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Introduction: Newcastle disease is one of the significant issues in the poultry industry, having catastrophic effects worldwide. The lung is one of the essential organs which harbours Bronchus-associated lymphoid tissue and plays a vital role in the immune response. Leghorn and Fayoumi breeds are known to have differences in resistance to Newcastle disease. Along with genes and long non-coding RNAs (lncRNAs) are also known to regulate various biological pathways through gene regulation. Methods: This study analysed the lung transcriptome data and identified the role of genes and long non-coding RNAs in differential immune resistance. The computational pipeline, FHSpipe, as used in our previous studies on analysis of harderian gland and trachea transcriptome was used to identify genes and lncRNAs. This was followed by differential expression analysis, functional annotation of genes and lncRNAs, identification of transcription factors, microRNAs and finally validation using qRT-PCR. Results and discussion: A total of 8219 novel lncRNAs were identified. Of them, 1263 lncRNAs and 281 genes were differentially expressed. About 66 genes were annotated with either an immune-related GO term or pathway, and 12 were annotated with both. In challenge and breed-based analysis, most of these genes were upregulated in Fayoumi compared to Leghorn, and in timepoint-based analysis, Leghorn challenge chicken showed downregulation between time points. A similar trend was observed in the expression of lncRNAs. Co-expression analysis has revealed several lncRNAs co-expressing with immune genes with a positive correlation. Several genes annotated with non-immune pathways, including metabolism, signal transduction, transport of small molecules, extracellular matrix organization, developmental biology and cellular processes, were also impacted. With this, we can understand that Fayoumi chicken showed upregulated immune genes and positive cis-lncRNAs during both the non-challenged and NDV-challenge conditions, even without viral transcripts in the tissue. This finding shows that these immune-annotated genes and coexpressing cis-lncRNAs play a significant role in Fayoumi being comparatively resistant to NDV compared to Leghorn. Our study affirms and expands upon the outcomes of previous studies and highlights the crucial role of lncRNAs during the immune response to NDV. Conclusion: This analysis clearly shows the differences in the gene expression patterns and lncRNA co-expression with the genes between Leghorn and Fayoumi, indicating that the lncRNAs and co-expressing genes might potentially have a role in differentiating these breeds. We hypothesise that these genes and lncRNAs play a vital role in the higher resistance of Fayoumi to NDV than Leghorn. This study can pave the way for future studies to unravel the biological mechanism behind the regulation of immune-related genes.
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Pollos , Perfilación de la Expresión Génica , Pulmón , MicroARNs , Enfermedad de Newcastle , Virus de la Enfermedad de Newcastle , Enfermedades de las Aves de Corral , ARN Largo no Codificante , Transcriptoma , Animales , Pollos/genética , Pollos/inmunología , Enfermedad de Newcastle/inmunología , Enfermedad de Newcastle/virología , Enfermedad de Newcastle/genética , Pulmón/inmunología , Pulmón/virología , Virus de la Enfermedad de Newcastle/inmunología , Virus de la Enfermedad de Newcastle/genética , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Enfermedades de las Aves de Corral/virología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/genética , MicroARNs/genética , MicroARNs/metabolismo , Regulación de la Expresión Génica , Resistencia a la Enfermedad/genética , Biología Computacional/métodos , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunologíaRESUMEN
Introduction: Newcastle disease is a highly infectious disease caused by the Newcastle Disease Virus (NDV) and has a devastating financial impact on the global chicken industry. It was previously established that Leghorn and Fayoumi breeds of chicken exhibit variable resistance against NDV infection. The harderian gland is the less studied tissue of the chicken, known to play an essential role in the immune response. Methods: Our previous study, we reported differential gene expression and long noncoding RNAs (lncRNAs) between challenged and non-challenged chickens in the Harderian gland transcriptomic data. Now, we report the analysis of the same data studying the differential expression patterns between Leghorn and Fayoumi and between different timepoints during disease. First, the pipeline FHSpipe was used for identification of lncRNAs, followed by differential expression analysis by edgeR (GLM), functional annotation by OmicsBox, co-expression analysis using WGCNA and finally validation of selected lncRNAs and co-expressing genes using qRT-PCR. Results: Here, we observed that Leghorn showed a higher number of upregulated immune-related genes than Fayoumi in timepoint-based analysis, especially during the initial stages. Surprisingly, Fayoumi, being comparatively resistant, showed little difference between challenged and non-challenged conditions and different time points of the challenge. The breed-based analysis, which compared Leghorn with Fayoumi in both challenged and non-challenged conditions separately, identified several immune-related genes and positive co-expressing cis lncRNAs to be upregulated in Fayoumi when compared to Leghorn in both challenged and non-challenged conditions. Discussion: The current study shows that Leghorn, being comparatively more susceptible to NDV than Fayoumi, showed several immune-related genes and positive co-expressing cis lncRNAs upregulated in challenged Leghorn when compared to non-challenged Leghorn and also in different timepoints during challenge. While, breed-based analysis showed that there were more upregulated immune genes and positive cis-lncRNAs in Fayoumi than Leghorn. This result clearly shows that the differences in the expression of genes annotated with immune-related GO terms and pathways, i.e., immune-related genes and the co-expressing cis-lncRNAs between Leghorn and Fayoumi, and their role in the presence of differences in the resistance of Leghorn and Fayoumi chicken against NDV. Conclusion: These immune-genes and cis-lncRNAs could play a role in Fayoumi being comparatively more resistant to NDV than Leghorn. Our study elucidated the importance of lncRNAs during the host defense against NDV infection, paving the way for future research on the mechanisms governing the genetic improvement of chicken breeds.
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The Australian Partnership for Preparedness Research on InfectiouS disease Emergencies (APPRISE) has developed a virtual biobank to support infectious disease research in Australia. The virtual biobank (https://apprise.biogrid.org.au) integrates access to existing distributed infectious disease biospecimen collections comprising multiple specimen types, including plasma, serum, and peripheral blood mononuclear cells. Through the development of a common data model, multiple collections can be searched simultaneously via a secure web portal. The portal enhances the visibility and searchability of existing collections within their current governance and custodianship arrangements. The portal is easily scalable for integration of additional collections.
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Bancos de Muestras Biológicas , Enfermedades Transmisibles , Humanos , Australia/epidemiología , Leucocitos Mononucleares , Manejo de EspecímenesRESUMEN
The complete genome (2298 nucleotides) of the economically important and immunosuppressive, chicken infectious anemia virus (CAV), from a disease outbreak in a layer flock is discussed. This is the first report of a complete genome sequence of CAV from India. The phylogenetic analyses grouped this isolate with CAV genogroup IIIb based on both complete genome and capsid protein (VP1) sequences. The analyses further revealed the presence of CAV genogroups II, IIIa and IIIb in India. The VP1 sequence identity ranged between 84.4 to 99.3% with that of the Indian isolates and carried a unique substitution at position 447 (serine instead of threonine). Two novel amino acid substitutions were observed at position 52 of VP1 (serine instead of proline) and at position 26 of VP2 (asparagine instead of serine). Sequence analyses of VP1, VP2 and VP3 suggested that the isolate could be attenuated. Comparison with CAV variants, isolated from mammalian species, showed similarities in the numbers of certain transcription factor binding sites in the non-coding regions. Recombination analysis detected no recombination events in this isolate. Further investigations are needed to understand the implications of the unique features of this isolate on viral virulence.
