral RNA synthesis at the early stage of NDV life-cycle.

Evaluation of Japanese encephalitis virus E and NS1 proteins immunogenicity using a recombinant Newcastle disease virus in mice.

Japanese encephalitis (JE) is the most important cause of acute encephalitis syndrome (AES). Japanese encephalitis virus (JEV), the prototype member of the JE serocomplex, belongs to the genus Flavivirus. The immunogenic proteins envelope (E) and non-structural protein 1 (NS1) of JEV are widely explored for the development of vaccines and diagnostics against JEV. However, there are underlying concerns such as the risk of reversion of live-attenuated vaccines to high virulence, the incomplete inactivation of pathogens in inactivated vaccines and partial vaccine coverage. Newcastle disease virus (NDV) is an efficient viral vaccine vector to express several human and animal immunogenic proteins. In the present study, we have developed a recombinant NDV (rNDV), individually expressing the E and NS1 proteins of JEV (rNDV-Ejev and rNDV-NS1jev). The recovered rNDV-Ejev and rNDV-NS1jev were characterized in 9-day-old SPF embryonated chicken eggs and in cell culture. The vaccination of rNDV-Ejev and rNDV-NS1jev showed effective immunity against JEV upon intranasal immunization in BALB/c mice. The rNDVs vaccination produced effective neutralization antibody titers against both NDV and JEV. The cytokine profiling of the vaccinated mice showed an effective Th1 and Th2 mediated immune response. The study also provided an insight that E, when used in combination with NS1 could reduce the efficacy of only E based immunization in mice. Our results suggested rNDV-Ejev to be a promising live viral vectored vaccine against JEV. This study implies an alternative and economical strategy for the development of a recombinant vaccine against JEV.

The emergence of porcine circovirus 2 infections in the Northeastern part of India: A retrospective study from 2011 to 2017.

Porcine circovirus (PCV) infection has emerged as an alarming threat to the pig population of India, especially in the Northeastern region (NER) over the last 10 years. The present study is a comprehensive report of the seroepidemiology of PCV2 and its incidences in the pig population from organized and unorganized farms of the entire NER of India from 2011 to 2017. A total of 5697 serum samples were screened by ELISA and the mean positivity of PCV2 antibodies in suspected sera was 31.27%. A total of 22 confirmed cases of PCV2 infection were recorded during the years 2014-2017. Seroprevalence of PCV2 infection in sows causing reproductive disorders in NER suggested its higher incidence in organized farms (65.7%) as compared to unorganized farms (17.6%). A detailed pathological and histopathological examination of the tissue samples collected from the affected animals indicated the presence of PCV2. Molecular characterization and phylogenetic analysis of four PCV2 isolates depicted the circulation of PCV2d genotype in the states of Meghalaya and Assam.

Emergence of a genotype I variant of avian infectious bronchitis virus from Northern part of India.

Infectious bronchitis virus (IBV) is one of the foremost causes of a persistent economic burden to poultry industries worldwide. IBV belongs to the genus Gammacoronavirus within the family Coronaviridae. The IBV infection leads to respiratory and nephrogenic symptoms in broiler chickens. In addition, its infection leads to reduced fertility and hatchability in layer birds. We determined the first complete genome sequence of a variant IBV from an outbreak in Haryana state of the Northern part of India using next generation sequencing. On phylogenetic analysis of the IBV isolate, it clustered with genotype I lineage 1 (GI-1). The deduced amino acid sequence of S gene of IBV isolates showed a high level of identity with strains isolated from Tamil Nadu and the reference vaccine strains. Our result suggests that the IBV virus isolated from unvaccinated chicken flocks in North India might be a revertant strain originally evolved from the live attenuated vaccine strains used in the region. Determination of the complete genome sequence of additional IBV isolates from India is necessary to understand the epidemiology of IBV in India.

Enhanced cytopathic effect of Japanese encephalitis virus strain SA14-14-2: Probable association of mutation in amino acid of its envelope protein.

Japanese encephalitis (JE) is a mosquito-borne viral disease. It is a global public health concern since it causes an acute encephalitis syndrome (AES). A large number of JE/AES cases are reported to occur in areas with established or developing JE vaccination program. Partial vaccine coverage and emergence of new variants of Japanese encephalitis virus (JEV) might be playing an important role. The envelope protein (E) of JEV is a major antigenic determinant and responsible for immunogenic responses as well as membrane fusion and virion assembly. In the present study, we have characterized the JEV live attenuated vaccine strain SA14-14-2 in baby hamster kidney cells (BHK-21). The vaccine strain showed enhanced replication following its passage in BHK-21 cells. Nucleotide sequence analysis of the E protein gene of the cell-culture adapted vaccine strain showed an important point mutation. The mutation in the E protein gene was identical to its wild-type parent strain SA14. This study suggests the possibility of reversion mutation and exaltation of vaccine strains following adaptation in the host cells.

Emerging variant of genotype XIII Newcastle disease virus from Northeast India.

Northeast India with its rich and diverse avifauna acts as a hotbed for emerging virulent Newcastle disease virus (NDV) strains. The present work describes the molecular and pathogenic characterization of NDV strain isolated from Pandu, Assam, India. Clinicopathological and genetic analysis showed the virulent nature of NDV strain Pandu. On molecular phylogenetic and evolutionary distance analysis, the NDV strain Pandu formed a distinct clade within the genotype XIII of class II NDV, suggesting a new sub-genotype XIIIc. The accumulation of mutations in the NDV strain Pandu makes it divergent enough to be considered as a new sub-genotype. The proposed NDV sub-genotype XIIIc consists of strains recently reported from eastern and northeastern India.

Molecular characterization of Newcastle disease virus strains isolated from different outbreaks in Northeast India during 2014-15.

Newcastle disease virus (NDV) isolates recovered from different outbreaks in chicken flocks in Assam during 2014-15 were genotypically and pathotypically characterized. Nucleotide sequence analysis of fusion (F) and hemagglutinin protein genes showed a close similarity with genotype XIII strains of NDV. Amino acid sequence of F protein showed a virulent cleavage site (112)R-R-Q-K-R-F(117). Furthermore, pathogenicity test in one-day-old chicks and embryonated chicken eggs showed a virulent pathotype of the isolated NDV strains. The study will help us to understand the biology of circulating strains of NDV in Northeastern part of India.