The international reference system for beta-particle emitting radionuclides: Validation through the pilot study CCRI(II)-P1.Co-60.

The Bureau International des Poids et Mesures (BIPM) is developing a new transfer instrument to extend its centralized services for assessing the international equivalence of radioactive standards to new radionuclides. A liquid scintillation counter using the triple/double coincidence ratio method is being studied and tested in the CCRI(II)-P1.Co-60 pilot study. The pilot study, involving 13 participating laboratories with primary calibration capabilities, validated the approach against the original international reference system based on ionization chambers, which has been in operation since 1976. The results are in agreement and an accuracy suitable for purpose, below 5×10-4, is achieved. The pilot study also reveals an issue when impurities emitting low-energy electrons are present in the standard solution, which have a different impact on liquid scintillation counting compared to other primary measurement methods.

Activity measurement of mixed complex radionuclide like 152Eu with different methods.

152Eu has been standardized by three independent 4π ß-γ coincidence counting systems with beta detectors as proportional counter, plastic scintillator and liquid scintillator along with the CIEMAT/NIST method. The average activity concentration by primary methods was linked to key comparison reference value (KCRV) by comparing it with that of 4π γ ionization chamber (GIC) whose calibration factor was determined from the KCRV (BIPM.RI(II)-K1.Eu-152 and CCRI(II)-K2.Eu-152) and deviates from GIC by ± 0.16% indicating good agreement within standard uncertainties.

Standardisation of 133Ba by efficiency extrapolation method and calibration of ionisation chamber.

133Ba has been standardised by direct measurements for the first time in the laboratory using two counting systems: (i) the 4πß (plastic scintillator) -γ coincidence, (ii) the 4πß (proportional counter) -γ coincidence. Furthermore, this standardisation experiment demonstrates the performance and applicability of the recently developed 4πß (plastic scintillator)-γ coincidence system for radionuclides decaying with complex decay schemes as well as for e, X-γ emitters. Additionally, 133Ba solution standards were prepared to calibrate the pressurized 4π γ ionisation chamber and determination of the calibration coefficient. The En score is a statistical indicator of the agreement between two independent estimations. Thus, the performance of the PS system was compared to the result obtained with the PC system using the En score as specified in the ISO13528:2015. The results of measurements are acceptable if En â‰¦ 1.0. An En score of 0.2 was obtained which indicates that, the 133Ba activity concentration obtained by the 4πß (plastic scintillator) -γ coincidence and 4πß (proportional counter) -γ coincidence systems are in agreement. This paper presents the standardisation procedure, the results obtained by the measurements and their comparison.

SURGICAL MANAGEMENT OF LARGE HEPATOCELLULAR CARCINOMA: THE FIRST SINGLE-CENTER STUDY FROM WESTERN INDIA.

BACKGROUND: Majority of patients with large size HCC (>10 cm) are not offered surgery as per Barcelona Clinic Liver Cancer (BCLC) criteria and hence, their outcomes are not well studied, especially from India, owing to a lower incidence. AIM: To analyze outcomes of surgery for large HCCs. METHODS: This retrospective observational study included all patients who underwent surgery for large HCC from January 2007 to December 2017. The entire perioperative and follow up data was collected and analyzed. RESULTS: Nineteen patients were included. Ten were non-cirrhotic; 16 were BCLC grade A; one BCLC grade B; and two were BCLC C. Two cirrhotic and three non-cirrhotic underwent preoperative sequential trans-arterial chemoembolization and portal vein embolization. Right hepatectomy was the most commonly done procedure. The postoperative 30-day mortality rate was 5% (1/19). Wound infection and postoperative ascites was seen in seven patients each. Postoperative liver failure was seen in five. Two cirrhotic and two non-cirrhotic patients had postoperative bile leak. The hospital stay was 11.9±5.4 days (median 12 days). Vascular invasion was present in four cirrhotic and five non-cirrhotic patients. The median follow-up was 32 months. Five patients died in the follow-up period. Seven had recurrence and median recurrence free survival was 18 months. The cumulative recurrence free survival was 88% and 54%, whereas the cumulative overall survival was 94% and 73% at one and three years respectively. Both were better in non-cirrhotic; however, the difference was not statistically significant. The recurrence free survival was better in patients without vascular invasion and the difference was statistically significant (p=0.011). CONCLUSION: Large HCC is not a contraindication for surgery. Vascular invasion if present, adversely affects survival. Proper case selection can provide the most favorable survival with minimal morbidity.

