Pritom Chowdhury

West Nile virus infection, Assam, India.

To the Editor: West Nile virus (WNV) is a mosquito-borne flavivirus. Sporadic infections with this virus have been found in Africa, Europe, Asia, and the United States. In humans, most infections with WNV cause subclinical or a mild influenza-like illness; encephalitis occurs in some (1). In India, antibodies against WNV were first detected in humans in Bombay in 1952 (2). Virus activity has been reported in southern, central, and western India. WNV has been isolated in India from Culex vishnui mosquitoes in Andhra Pradesh and Tamil Nadu, from Cx. quinquefasciatus mosquitoes in Maharashtra, and from humans in Karnataka State (3). Assam (26°–27°30′N, 89°58′–95°41′E) is the most populated state in northeastern India; it contains ≈50% of the 38.8 million inhabitants of northeastern India. Japanese encephalitis virus (JEV) has caused sporadic epidemics in Assam since 1976. Studies conducted during 2000–2002 in Assam showed that 187 (53.7%) of 348 persons with acute encephalitis syndrome were infected with JEV (4). JEV-negative persons also showed symptoms of neurotropic viral infection. Suspecting the presence of some other closely related flavivirus in this region, we screened samples from persons with acute encephalitis syndrome for WNV in 2006. To our knowledge, no study has been conducted on the prevalence of WNV in this region. We report WNV activity in the state of Assam in northeastern India. Ethical approval for this study was obtained from the institutional ethical committee, Regional Medical Research Center, Dibrugarh, India. A JEV vaccination campaign (SA14-14-2 vaccine) was started in Assam during May 2006. During its first phase, children 1–15 years of age in Dibrugarh and Sivasagar Districts were vaccinated. Mosquito surveillance in the study area and in an earlier study (5) identified Cx. vishnui mosquitoes. During the study period, 103 serum samples and 88 cerebrospinal fluid samples were obtained from 167 patients with acute encephalitis syndrome admitted to the Assam Medical College and Hospital in Dibrugarh, which administers to the health needs of >7 districts of Upper Assam and neighboring states of Arunachal Pradesh and Nagaland. Among the 167 patients, 124 (74.2%) were children <15 years of age. Among the 103 serum samples, 80 were positive for immunoglobulin (Ig) M against JEV (IgM monoclonal antibody–capture ELISA; National Institute of Virology, Pune, India) and 12 (11.6%) were positive for IgM against WNV (IgM antigen-capture ELISA; Panbio, Sinnamon Park, Queensland, Australia). These samples were from persons in 4 districts in Assam (Dibrugarh, Golaghat, Sivasagar, and Tinsukia) and negative for IgM against JEV (Table). Follow-up was conducted for 9 patients; 3 died, and 1 was lost to follow-up. Table Incidence of JEV and WNV infections among patients with acute encephalitis syndrome, Assam, India* Virus-neutralizing antibody titers against JEV and WNV were estimated in pig kidney epithelial cells by using JEV (isolate 733913) and WNV (isolate 68856) and a cytopathic-effect assay in 96-well tissue culture plates (6). Mouse polyclonal antibodies against JEV and WNV and nonimmune serum samples were included in the assay. Of 9 paired serum samples, 6 showed neutralizing antibody for WNV, of which 4 showed a 4-fold increase in antibody titer. The remaining 3 paired samples showed cross-reactivity with WNV (titer <80) and JEV (titer <40). All 12 WNV-infected patients had high fever and headache. Convulsions (6 patients), altered sensorium (7 patients), vomiting (5 patients), and neck rigidity (2 patients) were also observed. Signs and symptoms at the time of hospitalization and at follow-up for 6 months (at 3-month intervals) were similar for persons infected with JEV and those infected with WNV. Neurologic sequelae observed at <6 months follow-up were impaired memory (6 patients), irritable behavior (5 patients), impaired hearing (3 patients), incoherent speech and disorientation (1 patient), breathing difficulty (1 patient), impaired speech (1 patient), and quadriparesis (1 patient). We identified WNV in regions of Assam to which JEV is endemic. The finding indicates that WNV might be the cause of a substantial number of acute encephalitis syndrome cases in this region. Fever and headache were the most common signs and symptoms, as reported (7). There were 3 deaths (all children) in 13 patients. Our results corroborate a similar observation in the Kolar District of Karnataka (8). In contrast, in western countries, the attack rate and case-fatality rate for WNV infection are higher among immunocompromised elderly patients (9). Our findings may be caused by strain variations and host susceptibility to the virus. Identification of circulating genotypes of WNV and its vectors and epidemiologic studies are needed to obtain additional information on WNV infection in this region and identify WNV as a cause of acute encephalitis syndrome.

