Potential impact of SARS COV-2 infection on the performance of serological assays used to diagnose arboviral diseases.

BACKGROUND: The COVID-19 pandemic caused by SARS-CoV-2 was first described in December 2019, in China. In addition, there has also been an increase in arboviral infections in recent years. As both infections have similar symptoms, misdiagnosis may occur if both outbreaks occur at the same time. OBJECTIVE: Our objective was to assess the potential impact of SARS-CoV-2 infection on diagnostic assays used for arboviral diseases. MATERIALS AND METHODS: We conducted this study by testing samples obtained during the precovid phase (before November 2019) and during the covid period (after February 2020). Samples were further grouped as those with acute febrile illness (AFI) and those without. All samples were tested for anti SARS-CoV-2 Ab, Chikungunya and Dengue specific IgM antibodies to evaluate potential serological cross-reactions between COVID-19 and Arbovirus specific antibodies. RESULTS: One sample from the 62 cases of AFI during the pre-covid phase showed seropositivity for SARS-CoV-2 antibodies. Also, in asymptomatic individuals, arboviral seropositivity was significantly higher in the COVID period samples (22%) compared to pre-COVID samples (3%). CONCLUSION: Due to similar clinical symptoms and cross reactions in both infections, relying solely on serological testing for arboviral diagnosis may be less sensitive; other clinical and laboratory parameters may be required.

Pooled sample testing for COVID-19 diagnosis: Evaluation of bi-directional matrix pooling strategies.

In the on-going COVID-19 pandemic, pooled testing of samples by RT-PCR has been recommended at certain scenarios to increase labs' testing capacity and reduce cost of testing. This paper describes the evaluation of bi-directional matrix pooling strategies with clinical samples in a 5 × 5 and 10 × 10 matrix. Nasopharyngeal swab samples in viral transport medium (VTM) previously tested (positive or negative) by real time RT-PCR for SARS-CoV-2 were used for these experiments. Ten sets of 5 × 5 (250 samples) and ten sets of 10 × 10 (1000 samples) pooling of samples in both directions was done with known positive samples introduced at random positions. Extracted nucleic acid was tested for SARS-CoV-2 E-gene by RT-PCR. Sensitivity or concordance and feasibility of matrix pooling were assessed in comparison to direct RT-PCR testing. In comparison to direct testing, the overall concordance was 86.6% for 5 × 5 pooling, 73.3% for 10 × 10 with 200 µL extraction volume and 86.6% for 10 × 10 with 400 µL extraction volume. Bi-directional matrix pooling can be adopted with advantage over conventional direct or pool testing for COVID-19 by RT-PCR under the following conditions: i) sample positivity rate of ≤ 5%, ii) matrix pool size of 8-10 samples, iii) use of min. 40 µL VTM from each sample and iv) utilization of automated liquid handling equipment, if available, for sample addition to avoid human errors.

Epidemiological and molecular investigation of a hepatitis A outbreak in Tamil Nadu, Southern India.

INTRODUCTION: Hepatitis A virus causes an acute infection mainly in young children. The present study was carried out to characterize the nature of hepatitis A virus (HAV) involved in an outbreak of jaundice in children. METHODOLOGY: Serum and stool samples from five children were sampled from among 26 clinically diagnosed jaundice cases. HAV IgM ELISA and PCR were used for confirmatory diagnosis and molecular characterization by direct amplicon sequencing and analysis. RESULTS: All the serum samples collected from the symptomatic cases were found to be positive for Anti-HAV IgM ELISA as were all the serum samples and stool samples using semi-nested PCR. Phylogenetic analysis revealed that the HAV involved in the outbreak belonged to genotype IIIA. CONCLUSIONS: The infection was caused by HAV genotype IIIA. Improved access to clean drinking water, sanitation around drinking water sources and routine chlorination of drinking water in poor and developing countries are needed, as well as childhood HAV vaccination under regular immunization programs in endemic countries.

