Comparison of multiplex RT-PCR with virus isolation for detection, typing and sub-typing of influenza virus from influenza-like illness cases.

Purpose: Infl uenza epidemics and periodic pandemics occur worldwide resulting in signifi cant mortality, morbidity and economic loss. There is need for a sensitive, rapid and cost-effective assay to detect, type and sub-type infl uenza viruses, as cell culture has a long turnaround time. Materials and Methods: Nasopharyngeal swabs were collected from patients presenting with infl uenza-like illness (ILI) at AIIMS OPD and Primary Health Centre Ballabhgarh (Haryana). From June 2007 to January 2009 and then from September to November 2009, of 1567 specimens collected, 544 were randomly selected and were tested by virus culture using Madin-Darby Canine Kidney (MDCK) cells and by reverse transcription polymerase chain reaction (RT-PCR) for infl uenza A using primers for matrix gene and for infl uenza B using non-structural gene (NS) primers. All infl uenza A positives were sub-typed using primers for HA and NA genes of A/H1, A/H3. A separate multiplex RT-PCR having primers from matrix and HA genes of pandemic A (H1N1) pdm09 viruses was carried out on samples collected after September 2009. Results: Of the 544 samples, 136 (25%) were positive for infl uenza by RT-PCR. Further typing analysis revealed 86 (63.2%) were typed as infl uenza A and 47 (34.5%) as infl uenza B viruses and 3 (2%) samples showed dual infection with infl uenza A and B. Of the 86 infl uenza A positive samples 48 (55.8%) were identifi ed as seasonal infl uenza A/H1N1, 22 (25.6%) as A (H1N1) pdm09 and 16 (18.6%) as A/H3N2. Comparison of infl uenza positivity using virus culture revealed that only 97/136 (71.3%) were infl uenza positive. Sensitivity of viral detection was lowest for seasonal A/H1 (26/48; 54%), followed by H3N2 (11/16; 68.7%) and infl uenza B (38/47; 80.8%); all infl uenza A/H1N1pdm09 viruses were detected by both methods. Conclusion: RT-PCR is a sensitive, low cost and rapid screening test for diagnosing infl uenza infection during epidemics and pandemics. mRT-PCR increased the detection rates for infl uenza by 28.6% as compared with virus isolation and thus is a useful assay in both diagnostic and epidemiological settings in resource poor countries.

Biochemical Constituents in Malformed Tissues of Pearl Millet Cultivars Caused by Aggressive Pathotype of Sclerospora graminicola Causing Downy Mildew Disease.

Downy mildew (DM) of pearl millet [Pennisetum glaucum (L.) R. Br.] caused by Sclerospora graminicola is the most widespread and destructive disease. In DM affected plants disease symptoms appear suddenly with the emergence of green ear, which exhibits all possible degrees of proliferations and malformation of the panicle. The pathogen population at Jodhpur, India is more virulent among other prevalent pathotypes as highly resistant pearl millet lines turned susceptible at this location. Virulence of pathotype rapidly changes host physiology producing varied symptoms in leaves and ear heads. Biochemical components including carbohydrates, phenols, free proline, photosynthetic pigments and enzymes like polyphenol oxidase (PPO), peroxidase (POX), IAA oxidase (IAAO) and catalase were found considerably deranged in malformed tissues. Results indicated that in two highly susceptible cultivars (Nokha local and Eknath) high soluble sugars were recorded in DM necrotic/chlorotic leaves and malformed ear heads, whereas starch contents were reduced in infected ear heads. Total and O-dihydroxy phenols were higher in DM infected leaves as well as in the malformed ear heads. Free proline contents were increased manifold in DM infected leaves and in proliferated panicles. Total chlorophyll contents reduced drastically in DM infected leaves. In ear heads showing tufting and complete malformation, total chlorophyll and carotenoids were low when compared to healthy and diseased leaves. Activities of PPO, POX, IAAO and catalase were higher in DM affected leaves and suppressed and completely malformed ear heads in comparison to their healthy counterparts. The study suggests that accumulation of total phenols caused the hyperphenolicity in infected host tissues despite increased activities of POX, PPO, catalase and IAA oxidase.

Effect of Raw Cow Milk and Gliocladium virens in Pearl Millet against Downy Mildew Disease Caused by Sclerospora graminicola.

Pearl millet [Pennisetum glaucum (L.) R. Br.] downy mildew (DM) is caused by the fungus Sclerospora graminicola (SACC.) SCHRŐT. is the most widespread and destructive disease of pearl millet affecting yield and quality in all the millet cultivating tracts of India. Since pearl millet is a crop of low economic value grown by resource-poor farmers, conventional technological interventions are not cost feasible. Integration of indigenous knowledge with biocontrol agents appeared as a logical strategy in the present case. Studies were, therefore, undertaken to manage DM in rainfed crop of pearl millet using raw cow milk together with Gliocladium virens. Seed and soil treatments resulted in the lowest disease incidence. Biochemical constituents (metabolites and oxidative enzymes) were analysed to determine possible mode of action of Raw Cow Milk (RCM) and Gliocladium virens. A considerable increase in sugars, phenols and ortho-dihydroxy phenols (OD) in healthy and DM infected leaves of treated pearl millet plants was recorded when compared to untreated controls. A marked increase in all the photosynthetic pigments in both healthy and diseased treated plants was observed. The induction of resistance was accompanied by increased activities of defense related enzymes. It is assumed that the combination of RCM and G. virens is capable of stimulating different systemic responses in host plant.