Development of plastic scintillator based primary standard for activity measurements and its performance evaluation.

4πß-γ coincidence technique is a powerful tool and widely recognised method to determine the absolute activity concentration of radioactive solutions. A new plastic scintillator based coincidence system has been developed and established as a primary standard for radioactivity measurements. The performance of the system was evaluated by the standardisation of 60Co radioactive solution due to its simple decay scheme. The activity concentration results obtained by the new system were compared with the existing proportional counter and liquid scintillation based 4πß-γ coincidence systems. This paper discusses the design details of the new system and its performance evaluation.

Surgical management of large hepatocellular carcinoma: the first single-center study from western india / Manejo cirúrgico de grandes carcinomas hepatocelulares: primeiro estudo de um único centro da índia ocidental

ABSTRACT Background: Majority of patients with large size HCC (>10 cm) are not offered surgery as per Barcelona Clinic Liver Cancer (BCLC) criteria and hence, their outcomes are not well studied, especially from India, owing to a lower incidence. Aim: To analyze outcomes of surgery for large HCCs. Methods: This retrospective observational study included all patients who underwent surgery for large HCC from January 2007 to December 2017. The entire perioperative and follow up data was collected and analyzed. Results: Nineteen patients were included. Ten were non-cirrhotic; 16 were BCLC grade A; one BCLC grade B; and two were BCLC C. Two cirrhotic and three non-cirrhotic underwent preoperative sequential trans-arterial chemoembolization and portal vein embolization. Right hepatectomy was the most commonly done procedure. The postoperative 30-day mortality rate was 5% (1/19). Wound infection and postoperative ascites was seen in seven patients each. Postoperative liver failure was seen in five. Two cirrhotic and two non-cirrhotic patients had postoperative bile leak. The hospital stay was 11.9±5.4 days (median 12 days). Vascular invasion was present in four cirrhotic and five non-cirrhotic patients. The median follow-up was 32 months. Five patients died in the follow-up period. Seven had recurrence and median recurrence free survival was 18 months. The cumulative recurrence free survival was 88% and 54%, whereas the cumulative overall survival was 94% and 73% at one and three years respectively. Both were better in non-cirrhotic; however, the difference was not statistically significant. The recurrence free survival was better in patients without vascular invasion and the difference was statistically significant (p=0.011). Conclusion: Large HCC is not a contraindication for surgery. Vascular invasion if present, adversely affects survival. Proper case selection can provide the most favorable survival with minimal morbidity.

RESUMO Racional: A maioria dos pacientes com CHC de grande porte (>10 cm) não tem indicação cirúrgica conforme os critérios do Barcelona Clinic Liver Cancer (BCLC) e, portanto, seus resultados não são bem estudados, principalmente na Índia, devido a uma menor incidência. Objetivo: Analisar os resultados da cirurgia para HCCs de grande porte. Métodos: Este estudo observacional retrospectivo incluiu todos os pacientes submetidos à cirurgia para grandes CHC de janeiro de 2007 a dezembro de 2017. Todos os dados perioperatórios e de acompanhamento foram coletados e analisados. Resultados: Dezenove pacientes foram incluídos. Dez não eram cirróticos; 16 eram BCLC grau A; um BCLC grau B; e dois eram BCLC C. Dois cirróticos e três não-cirróticos foram submetidos à quimioembolização transarterial sequencial pré-operatória e embolização da veia porta. Hepatectomia direita foi o procedimento mais comumente realizado. A taxa de mortalidade pós-operatória em 30 dias foi de 5% (1/19). Infecção da ferida e ascite pós-operatória foram observadas em sete pacientes cada. Insuficiência hepática pós-operatória foi observada em cinco. Dois pacientes cirróticos e dois não cirróticos apresentaram vazamento de bile no pós-operatório. O tempo de internação foi de 11,9±5,4 dias (mediana de 12 dias). A invasão vascular estava presente em quatro pacientes cirróticos e cinco não cirróticos. O acompanhamento médio foi de 32 meses. Cinco pacientes morreram no período de acompanhamento. Sete tiveram recorrência e sobrevida mediana livre de recorrência foi de 18 meses. A sobrevida livre de recorrência cumulativa foi de 88% e 54%, enquanto a sobrevida global cumulativa foi de 94% e 73% em um e três anos, respectivamente. Ambos eram melhores em não-cirróticos; no entanto, a diferença não foi estatisticamente significante. A sobrevida livre de recidiva foi melhor nos pacientes sem invasão vascular e a diferença foi estatisticamente significante (p=0,011). Conclusão: CHC grande não é contraindicação para cirurgia. Invasão vascular, se presente, afeta adversamente a sobrevida. Seleção adequada de casos pode fornecer sobrevida mais favorável com morbidade mínima.