Pooled testing for COVID-19 diagnosis by real-time RT-PCR: A multi-site comparative evaluation of 5- & 10-sample pooling.

BACKGROUND & OBJECTIVES: Public health and diagnostic laboratories are facing huge sample loads for COVID-19 diagnosis by real-time reverse transcription-polymerase chain reaction (RT-PCR). High sensitivity of optimized real-time RT-PCR assays makes pooled testing a potentially efficient strategy for resource utilization when positivity rates for particular regions or groups of individuals are low. We report here a comparative analysis of pooled testing for 5- and 10-sample pools by real-time RT-PCR across 10 COVID-19 testing laboratories in India. METHODS: Ten virus research and diagnostic laboratories (VRDLs) testing for COVID-19 by real-time RT-PCR participated in this evaluation. At each laboratory, 100 nasopharyngeal swab samples including 10 positive samples were used to create 5- and 10-sample pools with one positive sample in each pool. RNA extraction and real-time RT-PCR for SARS-CoV-2-specific E gene target were performed for individual positive samples as well as pooled samples. Concordance between individual sample testing and testing in the 5- or 10-sample pools was calculated, and the variation across sites and by sample cycle threshold (Ct) values was analyzed. RESULTS: A total of 110 each of 5- and 10-sample pools were evaluated. Concordance between the 5-sample pool and individual sample testing was 100 per cent in the Ct value ≤30 cycles and 95.5 per cent for Ctvalues ≤33 cycles. Overall concordance between the 5-sample pooled and individual sample testing was 88 per cent while that between 10-sample pool and individual sample testing was 66 per cent. Although the concordance rates for both the 5- and 10-sample pooled testing varied across laboratories, yet for samples with Ct values ≤33 cycles, the concordance was ≥90 per cent across all laboratories for the 5-sample pools. INTERPRETATION & CONCLUSIONS: Results from this multi-site assessment suggest that pooling five samples for SARS-CoV-2 detection by real-time RT-PCR may be an acceptable strategy without much loss of sensitivity even for low viral loads, while with 10-sample pools, there may be considerably higher numbers of false negatives. However, testing laboratories should perform validations with the specific RNA extraction and RT-PCR kits in use at their centres before initiating pooled testing.

Human gingival derived neuronal cells in the optimized caffeic acid hydrogel for hemitransection spinal cord injury model.

Spinal cord injury induces scar formation causes axonal damage that leads to the degeneration of axonal function. Still, there is no robust conceptual design to regenerate the damaged axon after spinal injury. Therefore, the present study demonstrates that human gingival derived neuronal stem cells (GNSCs) transplants in the injectable caffeic acid bioconjugated hydrogel (CBGH) helps to bridge the cavity and promote the engraftment and repopulation of transplants in the injured spinal tissue. Our study reports that the bioluminescence imaging in vivo imaging system (IVIS) provides a satisfactory progression in CBGH-GNSCs transplants compare to lesion control and CBGH alone. Immune regulators interleukin-6 (IL-6), tumor necrosis factor-α, neutrophil elastase are decreased, IL-10 is increased. Likewise, immunostaining (TAU/TUJ-1, SOX-2/NeuN, MAP-2/PSD93, NSE, S100b, and GFAP) shown repopulated cells. Also, TRA-1-81 expression confirms the absence of immune rejection in the CBGH-GNSCs transplants. However, locomotor recovery test, gene (IL-6, CASPASE3, p14-ARF, VEGF, LCAM, BDNF, NT3, NGN2, TrKc, FGF2, Sox-2, TUJ-1, MAP-2, Nestin, and NeuN) and protein expression (TAU, TUJ-1, SOX-2 MAP-2, PSD93, NeuN, TRA-1-81, GFAP, TAU, and MBP) shows functional improvements in the CBGH-GNSCs group. Further, GABA and glutamine level demonstrates the new synaptic vesicle formation. Hence, the CBGH scaffold enhances GNSCs transplants to restore the injured spinal tissue.

Naringenin Decreases α-Synuclein Expression and Neuroinflammation in MPTP-Induced Parkinson's Disease Model in Mice.