Characterization of West Nile virus (WNV) isolates from Assam, India: Insights into the circulating WNV in northeastern India

West Nile virus (WNV) is a mosquito-borne flavivirus that causes subclinical symptoms, febrile illness with possible kidney infarction and encephalitis. Since WNV was first serologically detected in Assam during 2006, it has become recognized as an important etiological agent that causes acute encephalitis syndrome (AES) in addition to endemic Japanese encephalitis virus (JEV). Therefore, isolating and characterizing the currently circulating strain of WNV is important. The virus was isolated from the cerebrospinal fluid (CSF) of two patients that presented with AES. The genotyping of the isolates HQ246154 (WNIRGC07) and JQ037832 (WNIRTC08) based on the partial sequencing of 921 nucleotides (C-prM-E) of the genome placed them within lineage 5 along with other Indian strains isolated prior to 1982, but the present circulating virus formed a distinct subclade. The derived amino acid sequence alignment indicated substitution in A81T and A84P of the capsid region in HQ246154. A cross-neutralization assay suggested substantial antigenic variation between isolates. The pathogenesis in mice that suggested the circulating WNV was neuroinvasive and comparatively more pathogenic than previous strains from India.

Co-infection of arboviruses presenting as Acute Encephalitis Syndrome.

A 35-year male patient presenting as Acute Encephalitis yndrome (AES) was admitted to the Assam Medical College & ospital, Dibrugarh, Assam, India. The patient had acute onset f fever, altered consciousness, convulsion and neck rigidity. erum and cerebrospinal fluid (CSF) were collected on the day of dmission with written consent of the patient for diagnosis of the isease as a routine procedure; a clinical history of 5 days was eported. Laboratory examination showed low hemoglobin level 9.1 gm/dl). White blood cell count (WBC), blood urea level, total ilirubin count, serum aspartate and alanine transaminase were ecorded as 17,800 cells/ l, 61mg/dl, 3.6mg/dl, 350 and 336 IU/l espectively. The patient had no record of travel history prior o onset of symptoms. X-ray examination revealed ill-defined

Characterization of Japanese encephalitis virus (JEV) genotype III clinical isolates in northeast India.

BACKGROUND Japanese encephalitis virus (JEV) is one of the major etiological agents responsible for causing large numbers of acute encephalitis syndrome (AES) cases in the northeastern region of India. This study was carried out to establish and characterize the circulating strain of JEV in the region in order to understand the disease epidemiology. METHODS Virus isolation was attempted from 121 patients that presented with AES. Phylogenetic analysis was done using the Kimura-2-Parameter model based on envelope and pre-membrane gene sequence. A pathogenecity study was done in the Swiss albino mice model and assessed by Kaplan-Meier survival analysis. RESULTS The phylogenetic analysis of the two JEV isolates obtained placed them within genotype (G)III, where they form a subclade within the Vellore group of Indian JEV strains. Neutralization assays suggested similarity between the study isolates and prototype Vellore JEV strain P20778. Pathogenesis in mice suggested that the circulating GIII JEV strains were neuroinvasive. CONCLUSIONS This study showed that a pathogenic GIII JEV strain was circulating in the northeastern region of India. This finding is important as it is contrary to the belief that GI is gradually replacing GIII as the dominant genotype in Asia. GenBank accession numbers: HQ270470, JQ434468, HQ246155, JX018170.

Pharmaceutical and biotech product patents in India: doldrums or blissfulness?

Confusion reigns over pharmaceutical and biotech-related patents in India owing to uncertain application of patent law and pressure from generics.

Genetic diversity and gene structure of mitochondrial region of Anopheles minimus (Diptera: Culicidae) - major malaria vector of North east India.