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Virus de la Anemia del Pollo , Infecciones por Circoviridae , Enfermedades de las Aves de Corral , Animales , Virus de la Anemia del Pollo/genética , Pollos , Infecciones por Circoviridae/epidemiología , Infecciones por Circoviridae/veterinaria , Brotes de Enfermedades , Genotipo , Mamíferos , Filogenia , SerinaRESUMEN
Marek's disease (MD) is a re-emerging viral disease of chickens and a serious economic threat to the poultry industry worldwide. Continuous surveillance with molecular investigation is essential to monitor the emergence of virulent Marek's disease virus (MDV) strains and to devise any appropriate vaccination strategy and implement bio-security programmes. In the present study, we investigated the cases of MD outbreaks in vaccinated poultry flocks. The MD outbreak was confirmed through necropsy (mainly visceral tumours), histopathology and viral gene specific PCR. The pathotypes of the field MDV strains were assessed by molecular analysis of three virulence-associated genes, meq, pp38 and vIL-8. The Meq sequence of the field strains analyzed in this study lacked the 59 aa unique to mild strains, indicating that they are potentially virulent strains. Mutation at position 71 and the presence of five proline rich repeats in the transactivation domain, both associated with virulence were observed in these strains; however, the signature sequences specific to very virulent plus strains were absent. Phylogenetic analysis of meq oncogene sequences revealed clustering of the field strains with North Indian strains and with a very virulent plus ATE 2539 strain from Hungary. Analyses of pp38 protein at positions 107 and 109 and vIL-8 protein at positions 4 and 31 showed signatures of virulence. Sequence and phylogenetic analysis of oncogene and virulence-associated genes of field MDVs from vaccinated flock indicated that these strains possessed molecular features of virulent strains.
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Herpesvirus Gallináceo 2 , Enfermedad de Marek , Enfermedades de las Aves de Corral , Animales , Pollos , Genotipo , Herpesvirus Gallináceo 2/genética , Enfermedad de Marek/epidemiología , Enfermedad de Marek/prevención & control , Filogenia , Aves de Corral , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/prevención & control , Virulencia/genéticaRESUMEN
Oncogenic tumour diseases are major threat to poultry industry. Marek's disease (MD), avian leukosis (ALV) and reticulosendotheliosis virus (REV) are the major tumour causing immunosuppressive viral diseases of chicken. A total of 120 tissue samples presented with tumour lesions from different chicken flocks of coloured broiler, layer breeders and native chicken breeds were screened for MDV, ALV and REV by histopathology and virus specific PCRs individually. Presence of oncogenic viruses in the samples were screened by virus specific PCR. A total of 47 samples were detected either with single infection or dual infection with these viruses. Out of 47, 17 were detected with either one of the viruses and remaining 30 with any of the two viruses. REV was the major cause of tumour in the present samples followed by MDV. ALV was not detected alone, it was either with MD or REV. All 5 ALV positive samples were detected with ALV-E subtype. REV was detected predominantly (22 out of 25 positives) as single infection rather than co-infection.
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Infectious bronchitis virus isolate (IND/AHL/16/01) from a disease outbreak characterized by nephritis, gout and mortality in coloured layer pureline at Directorate of Poultry Research, India was characterized as nephropathogenic strain by S1 genotyping and phylogenetic analysis. Serotyping with homologous and heterologous serum (M41) by virus neutralization assay in embryonated chicken eggs (ECE) showed indices of 7.3 and 2.3 respectively. Pathogenesis, tissue tropism and host immune response induced by this isolate were investigated in experimentally infected chicken. A total of 150, twenty days old seronegative Vanaraja birds were inoculated through intranasal and intravenous route using 104.7 Embryo infective dose50 (EID50/ml). Infected chickens were sacrificed at 4 h, 1, 2, 3, 5, 7, 11, 13, 15- and 20-days post-infection (dpi) for necropsy. Tissues were collected for histopathology and virus detection by isolation in ECE and by reverse transcription- PCR (RT-PCR). Serum was also collected at these intervals to investigate the specific antibody response induced. The symptoms started as early as 3 dpi and included primarily wet droppings, diarrhoea, dehydration rather than respiratory symptoms. Gross lesions were prominent in kidneys including mottling and congestion. Virus isolation and RT-PCR detection indicated the presence of virus as early as 4 h post-infection in trachea and 24 h in kidney and lungs and from 2 dpi in caecal tonsil. The host antibody response after experimental infection in serum by ELISA indicated that the protective titres were induced from 13 dpi and peaked at 35 dpi and declined thereafter. Overall, this isolate is nephropathogenic and capable of inducing severe nephritis and production loss in broilers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00693-4.