The present study was designed to ascertain the role of naringenin (NGN), a citrus fruit flavanone, against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced α-synuclein (SYN) pathology and neuroinflammation in a mouse model. NGN was administered to C57BL/6J mice once a day for 5 consecutive days prior to the MPTP intoxication. On day 5, 40-50 min after the NGN or vehicle administration, MPTP was injected in two divided doses (2× 40 mg/kg, i.p. at 16 h apart). The animals were observed for motor functions 48 h after the first MPTP injection. The animals were then euthanized, the brains collected to analyze SYN pathology, cytokines, and oxidative stress levels in the substantia nigra region. The NGN significantly downregulated SYN and upregulated dopamine transporter (DAT) and tyrosine hydroxylase (TH) protein expressions. It also downregulated tumor necrosis factor-α (TNFα) and interleukin 1ß (IL1ß) mRNA expressions and improved superoxide dismutase levels. It also reduced glutathione levels when compared to vehicle-treated PD animals. The upregulation of TH corroborates to an increase in dopamine, DOPAC, and homovanillic acid turnover and motor functions with NGN treatment. To summarize, NGN, a dietary flavone, has the potential to counteract MPTP-induced dopaminergic degeneration by regulating SYN pathology, neuroinflammation, and oxidative stress. This warrants the investigation of NGN's potential effects in a genetic model of PD.

Troxerutin with copper generates oxidative stress in cancer cells: Its possible chemotherapeutic mechanism against hepatocellular carcinoma.

Troxerutin (TXER) a rutin derivative is known for its anticancer effect against hepatocellular carcinoma (HCC). As part of large study, recently we have shown TXER interact with genetic material and its anti-mutagenic property. In the present study we have explored its possible mode of action in HCC. Since TXER alone did not show significant anticancer effect on Huh-7 cells, in vitro biochemical assays were performed for determining anticancer efficacy of TXER + metal complex using transition metals such as Cu, Zn, and Fe. The anticancer efficacy of TXER + Cu on Huh-7 cells were evaluated using MTT assay, DCFDA, JC-1 staining, comet assay, cell cycle analysis, immunocytochemistry, and Western blotting. Non-toxic nature of TXER was analyzed on primary rat hepatocytes. The in vivo efficacy of TXER was tested in N-nitrosodiethylamine initiated and γ-benzene hexachloride and partial hepatectomy promoted rat liver cancer. Liver markers, transition metal levels, histopathological examination, and expression levels of GST-P, 8-OHdG and Ki-67 were studied to assess the in vivo anticancer effect of TXER. We observed that TXER + Cu induced extensive cellular death on Huh-7 cells through generating free radicals and did not possess any toxic effect on normal hepatocytes. The in vivo studies revealed that TXER possess significant anti-cancer effect as assessed through improved liver markers and suppressed GST-P, 8-OHdG, and Ki-67 expression. TXER treatment reduced the hepatic Cu level in cancer bearing animals. Current study brings the putative mechanism involved in anti-cancer effect of TXER, further it will help to formulate phytoconstituents coupled anti-cancer drug for effective treatment of HCC.

Molecular characterization, isolation, pathology and pathotyping of peafowl (Pavo cristatus) origin Newcastle disease virus isolates recovered from disease outbreaks in three states of India.

Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.

Influences of Chronic Mild Stress Exposure on Motor, Non-Motor Impairments and Neurochemical Variables in Specific Brain Areas of MPTP/Probenecid Induced Neurotoxicity in Mice.