Standardisation of Rhenium-188 and determination of calibration factors for secondary standard and radionuclide calibrator.

The use of Rhenium-188 for various therapeutic applications in the field of nuclear medicine has increased in recent years due to its favourable properties like decay scheme, cost effective availability and easy chemistry. Two independent measuring setups were used to standardise 188Re radioactive solution. The modus operandi of standardisation was 4πß-γ coincidence technique where the beta detection was done by proportional counting and liquid scintillation counting and the gamma detection was done by using NaI(Tl) detectors. The secondary standard, high pressure ionisation chamber type Centronic IG12, 20A was calibrated with the standardised 188Re solution and the sensitivity coefficient (pA MBq-1) was determined. To enhance the accuracy of the commercial radionuclide calibrator and to ensure that patients receive optimum dose of these radiopharmaceuticals, calibration number of the Capintec CRC-15ß radionuclide calibrator was also verified. This paper presents the standardisation of 188Re radioactive solution by primary methods and calibration of BARC secondary standard ionisation chamber system and a Capintec CRC-15ß radionuclide calibrator.

63Ni activity measurements a bilateral intercomparison.

To assess the accuracy and capabilities of BARC, for standardization of 63Ni before participating in larger scale trial exercise to implement and test the methods for the extension of the SIR to ß emitters, a bilateral intercomparison was organised with National Metrology Institute of Japan (NMIJ), Japan. Standardization of 63Ni was carried out and the results were compared with those obtained from NMIJ to assess the accuracy and capabilities of the laboratories. The CIEMAT/NIST efficiency tracing technique based on 3H standard was used for measurement of 63Ni activity concentration at BARC, India whereas Triple to Double Coincidence Ratio (TDCR) method was used at NMIJ, Japan. The procedures adopted for the standardization of 63Ni by CIEMAT/NIST method at BARC and TDCR method at NMIJ are presented. The percentage deviation in activity concentration of 63Ni between BARC, India and NMIJ, Japan is 0.27%. To evaluate the performance of techniques used at both the laboratories, En score (k = 2) and degrees of equivalence was calculated. The En score of -0.12 and degrees of equivalence -0.06 kBq g-1 clearly indicates that the activity concentration of 63Ni measured at BARC, India and NMIJ, Japan are in excellent agreement and comparable within uncertainty limits and demonstrates the degrees of equivalence of the standards maintained at BARC, India and NMIJ Japan.

Porcine reproductive and respiratory syndrome virus induces concurrent elevation of High Mobility Group Box-1 protein and pro-inflammatory cytokines in experimentally infected piglets.

Porcine Reproductive and Respiratory Syndrome (PRRS), caused by PRRS virus (PRRSV), is one of the most important devastating diseases of pigs, characterized by reproductive failure in sows, and respiratory disease with heavy mortality in piglets. PRRS virus has been reported to elevate the levels of proinflammatory cytokines in the serum of infected pigs. High Mobility Group Box-1 (HMGB-1) protein is a cellular biomolecule belonging to the Danger Associated Molecular Patterns (DAMP) family, which stimulates immune cells to release pro-inflammatory cytokines upon release out of cells. The role of HMGB-1 in the pathogenesis of PRRSV remains largely unknown. In the present study, HMGB-1 levels in serum samples collected from six-week-old piglets infected intra-nasally with 2 × 105.75 TCID50/mL of Indian PRRSV (Ind-297221/2013) was estimated by ELISA up to 21 days post infection (dpi). Pro-inflammatory cytokine mRNA (IL-1ß, IL-6 and TNF- α) expression in PBL was estimated by SYBR green based real time PCR. Mean HMGB-1 concentration in serum was found to be significantly elevated in PRRSV infected piglets on 6 dpi as compared to uninfected control piglets. At mRNA level, significant increase in expression of HMGB-1 was observed from 4 to 5 dpi and from 11 to 13 dpi. IL-1ß and IL-6 mRNA were significantly upregulated between 4 and 6 dpi. Significant increase in TNF-α gene expression was seen only on 7 and 9 dpi. Higher levels of pro-inflammatory cytokines and HMGB-1 could be correlated with fever which was observed within 7 dpi in all the infected piglets and additionally around 13 dpi in the animal that died on 17 dpi. Thus, elevated HMGB-1 level in PRRSV infected piglets could be correlated with concurrent increase in pro-inflammatory cytokine (IL-6) mRNA. In-vitro studies were conducted in PRRSV infected Porcine Pulmonary Alveolar Macrophages (PAM) to ascertain HMGB-1 role in PRRS pathogenesis. The results of both in-vivo and in-vitro studies showed that HMGB-1 plays an important role in mediating the pro-inflammatory cytokine responses in PRRS pathogenesis.