OBJECTIVE To depict mitochondrial genetic variation for the first time among Anopheles minimus (An.minimus) (Diptera: Culicidae) species from two malaria endemic states of NE India. METHODS Phylogeographic analysis was carried at 9 out of 12 sites of An.minimus confirmed malaria endemic places. RESULTS All sequences were Adenine-Thymine rich regions. Transitions were observed in 6 sequences where 5 mutations were synonymous substitutions and in 1 case non synonymous mutation was observed. Three distinct clusters of haplotypes were generated. Haplotype diversity and low nucleotide diversity were studied. Overall negative values obtained from Tajimas D test and FusFS test indicate a recent genetic population expansion. Network analysis has explained sequence diversity that was also shown by mutations in 6 sequences. CONCLUSIONS High genetic diversity observed within the populations of An.minimus species has several possible implications for vector control in the region.

Intellectual Property Rights for Nanotechnology in Agriculture

Nanoscience studies biological properties of materials at the nanoscale. Nanotechnology research develops improved materials, devices, systems and therapeutics. Nanomaterials can be protected for their intellectual property rights by innovators. However, due to the interdisciplinary nature of nanotechnology, there is a risk of overlapping patent claims and lack of distinction between nano-based and traditional patents. Scientists also must solve ethical and social issues, from health to environmental risk and consumer perception.

Comparison of β-Propiolactone and Formalin Inactivation on Antigenicity and Immune Response of West Nile Virus

West Nile Virus (WNV) is a pathogenic arbovirus that belongs to genus Flavivirus under family Flaviviridae. Till now there are no approved vaccines against WNV for human use. In this study, the effect of two alkylating agents, formaldehyde and β-PL, generally used for inactivated vaccine preparation, was assessed on the basis of antigenic and immunogenic potential of the inactivated WNV. Lineage 5 WNV isolates were inactivated by both formalin and β-PL treatments. Inactivation was confirmed by repeated passage in BHK-21 cell line and infant mice. Viruses inactivated by both the treatments showed higher antigenicity. Immune response in mice model showed serum anti-WNV antibody titre was moderately higher in formalin inactivated antigen compared to β-PL inactivated antigen. However, no significant differences were observed in neutralization antibody titre. In conclusion, we can state that both formaldehyde and β-PL inactivation processes were found to be equally efficient for inactivation of WNV. However, they need to be compared with other inactivating agents along with study on cell mediated immune response.

Nanotechnology applications and intellectual property rights in agriculture

Nanotechnology research uses specific properties of materials at the nanoscale to develop improved materials, devices, systems and therapeutics. There is a risk of overlapping patent claims and lack of distinction between nano-based and traditional patents due to the interdisciplinary nature of nanotechnology. There is an increasing trend of granted patents. The World Intellectual Property Organization and World Health Organization, regulatory and policy bodies, are working to make a comprehensive property right regulation for nanotechnology products. The USA, the leader of nanotechnology products, has made guidelines to make patent search easier for nano-based products. The European Patent Office has also created a new classification for nano-based inventions. Here we review the status of intellectual property rights protection of nanomaterial, environmental implications and application of nanotechnology in agriculture.

Theaflavins, polyphenols of black tea, inhibit entry of hepatitis C virus

The treatment of hepatitis C virus (HCV) infection by combination of direct acting antivirals (DAA), with different mode of action, has made substantial progress in the past few years. However, appearance of resistance and high cost of the therapy is still an obstacle in the achievement of the therapy, more specifically in developing countries. In this context, search for affordable antivirals with new mechanisms of action is still needed. Tea, after water, is the most popular drink worldwide. Polyphenols extracted from green tea have already shown anti-HCV activity as entry inhibitors. Here, three different theaflavins, theaflavin (TF1), theaflavin-3’-monogallate (TF2), and theaflavin-3-3’-digallate (TF3), which are major polyphenols from black tea, were tested against HCV in cell culture. The results showed that all theaflavins inhibit HCV infection in a dose-dependent manner in an early step of infection. Results obtained with HCV pseudotyped virions confirmed their activity on HCV entry and demonstrated their pan-genotypic action. No effect on HCV replication was observed by using HCV replicon. Investigation on the mechanism of action of black tea theaflavins showed that they act directly on the virus particle and are able to inhibit cell-to-cell spread. Combination study with inhibitors most widely used in anti-HCV treatment regimen demonstrated that TF3 exerts additive effect. In conclusion, theaflavins, that are present in high quantity in black tea, are new inhibitors of HCV entry and hold promise for developing in therapeutic arsenal for HCV infection.