Parkinson's disease (PD) is regarded as a movement disorder mainly affecting the elderly population and occurs due to progressive loss of dopaminergic (DAergic) neurons in nigrostriatal pathway. Patients suffer from non-motor symptoms (NMS) such as depression, anxiety, fatigue and sleep disorders, which are not well focussed in PD research. Depression in PD is a predominant /complex symptom and its pathology lies exterior to the nigrostriatal system. The main aim of this study is to explore the causative or progressive effect of chronic mild stress (CMS), a paradigm developed as an animal model of depression in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (25 mg/kg. body wt.) with probenecid (250 mg/kg, s.c.) (MPTP/p) induced mice model of PD. After ten i.p. injections (once in 3.5 days for 5 weeks) of MPTP/p or exposure to CMS for 4 weeks, the behavioural (motor and non-motor) impairments, levels and expressions of dopamine (DA), serotonin (5-HT), DAergic markers such as tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporters-2 (VMAT 2) and α-synuclein in nigrostriatal (striatum (ST) and substantia nigra (SN)) and extra-nigrostriatal (hippocampus, cortex and cerebellum) tissues were analysed. Significantly decreased DA and 5-HT levels, TH, DAT and VMAT 2 expressions and increased motor deficits, anhedonia-like behaviour and α-synuclein expression were found in MPTP/p treated mice. Pre and/or post exposure of CMS to MPTP/p mice further enhanced the MPTP/p induced DA and 5-HT depletion, behaviour abnormalities and protein expressions. Our results could strongly confirm that the exposure of stress after MPTP/p injections worsens the symptoms and neurochemicals status of PD.

Detection and partial genetic characterisation of a novel variant of Avian nephritis virus in Indian poultry flocks showing diverse clinical signs.

Avian nephritis virus (ANV) infects poultry flocks worldwide, but no confirmed cases have been reported from India so far. In the current study, disease investigation was carried out in 21 broiler flocks at different parts of India with clinical signs of nephritis, uneven and stunted growth, diarrhoea, reduced body weight, and mortality up to 9.72%. Out of the 21 flocks screened, two were found positive for ANV in RT-PCR assay. BLAST analysis revealed that the ANV of Indian origin was closely related to ANV-1 strains reported from Japan, Hungary and China. However, comparison of a small portion (~12% of nucleotides, i.e. ~60 nts, common site for ANV-1 and ANV-3, position 2200-2260 of ORF 1a gene) of the Indian ANV sequence with ANV-3 sequences revealed 89-93% identities with different ANV-3 isolates. Phylogenetically, ANV-1 forms three clades, and the Indian ANV clustered under clade II. This study confirms the existence of ANV in Indian poultry flocks and is the first report on the molecular detection and genetic characterisation of ANV from India.

Molecular detection and characterization of infectious laryngotracheitis virus (Gallid herpesvirus-1) from clinical samples of commercial poultry flocks in India.

Although the existence of infectious laryngotracheitis virus (ILTV) in India was first reported in 1964, no reports are available regarding its molecular detection and characterization. The present study was aimed to detect and characterize ILTV from recent respiratory disease complex (RDC) outbreaks of commercial poultry flocks in different parts of the country by using envelope glycoprotein G gene (US4 gene) based PCR and sequencing. A total of thirty two flocks with a history of RDC were investigated. Overall, all the strains/breeds of birds and all ages of birds are equally susceptible and depending on the severity, the clinical signs and gross lesions were varied. Out of 32 flocks investigated 10 were found positive for ILTV infection by PCR. The phylogenetic analyses of eight representative sequences in the present study deciphered that Indian ILT viruses are closely related to chicken embryo origin vaccine strains of Italy, USA, China and Brazil.

Emergence of Avian Infectious Bronchitis Virus and its variants need better diagnosis, prevention and control strategies: a global perspective.