Evaluation of antiviral activity of Ocimum sanctum and Acacia arabica leaves extracts against H9N2 virus using embryonated chicken egg model.

BACKGROUND: In the view of endemic avian influenza H9N2 infection in poultry, its zoonotic potential and emergence of antiviral resistance, two herbal plants, Ocimum sanctum and Acacia arabica, which are easily available throughout various geographical locations in India were taken up to study their antiviral activity against H9N2 virus. We evaluated antiviral efficacy of three different extracts each from leaves of O. sanctum (crude extract, terpenoid and polyphenol) and A. arabica (crude extract, flavonoid and polyphenol) against H9N2 virus using in ovo model. METHODS: The antiviral efficacy of different leaves extracts was systematically studied in three experimental protocols viz. virucidal (dose-dependent), therapeutic (time-dependent) and prophylactic (dose-dependent) activity employing in ovo model. The maximum non-toxic concentration of each herbal extracts of O. sanctum and A. arabica in the specific pathogen free embryonated chicken eggs was estimated and their antiviral efficacy was determined in terms of reduction in viral titres, measured by Haemagglutination (HA) and real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays. RESULTS: All the extracts of O. sanctum (crude extract, terpenoid and polyphenol) and A. arabica (crude extract, flavonoid and polyphenol) showed significant virucidal activity, however, crude extract ocimum and terpenoid ocimum showed highly significant to significant (p < 0.001-0.01) decrease in virus genome copy numbers with lowest dose tested. Similarly, therapeutic effect was observed in all three extracts of O. sanctum in comparison to the virus control, nevertheless, crude extract ocimum and terpenoid ocimum maintained this effect for longer period of time (up to 72 h post-incubation). None of the leaves extracts of A. arabica had therapeutic effect at 24 and 48 h post-incubation, however, only the crude extract acacia and polyphenol acacia showed delayed therapeutic effect (72 h post-inoculation). Prophylactic potential was observed in polyphenol acacia with highly significant antiviral activity compared to virus control (p < 0.001). CONCLUSIONS: The crude extract and terpenoid isolated from the leaves of O. sanctum and polyphenol from A. arabica has shown promising antiviral properties against H9N2 virus. Future investigations are necessary to formulate combinations of these compounds for the broader antiviral activity against H9N2 viruses and evaluate them in chickens.

Pathogenic characterization of porcine reproductive and respiratory syndrome virus of Indian origin in experimentally infected piglets.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important transboundary viral disease of pigs confronting the swine industry worldwide. This study was aimed to assess the pathogenic potential of PRRS virus belonging to genotype 2 that emerged in India in 2013. Nine 6-week-old piglets were inoculated intranasally with 2 × 105.75  TCID50 /ml of PRRSV (Ind-297221/2013). Three piglets were kept as uninfected controls. Blood and nasal swabs were collected daily up to 7 days post-infection (dpi) and on alternate days subsequently. Piglets were necropsied for tissue sample collection either on death or after euthanasia on 7, 14 or 21 dpi (one uninfected control and three PRRSV-infected piglets per interval). The virus caused high fever, typical blue ear, weight loss, respiratory distress, diarrhoea and leucopenia between 2 and 8 dpi. Two infected piglets died (on 3 and 17 dpi) during the course of study. The presence of virus in serum and nasal secretion was observed up to 19 and 17 dpi, respectively, with the maximum load between 4 and 7 dpi. Seroconversion started 6 dpi and the mean PRRSV antibody titre reached up to 640 by 21 dpi. Virus load was highest in tonsils at all the intervals, whereas in spleen and lymph nodes load was higher in later intervals. Major microscopic lesions in PRRSV-infected piglets included moderate to severe interstitial pneumonia, lymphoid depletion in tonsils and lymph nodes (cystic), thymic atrophy, reactive hyperplasia followed by lymphoid depletion in spleen. PRRSV antigen was consistently demonstrated by immunoperoxidase test in the lungs, spleen, tonsils and lymph nodes. Antigen distribution was more widespread on 7 and 14 dpi than on 21 dpi. The findings establish that the Indian PRRSV is highly pathogenic to piglets.

