Evaluation of dynamics of immune responses and protective efficacy in piglets immunized with an inactivated porcine reproductive and respiratory syndrome vaccine candidate.

Porcine Reproductive and Respiratory Syndrome (PRRS) is an important viral disease of swine that causes significant mortality in piglets and production losses in adult pigs. In this study, we investigated the protective efficacy of an inactivated PRRS virus vaccine candidate and evaluated the differences in PRRSV specific anamnestic response in piglets when challenged with live PRRSV at two different intervals post-immunization. Six-week-old piglets were immunized intramuscularly with an inactivated, Montanide ISA-206 adjuvanted Indian PRRSV isolate, followed by a booster dose at 21 days post-immunization. Homologous live PRRS virus challenge was done on 60 and 180 days post-booster (dpb). We assessed humoral and cell-mediated immune responses at various intervals post-immunization and after challenge. Viraemia, virus shedding in nasal secretions and lung lesion scores were studied to assess the efficacy of the vaccine candidate. All the immunized pigs developed PRRSV-specific antibodies upon booster dose administration. Neutralizing antibody (NA) titres before challenge, in most animals, ranged between 0 and 4. Potentially protective NA titre of 8 was observed in serum of seven out of the 12 immunized piglets after challenge, across the immunized groups. A significant increase in the mean T-helper, T-cytotoxic, memory or activated T-helper and NK cell populations was observed in immunized piglets challenged at 180 dpb, from 4 to 11 dpc, 5 to 11 dpc, 5 to 7 dpc and 6 to 11 dpc, respectively as compared to the challenge controls. Protective efficacy of the inactivated PRRSV antigen against the homologous virus challenge was evidenced by earlier onset of PRRSV specific virus neutralizing antibodies and cell mediated immune responses, reduced viremia, nasal virus shedding and severity of lung lesions in immunized piglets as compared to unimmunized controls post-challenge. Our results indicated that the inactivated PRRSV antigen elicited better virus specific anamnestic immune responses in piglets when challenged at six months after the single booster dose, due to age related increase in antigen-specific memory T helper cell responses, as compared to those challenged at 2 months post booster.

Growth kinetics of an Indian isolate of highly pathogenic porcine reproductive and respiratory syndrome virus in MARC-145 cells.

The aim of the present study was to understand the replication kinetics of an Indian isolate of highly pathogenic porcine reproductive and respiratory syndrome (PRRS) virus (Ind-297221) in MARC-145 cells infected at different multiplicity of infection (MOI) of 1.0, 0.1, 0.01 and 0.001. PRRSV titre in the infected cell fraction and the culture supernatant harvested at different intervals (12, 36, 48, 72, 96 and 120 h) post infection (hpi) was estimated by immunoperoxidase monolayer assay. Viral RNA copy numbers were quantified by TaqMan RT-PCR. PRRS virus could be detected first in intracellular fraction at 12 hpi in cells infected at 1.0 MOI, whereas in the extracellular fraction, earliest detection was at 36 hpi. Highest PRRSV titre of 1.3 × 105.0 TCID50/mL was achieved in 0.01 and 0.001 MOI groups at 96 hpi. Infection with 0.01 MOI resulted in the maintenance of maximum titre up to 120 hpi. The maximum viral copy numbers observed was 3.15 × 107.0 in 0.1 MOI group at 120 hpi in culture medium. The results of the study showed that MARC-145 cells infected with Indian PRRSV at 0.01 MOI and harvested in 96-120 hpi was found to be optimum for obtaining maximum virus yield and hence can be used for bulk propagation of the virus.

Immunogenicity and cross-protective efficacy of recombinant H5HA1 protein of clade 2.3.2.1a highly pathogenic H5N1 avian influenza virus expressed in E.coli.

The global spread of H5N1 highly pathogenic avian influenza virus (HPAIV) in poultry has caused great economic loss to the poultry farmers and industry with significant pandemic threat. The current study involved production of recombinant HA1 protein of clade 2.3.2.1a H5N1 HPAIV (rH5HA1) in E.coli and evaluation of its protective efficacy in chickens. Purification under denaturing conditions and refolding by dialysis against buffers containing decreasing concentrations of urea was found to preserve the biological activity of the expressed recombinant protein as assessed by hemagglutination assay, Western blot and ELISA. The Montanide ISA 71 VGA adjuvanted rH5HA1 protein was used for immunization of chickens. Humoral response was maintained at a minimum of 4log2 hemagglutination inhibition (HI) titre till 154 days post 2nd booster. We evaluated the protective efficacy of rH5HA1 protein in immunized chickens by challenging them with homologous (2.3.2.1a) and heterologous (2.3.2.1c) clades of H5N1 HPAIV. In both the groups, the HI titre significantly increased (P < 0.05) after challenge and the virus shedding significantly (P < 0.05) reduced between 3rd and 14th day post challenge. The virus shedding ratio in oro-pharyngeal swabs did not differ significantly between both the groups except on 7 days post challenge and during the entire experimental period in cloacal swabs. These results indicate that rH5HA1 was able to induce homologous and cross protective immune response in chickens and could be a potential vaccine candidate used for combating the global spread of H5N1 HPAIV threat. To our knowledge, this is the first study to report immunogenicity and protective efficacy of prokaryotic recombinant H5HA1 protein in chicken.