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A real time polymerase chain reaction (real-time PCR) assay was developed to detect and quantify the chicken infectious anemia virus (CIAV). The two sets of primers specific to VP1 region of CIAV were designed and their sensitivity and efficacy were studied. Both the primers designed in this study were highly sensitive and were able to detect upto 0.01 fg/µl or 82 × 102 copy number of plasmid DNA. The efficiency of the real time PCR was 100.9%. The results have also shown that the present qPCR assay is 100 times more sensitive than regular qualitative PCR. Both primer sets were validated using 28 field poultry samples and showed good results. The optimized real-time quantitative PCR will be useful in quick detection of field outbreaks, sub-clinical infection in poultry flocks, virus pathogenesis studies and for detecting vaccine contamination.
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Toll-like receptor (TLR) agonists are emerging as promising vaccine adjuvants and immunomodulators in poultry against many diseases. Infectious bursa disease (IBD) still remains as a major threat to poultry industry. Improving the vaccine mediated immune response would help in better protection against IBD virus infection. Adjuvant potential of TLR3 agonist, Polinosinic polycytidylic acid (Poly I:C) with different IBD vaccines has been analyzed in chicken in the present study. Intermediate, intermediate plus IBD vaccine, bursaplex vaccine and their respective poly I:C combinations were used for immunization of chicken. IBD specific antibody titers, bursa to body weight ratio, body weight gain and bursal lesion scores were evaluated at weekly interval in different immunization groups. Fold changes in cytokines IL-1ß and IFN-γ mRNA expression levels in spleen were also analyzed in different groups. Intermediate plus IBD vaccine induced significantly (P ≤ 0.05) higher IBD specific antibody response at 35 days of age than other groups with comparatively lower body weight gain and moderate bursal lesion score. Poly I:C co-administration with intermediate IBD vaccine and bursaplex vaccine improved the IBD specific antibody titers, better body weight gain and moderately less bursal lesion score. However, Poly I:C combination with intermediate plus IBD vaccine did not improve the specific immune response. IL-1ß levels were up-regulated in intermediate plus and bursaplex group, whereas IFN-γ m RNA expression levels were upregulated in intermediate IBD with Poly I:C group. In conclusion, poly I:C co-administration with intermediate IBD and bursaplex vaccine was beneficial and improved the specific immune response with least immunosuppression and bursal damage.
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Infecciones por Birnaviridae/veterinaria , Pollos , Inmunidad , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Poli I-C/administración & dosificación , Enfermedades de las Aves de Corral/prevención & control , Receptor Toll-Like 3/agonistas , Vacunas Virales/administración & dosificación , Animales , Infecciones por Birnaviridae/prevención & control , Infecciones por Birnaviridae/virología , Enfermedades de las Aves de Corral/virologíaRESUMEN
Indigenous chicken breeds are considered to be more disease tolerant than exotic chicken breeds especially for the bacterial diseases. Nicobari and Vanaraja chicken were evaluated for the survivability/mortality patterns and host immune response after experimental infection with P. multocida A1 isolate. The birds were inoculated with 1.9 × 105 CFU/mL through intraperitoneal (I/P) and intranasal (I/N) routes at 2 different age groups viz., 12 wk and 18 wk. Symptoms, mortality rates, lesions in dead birds were observed; Serum from surviving birds of different groups from both breeds were collected at 5, 14, 21, 28, 35 and 42nd d and specific antibody titers were measured by indirect ELISA. At 12 wk of age, the mortality rates were 100% and 16% in birds inoculated by I/P and I/N routes respectively in Vanaraja birds; whereas the mortality rates were 50% and 16% I/P and I/N routes respectively in Nicobari birds. At 18 wk of age the mortality rates were 16% and 50% for I/P routes in Nicobari and Vanaraja birds respectively. The mortality rates were 16% for I/N route in both Nicobari and Vanaraja birds. Lesions such as necrotic foci on liver, congestion in the liver were observed in dead birds. Serum titers were significantly (P < 0.05) higher in surviving Nicobari birds inoculated through I/P route followed by I/N route. The peak titers were reached on 14th d postinfection and declined thereafter. However, no significant difference was found in I/N route of inoculation between 2 breeds. Nicobari chicken breed showed significantly higher survivability and longer mean death time than Vanaraja germplasm to experimental Pasteuralla infection at both the ages however the survivability rate in both breeds improved at later ages.