An automated LS(ß)- NaI(Tl)(γ) coincidence system as absolute standard for radioactivity measurements.

4πß-γ coincidence method is a powerful and widely used method to determine the absolute activity concentration of radioactive solutions. A new automated liquid scintillator based coincidence system has been designed, developed, tested and established as absolute standard for radioactivity measurements. The automation is achieved using PLC (programmable logic controller) and SCADA (supervisory control and data acquisition). Radioactive solution of 60Co was standardized to compare the performance of the automated system with proportional counter based absolute standard maintained in the laboratory. The activity concentrations determined using these two systems were in very good agreement; the new automated system can be used for absolute measurement of activity concentration of radioactive solutions.

Results of an international comparison of activity measurements of 68Ge.

An international key comparison, identifier CCRI(II)-K2.Ge-68, has been performed. The National Institute of Standards and Technology (NIST) served as the pilot laboratory, distributing aliquots of a 68Ge/68Ga solution. Results for the activity concentration, CA, of 68Ge at a reference date of 12h00 UTC 14 November 2014 were submitted by 17 laboratories, encompassing many variants of coincidence methods and liquid-scintillation counting methods. The first use of 4π(Cherenkov)ß-γ coincidence and anticoincidence methods in an international comparison is reported. One participant reported results by secondary methods only. Two results, both utilizing pure liquid-scintillation methods, were identified as outliers. Evaluation using the Power-Moderated Mean method results in a proposed Comparison Reference Value (CRV) of 621.7(11)kBqg-1, based on 14 results. The degrees of equivalence and their associated uncertainties are evaluated for each participant. Several participants submitted 3.6mL ampoules to the BIPM to link the comparison to the International Reference System (SIR) which may lead to the evaluation of a Key Comparison Reference Value and associated degrees of equivalence.

Kinetic energy offsets for multicharged ions from an electron beam ion source.

Using a retarding field analyzer, we have measured offsets between the nominal and measured kinetic energy of multicharged ions extracted from an electron beam ion source (EBIS). By varying source parameters, a shift in ion kinetic energy was attributed to the trapping potential produced by the space charge of the electron beam within the EBIS. The space charge of the electron beam depends on its charge density, which in turn depends on the amount of negative charge (electron beam current) and its velocity (electron beam energy). The electron beam current and electron beam energy were both varied to obtain electron beams of varying space charge and these were related to the observed kinetic energy offsets for Ar4+ and Ar8+ ion beams. Knowledge of these offsets is important for studies that seek to utilize slow, i.e., low kinetic energy, multicharged ions to exploit their high potential energies for processes such as surface modification. In addition, we show that these offsets can be utilized to estimate the effective radius of the electron beam inside the trap.

Genome-wide gene expression pattern underlying differential host response to high or low pathogenic H5N1 avian influenza virus in ducks.