Growth in poultry sector is being challenged due to increased incidence and re-emergence of diseases caused due to evolution of several viral pathogens and use of live vaccines. Piles of economic losses are encountered due to these diseases. Avian Infectious Bronchitis (IB), caused by Corona virus, is OIE-listed disease and characterized by respiratory, renal and urogenital involvements, causing high mortality. Economic losses are encountered due to loss of productive performance of both egg and meat-type chickens. Variant viruses evolve due to spontaneous mutations and recombinations, causing disease in vaccinated flocks of all ages. Serotyping and genotyping are the common methods of classification of IBV strains. The virus has 4 clusters, grouped into 7 serotypes and the most important strains are Massachusetts, Connecticut, Arkansas, Gray, Holte and Florida along with numerous others, distributed round the globe. Several conventional and molecular diagnostic methods have been described for the diagnosis of IB in chickens. 'All-in/all-out' operations of rearing along with good biosafety measures forms the basis of prevention, whereas vaccination forms the backbone of IB control programme. Both live and inactivated (oil emulsified) conventional vaccines are available. The new generation vaccines (recombinant and vector-based) developed against locally prevailing IBV strains may be more helpful and avoid the reversion of virulence in live vaccine viruses. The present review deals with all these perspectives of this important emerging poultry pathogen.

Upregulated Myc expression in N-methyl nitrosourea (MNU)- induced rat mammary tumours.

BACKGROUND: The most common incident cancer and cause of cancer-related deaths in women is breast cancer. The Myc gene is upregulated in many cancer types including breast cancer, and it is considered as a potential anti-cancer drug target. The present study was conducted to evaluate the Myc (gene and protein) expression pattern in an experimental mammary tumour model in rats. MATERIALS AND METHODS: Thirty six Sprague Dawley rats were divided into: Experimental group (26 animals), which received the chemical carcinogen N-methyl nitrosourea (MNU) and a control group (10 animals), which received vehicle only. c-Myc oncoprotein and its mRNA expression pattern were evaluated using immunohistochemistry (IHC) and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), respectively, in normal rat mammary tissue and mammary tumours. The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as internal control for semi-quantitative RT-PCR. RESULTS: Histopathological examination of mammary tissues and tumours from MNU treated animals revealed the presence of premalignant lesions, benign tumours, in situ carcinomas and invasive carcinomas. Immunohistochemical evaluation of tumour tissues showed upregulation and heterogeneous cellular localization of c-Myc oncoprotein. The expression levels of c-Myc oncoprotein were significantly elevated (75- 91%) in all the tumours. Semi-quantitative RT-PCR revealed increased expression of c-Myc mRNA in mammary tumours compared to normal mammary tissues. CONCLUSIONS: Further large-scale investigation study is needed to adopt this experimental rat mammary tumour model as an in vivo model to study anti-cancer strategies directed against Myc or its downstream partners at the transcriptional or post-transcriptional level.

Detection and differentiation of pigeon paramyxovirus serotype-1 (PPMV-1) isolates by RT-PCR and restriction enzyme analysis.

Detection and pathotyping of Newcastle disease virus (NDV) is extremely important because the appearance of virulent virus has significant economic consequences. During 1981 to 1985, infections of racing and show pigeons with an avian paramyxovirus serotype-1 (APMV-1) hit worldwide, and a panzootic occurred due to a variant form of classical NDV. On the basis of pathogenicity and monoclonal antibody binding studies, the virus was termed 'pigeon PMV-1' (PPMV-1). In the past, number of Newcastle disease outbreaks in poultry and other birds has been attributed to PPMV-1. PPMV-1 viruses are known to present difficulty when assessed by conventional in vivo pathogenicity tests. In this study, the technique of reverse transcription-polymerase chain reaction (RT-PCR) and restriction enzyme (RE) analysis was used to detect and differentiate PPMV-1 isolates of Indian origin. Restriction enzyme digestion analysis of RT-PCR-amplified fusion protein (F) gene, encoding for the cleavage activation sites of fusion protein, was carried out with restriction enzymes BglI, HhaI, HaeIII, HinfI, MboI, MspI, PvuII and StyI. A set of only four enzymes HhaI, MspI or HaeIII, MboI and BglI alone were sufficient to differentially detect APMV-1 and PPMV-1 viruses and their pathotypes. In conclusion, RT-PCR followed by RE analysis proved to be useful for detection and differentiation of APMV-1 and PPMV-1 isolates at genomic level.

Fungal/mycotic diseases of poultry-diagnosis, treatment and control: a review.