First report on co-isolation and whole-genomic characterisation of mammalian orthorubulavirus 5 and mammalian orthoreovirus type 3 from domestic pigs in India.

During a surveillance study to monitor porcine epidemic diarrohoea virus and transmissible gastroenteritis virus in India, a total of 1043 swine samples including faeces (n = 264) and clotted blood (n = 779) were collected and tested. Five samples (four faecal and one serum) showed cytopathic effects in Vero cells. Transmission electron microscopy of infectious cell supernatant revealed the presence of two types of virions. Next-generation sequencing (de novo) allowed the complete genome sequence of mammalian orthorubulavirus 5 (MRuV5; 15246 bp) and that of all 10 gene segments of mammalian orthoreovirus to be determined. Genetic analysis of MRuV5 revealed grouping of the Indian MRuV5 with isolates from various mammalian species in South Korea and China, sharing more than 99% nucleotide sequence identity. The deduced amino acid sequences of the HN, NP, and F genes of MRuV5 isolates showed three (92L, 111R, 447H), two (86S, 121S), and two (139T, 246T) amino acid substitutions, respectively, compared to previously reported virus strains. Phylogenic analysis based on S1 gene sequences showed the Indian MRV isolates to be clustered in lineage IV of MRV type 3, with the highest nucleotide sequence identity (97.73%) to MRV3 strain ZJ2013, isolated from pigs in China. The protein encoded by the MRV3 S1 gene was found to contain the amino acid residues 198-204NLAIRLP, 249I, 340D, and 419E, which are known to be involved in sialic acid binding and neurotropism. This is the first report of co-isolation and whole-genomic characterisation of MRuV5 and MRV3 in domestic pigs in India. The present study lays a foundation for further surveillance studies and continuous monitoring of the emergence and spread of evolving viruses that might have pathogenic potential in animal and human hosts.

Complete genome analysis of African swine fever virus isolated from domestic pigs during the first ASF outbreaks in India.

African swine fever (ASF), considered as the most dreadful swine disease due to its very high mortality, emerged in India in 2020. The complete genome analysis of ASF viruses isolated during the first outbreaks in India showed a few unique non-synonymous mutations in MGF 369-11L, MGF 505-4R, K205R and B263R genes. Frame shifts in the protein coding sequences were observed in DP60R, ASFV-G_ACD 00190, MGF 110-10-L-MGF110-14L fusion, MGF 360-14L and I267L genes of Indian ASF viruses as compared to ASFV/Georgia/2007. Complete genome based phylogenetic analysis of p72-genotype-II viruses showed the clustering of Indian isolates with ASFV/Wuhan/2019 in a separate clade. Phylogenetic analysis of concatenated sequences of 14 open reading frames (ORF) having single nucleotide polymorphisms (SNP) showed distinct grouping of Indian ASFVs with other Asian ASFVs. This is the first complete genome characterization of ASF viruses isolated from domestic pigs in India. The results indicate that number of Tandem Repeat Sequence (TRS) in the intergenic region between I73R and I329L genes, and the 14 ORFs with SNP reported in this study could be the genetic determinants to differentiate the closely related p72-genotype II viruses circulating in Asia.

Role of expression of host cytokines in the pathogenesis of H9N2-PB2 reassortant and non-reassortant H5N1 avian influenza viruses isolated from crows in BALB/c mice.

The present experiment was conducted to study the role of cytokine, chemokine and TLRs responses of H9N2-PB2 reassortant H5N1 virus as compared to non-reassortant H5N1 virus isolated from crows in BALB/c mice. Two groups (12 mice each) of 6-8 weeks old BALB/c mice were intranasally inoculated with 106 EID50/ml of viruses A/crow/India/03CA04/2015 (H9N2-PB2 reassortant H5N1) and A/crow/India/02CA01/2012 (non-reassortant H5N1). At each interval, brain, lung and spleen were collected and relative quantification of cytokines, chemokines and TLRs was done by qPCR. The H9N2-PB2 reassortant H5N1 infected mice brain, the transcripts of TLR7 were significantly higher than other cytokines at 3dpi and KC was significantly upregulated at 7dpi. In non-reassortant H5N1 infected mice brain showed, TLR 7 and IFNα upregulation at 3dpi and IFNγ and TLR7 upregulation at 7dpi. The H9N2-PB2 reassortant H5N1 infected mice lung revealed, IL2 and TLR7 significant upregulation at 3dpi and in non-reassortant H5N1 infected mice, IL6 was significantly upregulated. At 7dpi in H9N2-PB2 reassortant H5N1 virus infected group mice, IL1 and TLR 3 were significantly upregulated in lungs and in non-reassortant group mice, IL1 and TLR7 were significantly upregulated. At 3dpi in H9N2-PB2 reassortant H5N1 virus infected mice spleen, IL4, IFNα, IFNß were significantly downregulated and TLR7 transcript was significantly upregulated. In non-reassortant group mice, IL6, IFNα, IFNß and TLR 3 were significantly upregulated. At 7dpi in H9N2-PB2 reassortant H5N1 virus infected mice spleen, IFNα, IFNß and TLR7 were significantly lower than other cytokines and in non-reassortant group mice, IFNα and IFNß were significantly downregulated. This study concludes that dysregulation of cytokines in lungs and brain might have contributed to the pathogenesis of both the viruses in mice.