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Infecciones por Pasteurella , Pasteurella multocida , Enfermedades de las Aves de Corral , Animales , Pollos , Resistencia a la Enfermedad , Tolerancia Inmunológica , Infecciones por Pasteurella/veterinariaRESUMEN
BACKGROUND: High-frequency 10-kHz spinal cord stimulation (10-kHz SCS) has shown promise in multicenter prospective trials for the management of chronic back and leg pain. Traditional spinal cord stimulation (t-SCS) has a long history of effectiveness in chronic neuropathic syndromes but not uncommonly can fail to provide long-term relief, leaving a significant group of patients with unsatisfactory outcomes. There is mounting evidence that 10-kHz SCS may offer relief in this subset of patients. METHODS: The purpose of this retrospective analysis was to report a single-institution long-term experience of 10-kHz SCS in patients who did not get adequate pain relief with prior t-SCS devices. A temporary trial of 10-kHz SCS was carried out for 7 days, and those experiencing an average of 50% reduction in pain intensity underwent implantation. Patients were classified as moderate responders if relief was 31% to 50% and excellent responders if pain relief exceeded 50%. RESULTS: Thirty-one patients who had experienced failed t-SCS primarily from poor paresthesia coverage underwent a trial of 10-kHz SCS and 29 underwent implantation. Twenty-eight patients were available for analysis, with 57.1% experiencing 30% response and 46.4% experiencing excellent response at a median follow-up of 21.2 (±8.4) months. CONCLUSIONS: This small single-institution study suggests that a significant proportion of patients with previously failed t-SCS may achieve clinically meaningful and durable pain relief with 10-kHz SCS.
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Dolor Crónico/terapia , Manejo del Dolor/métodos , Terapia Recuperativa/métodos , Estimulación de la Médula Espinal/métodos , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
The aim of the present study was to develop a questionnaire in Kannada language which assesses the handicapping consequences of dizziness. A cross sectional study design and a convenient type of sampling was used to recruit the participants. A total of 36 participants in age range of (18-60 years of age) who reported to have dizziness or vertigo for at least three months of period and who knew to read and write in kannada language participated. The overall questionnaire was found to have an internal consistency α = 0.935 on cronbach's alpha test and for test retest reliability (r = 0.988) on intra-class correlation coefficient measure. The present studies provide International Classification of Functioning, Disability and Health based questionnaire in kannada which can be used in the clinical set up to assess the quality of life (QOL) in individuals with Vertigo or Dizziness. It will also help to understand the impact of dizziness on QOL from individual's perspective.
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Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) that mediate first line of host defence to pathogens. TLR agonists are potent immunostimulatory agents that help to prime a robust adaptive immune response. In the present study, adjuvant potential of Poly I:C and lipopolysaccharide (LPS) were evaluated with live Newcastle disease virus (NDV) vaccine. Cornish chickens were immunized with live Newcastle disease virus (NDV) vaccine (R2B-mesogenic strain) adjuvanted either with Poly I:C (TLR3 agonist) or LPS-TLR4 agonist and both. Humoral Immune response to ND vaccine was evaluated through haemagglutination inhibition (HI) test and ELISA, while the cellular immune response (CMI) was quantified by lymphocyte transformation test (LTT). IL-1ß cytokine mRNA levels in spleen tissue were also quantified by real time PCR. The results suggest that TLR3 and TLR4 agonists are an efficient immune-stimulators separately, as LPS co-administered group has shown significantly higher serum titre on second week post-immunization and Poly I:C group on third week post-immunization both by HI and ELISA (P < 0.01), however, the combined administration of both LPS and Poly I:C did not give any complementary effect on serum titre. There were no significant differences in stimulation indices (SI) and IL-1ß cytokine levels between groups at different intervals post-immunization. Hence, TLR agonists LPS followed by Poly I:C could be used as adjuvant to enhance the immune response to NDV vaccine in chicken.