Fungal/mycotic diseases cause significant economic losses to the poultry industry either due to their direct infectious nature or due to production of mycotoxins, the secondary fungal metabolites produced in grains or poultry feed. Several fungi have created havoc in the poultry industry and some of them cause direct harm to human health due to their zoonotic implications. They are responsible for high morbidity and mortality, especially in young birds and cause stunted growth and diarrhea; and fatal encephalitis. Mycotic dermatitis is a possible health hazard associated with poultry houses. Mycotoxins are the leading cause of producing immunosuppression in birds, which makes them prone to several bacterial and viral infections leading to huge economic losses to the poultry industry. In comparison to bacterial and viral diseases, advances in diagnosis, treatment, prevention and control of fungal diseases in poultry has not taken much attention. Recently, molecular biological tools have been explored for rapid and accurate diagnosis of important fungal infections. Effective prevention and control measures include: appropriate hygiene, sanitation and disinfection, strict biosecurity programme and regular surveillance/monitoring of fungal infections as well as following judicious use of anti-fungal drugs. Precautionary measures during crop production, harvesting and storing and in feed mixing plants can help to check the fungal infections including health hazards of mycotoxins/mycotoxicosis. The present review describes the fungal pathogens causing diseases in poultry/birds, especially focusing to their diagnosis, prevention and control measures, which would help in formulating appropriate strategies to have a check and control on these unwanted troubles to the poultry producers/farmers.

Isolation and molecular characterization of infectious bronchitis virus from recent outbreaks in broiler flocks reveals emergence of novel strain in India.

In this study, two isolates of infectious bronchitis virus (IBV) from field outbreaks in 2008 (India/LKW/56/IVRI/08) and 2010 (India/NMK/72/IVRI/10) from broiler chickens in India were isolated and characterized. Reverse transcription polymerase chain reaction-restriction fragment length polymorphism of the entire S1 gene revealed that these isolates belong to two different genotypes, India/LKW/56/IVRI/08 as Mass strain whereas India/NMK/72/IVRI/10 as of different genotype. Nucleotide sequencing analysis showed that India/LKW/56/IVRI/08 shared 99 % homology with THA280252 (Thailand) and India/NMK/72/IVRI/10 shared greater than 99 % homology with 4/91 pathogenic strain (UK), JP/Wakayama/2/2004 (Japan) and TA03 (China), while the two Indian IBV isolates shared 73 % identity between them. Phylogenetic data allowed classification of two Indian isolates, India/LKW/56/IVRI/08 as having unique lineage within Mass genotype and India/NMK/72/IVRI/10 as of 4/91 genotype. Our study confirmed the presence of 4/91 (793/B) IBV nephropathogenic strain for the first time in India by virus isolation and sequencing.

Recent trends in diagnosis and control of Marek's disease (MD) in poultry.

Marek's Disease (MD), caused by Marek's Disease Virus (MDV) is a highly contagious oncogenic and neuropathic disease of chickens responsible for great economic losses to the poultry industry all around the world and characterized by development of CD4+T cell lymphomas as well as infiltration of nerves and visceral organs by lymphocytes. MD is one of the most common lymphoproliferative diseases of chickens which cause mononuclear cell infiltration in one or more of the following tissues: peripheral nerves, gonads, lymphoid organs, iris, muscle, skin and other visceral organs resulting into development of tumours in visceral organs, paralysis of legs, wings and neck, grey eye (iris) or irregular pupil, vision impairment, blindness, skin lesions and immunosuppression, all of which can be accompanied by non-specific signs such as anorexia, weight loss and poor performance. Today there are evolving highly pathogenic isolates of MDV around the world capable of overwhelming the protection from currently employed vaccines. Thus MD poses a big challenge to the welfare and wellbeing of the poultry with increased condemnation of carcass, loss of productivity and quality products, leading to huge economic losses. It is also an immunosuppressive disease and causes increased susceptibility to other infections. The present review discusses in brief about the Marek's disease, its etiology, conventional and advance tools and techniques being used for its diagnosis, prevention and control strategies in poultry.