Widespread Prevalence of Antibodies Against Swine Influenza A (pdm H1N1 09) Virus in Pigs of Eastern Uttar Pradesh, India.

Swine influenza virus (SIV) belongs to family Orthomyxoviridae and can cause acute respiratory infection in pigs. Several pandemic H1N1 human fatal influenza cases were reported in India. Though pigs are predisposed to both avian and human influenza virus infections with the potential to generate novel reassortants, there are only a few reports of SIV in Indian pigs. We conducted a serological survey to assess the status of H1N1 infection in pigs of various states in India, between 2009 and 2016. Based on Haemagglutination inhibition (HI) assay, seroprevalence rate of H1N1 virus ranged between 5.2% (2009) and 36.3% (2011). Widespread prevalence of antibody was observed in eastern Uttar Pradesh from 6.2 to 37.5% during the study period. Co-circulation of seasonal H1N1 virus along with pandemic H1N1 virus was indicated by the presence of specific antibodies against seasonal H1N1 virus in eastern part of Uttar Pradesh. Seroprevalence rate in pigs and influenza infection trend in human shows the possible spill over transmission of influenza to pigs from human. Hence, besides serological surveillance, continuous and systematic molecular surveillance should be implemented in pig population to reduce/quantify the risk and emergence of pandemic influenza.

Genetic characterization of African swine fever virus from domestic pigs in India.

African swine fever (ASF) is the most dreaded disease of pigs, which can cause mortality of up to 100%. Following disease outbreaks with high mortality in pigs in two states of north-east India, namely Arunachal Pradesh and Assam in early 2020, we confirmed the first occurrence of African swine fever (ASF) in domestic pigs in India by real-time PCR, virus isolation and nucleotide sequencing. Genetic analyses in three independent genomic regions (B646L gene encoding the p72 protein, E183L gene encoding the p54 protein and the central variable region (CVR) of B602L gene) showed that the Indian ASF viruses are similar to the post-2007-p72-genotype II viruses reported from Asia and Europe, suggesting the transboundary expansion of ongoing ASF outbreaks in the region.

Diabetic retinopathy screening model for rural population: awareness and screening methodology.

CONTEXT: Worldwide, the prevalence of diabetic retinopathy is increasing at an alarming rate. WHO has predicted that in India the number of adults with diabetes will be the highest in the world: from 19 million in 1995 to 80 million in 2030. Although originally thought to be a disease of an urban population, the prevalence of diabetes mellitus is increasing in rural areas as well. The socioeconomic burden resulting from visual impairment or blindness caused by diabetic retinopathy, particularly in the working age group, is a serious concern. ISSUE: In order to combat diabetic retinopathy related blindness, Sankara Nethralaya, the premier eye institute of India, in collaboration with the Lions Clubs International Foundation (LCIF) and the RD Tata Trust, Mumbai, India launched a major diabetic retinopathy screening program in the rural community of South India. The objectives were to create awareness among the rural population of diabetic retinopathy with emphasis on early detection, to conduct diabetes and diabetic retinopathy screening camps, and to bring to the base hospital patients who have sight-threatening diabetic retinopathy, for ancillary investigations such as fluorescein angiography, ultrasound and to perform laser photocoagulation or vitreous surgery, or both. Other objectives included training general ophthalmologists and general physicians in order to develop an integrated diabetic retinopathy model. To address the question as to why certain individuals run the risk of developing sight threatening diabetic retinopathy, biochemical and genetic factors were also studied. The program was launched in June 2003 and 3 rural districts have been screened. To the time of writing, 128 screening camps had been organized, 103 awareness meetings conducted, 23 ophthalmologists trained and 43 general physicians attended the continuing medical education program on diabetic retinopathy. LESSONS: The key elements in the successful implementation of this program have been a team approach, involvement of community leaders and voluntary organizations, and support of the district and state administrators.