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Inmunidad Humoral/efectos de los fármacos , Lipopolisacáridos/farmacología , Enfermedad de Newcastle/inmunología , Poli I-C/farmacología , Receptor Toll-Like 3/agonistas , Receptor Toll-Like 4/agonistas , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Animales , Pollos , Ensayo de Inmunoadsorción Enzimática , Inmunidad Celular/efectos de los fármacos , Lipopolisacáridos/administración & dosificación , Virus de la Enfermedad de Newcastle/inmunología , Poli I-C/administración & dosificaciónRESUMEN
This article reviews Nevro Corporation's (CA, USA) Senza System high-frequency spinal cord stimulator (HF10 SCS). HF10 therapy has been shown to be noninferior to traditional low-frequency spinal cord stimulation (SCS), and in some studies, has been shown to have long-term effects that are superior compared with traditional low-frequency SCS in treating both back and leg pain. In addition, safety profiles for the HF10 SCS system were equivalent to traditional SCS. HF10 SCS is based on spinal anatomic mapping and does not require intraoperative paresthesia mapping for placement, thereby improving procedural experience for patients and reducing operating room time. HF10 SCS has altered the nature of SCS therapy in a manner that treats more individuals with chronic, refractory leg and back pain than in the past, and is widely used in the USA, Europe and Australia.
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Dolor Crónico/terapia , Estimulación de la Médula Espinal/instrumentación , Estimulación de la Médula Espinal/métodos , Dolor de Espalda/terapia , Humanos , Pierna , ParestesiaRESUMEN
Early and rapid detection of dengue virus (DENV) infection during the acute phase of illness is crucial for proper patient management and prevention of the spread of the infection. In the present study, the standardization and validation of a one step, four tube reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) for rapid detection and serotyping of the DENV targeting NS1 gene using the Genie® II flourometer was carried out. The performance of the RT-LAMP was compared to RT-PCR, CDC 1-4 Real time PCR and the NS1 antigen ELISA, IgM and IgG anti DENV antibodies. Acute DENV infection was confirmed in 250/300 patients suspected clinically of DENV infection. RT- LAMP and CDC 1-4 Real time PCR assay was positive in 148/250 patients, while 92/250 patients were positive for anti- Dengue IgM and IgG antibodies. The RT-LAMP assay and the CDC real-time RT-PCR assay showed high concordance (k=1.0). The detection rate of acute DENV infection improved to 96% (240/250) when the results of RT-LAMP were combined with NS1 Ag, IgM and IgG ELISA. The RT-LAMP had a detection limit of 100 copies for DEN-1 and DEN-2, 10 copies for DEN-3 and DEN-4 compared to 1000 copies for DEN-1 and DEN-2, 100 copies for DEN-3 and DEN-4 by the conventional RT-PCR. The assay showed 100% specificity. The RT-LAMP assay developed in this study has potential use for early clinical diagnosis, serotyping and surveillance of DENV infection in endemic countries such as India.
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Virus del Dengue/clasificación , Virus del Dengue/aislamiento & purificación , Dengue/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Transcripción Reversa , Dengue/virología , Virus del Dengue/genética , Humanos , India , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico/normas , ARN Viral/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Serogrupo , Centros de Atención Terciaria , Factores de Tiempo , Proteínas no Estructurales Virales/genéticaRESUMEN
Dengue is the most rapidly spreading mosquito-borne viral disease in the world, and as a larger proportion of the population is being affected, more unusual manifestations are being reported. Very few studies have documented unusual manifestations of dengue in South India. This prospective study was undertaken from July 2011 to June 2013 to document rare manifestations of dengue fever in 175 hospitalized patients. The clinical diagnosis was confirmed by the detection of NS1Ag, dengue IgM, or IgG by ELISA and/or a RT-PCR and CDC real-time PCR for dengue virus (DENV) RNA. The daily profiles of the hematological and biochemical investigations were followed and recorded. Unusual and rare manifestations of dengue were documented for 115 patients (66 %). Hepatitis was observed in 70 % of the cases. Pleural effusion was seen in 11 %, acute renal failure in 10 %, neurological complications such as encephalitis in 7.4 %, myocarditis in 9 %, and bleeding gastric ulcers in 3.4 % of the cases. DENV serotype 2 was more prevalent in patients with unusual manifestations of dengue in our study. The WHO classification system does not include unusual and rare manifestations; hence, it is essential to be aware of these manifestations and closely monitor them for better clinical management and outcome of patients.