The differences in the influenza viral pathogenesis observed between different pathogenic strains are associated with distinct properties of virus strains and the host immune responses. In order to determine the differences in the duck immune response against two different pathogenic strains, we studied genome-wide host immune gene response of ducks infected with A/duck/India/02CA10/2011 and A/duck/Tripura/103597/2008 H5N1 viruses using custom-designed microarray. A/duck/India/02CA10/2011 is highly pathogenic virus (HP) to ducks, whereas A/duck/Tripura/103597/2008 is a low pathogenic (LP) virus strain. Comparative lung tissue transcriptome analysis of differentially expressed genes revealed that 686 genes were commonly expressed, 880 and 1556 genes are expressed uniquely to infection with HP and LP virus, respectively. The up-regulation of chemokines (CCL4 and CXCR4) and IFN-stimulated genes (IFITM2, STAT3, TGFB1 and TGFB3) was observed in the lung tissues of ducks infected with HP virus. The up-regulation of other immune genes (IL17, OAS, SOCS3, MHC I and MHC II) was observed in both infection conditions. The expression of important antiviral immune genes MX, IFIT5, IFITM5, ISG12, ß-defensins, RSAD2, EIF2AK2, TRIM23 and SLC16A3 was observed in LP virus infection, but not in HP virus infection. Several immune-related gene ontology terms and pathways activated by both the viruses were qualitatively similar but quantitatively different. Based on these findings, the differences in the host immune response might explain a part of the difference observed in the viral pathogenesis of high and low pathogenic influenza strains in ducks.

Standardization of 68Ge-68Ga using 4πß(LS)-γ coincidence counting system for activity measurements.

68Ga has great scope for use in future for positron emission tomography (PET) imaging due to its very fast blood clearance and fast target localization, even though at present 18F is widely used. 68Ge in equilibrium with 68Ga (68Ge-68Ga) can also be used as a surrogate for 18F calibration, as 18F source standardization can be done at national metrology institute (NMI) but, these standards cannot be sent to nuclear medicine centers (NMCs) across India for calibration of isotope calibrators, due to the short half-life of 18F (110min). Providing 68Ge-68Ga standards to NMCs requires that first standardization must be carried out at NMI (BARC in India) to provide traceability to the measurements carried out at NMCs. In the present work, standardization of 68Ge-68Ga was carried out using 4πß(LS)-γ coincidence counting system and CIEMAT/NIST efficiency tracing technique. The decay scheme correction factors for two gamma windows were calculated by Monte Carlo technique using general purpose code FLUKA. The activity concentration values were normalized by the activity concentration obtained by 4πß(LS)-γ coincidence counting system using window-1. The final result reported to BIPM for 4πß(LS)-γ coincidence counting was calculated by taking arithmetic mean of activity concentrations obtained for two gamma windows. The normalized activity concentration obtained by 4πß(LS)-γ coincidence counting was 0.998±0.005 and that obtained using CIEMAT/NIST efficiency tracing was 1.002±0.007 which are in excellent agreement within uncertainty limits.

Intra-operative assessment of excised breast tumour margins using ClearEdge imaging device.

INTRODUCTION: Breast conserving surgery (BCS) aims to remove a breast cancer completely and obtain clear margins. Complete excision is essential to reduce the risk of local recurrence. The ClearEdge™ (CE) imaging device examines margins of excised breast tissue intra-operatively. The aim of this study was to investigate the potential of the device in detecting margin involvement in patients having BCS. METHODS: In Phase-1 58 patients underwent BCS and had 334 margins assessed by the device. In Phase-2 the device was used in 63 patients having BCS and 335 margins were assessed. Patients with margins considered close or involved by the CE device were re-excised. RESULTS: The margin assessment accuracies in Phase-1 and Phase-2 compared to permanent section pathology were very similar: sensitivity (84.3% and 87.3%), specificity (81.9% and 75.6%), positive predictive value (67.2% and 63.6%), and negative predictive value (92.2% and 92.4%). The false positive rate (18.1% and 24.4%) and false negative rate (15.7% and 12.7%) were low in both phases. In Phase-2 re-excision rate was 37%, but in the 54 where the CE device was used appropriately the re-excision rate was 17%. Had all surgeons interpreted all images appropriately and re-excised margins detected as abnormal by the device in Phase-2 then the re-excision rate would have been 7%. CONCLUSION: This study shows that the CE device has potential to reduce re-excision after BCS and further randomized studies of its value are warranted.

Reverse genetics based rgH5N2 vaccine provides protection against high dose challenge of H5N1 avian influenza virus in chicken.

An inactivated vaccine was developed using the rgH5N2 virus (6 + 2 reassortant) generated by plasmid based reverse genetics system (RGS) with WSN/33/H1N1 as backbone virus. Following mutation of the basic amino acid cleavage site RRRKKR*GLF to IETR*GLF, the H5-HA (haemagglutinin) gene of the selected donor H5N1 virus (A/chicken/West Bengal/80995/2008) of antigenic clade 2.2 was used along with the N2-NA gene from H9N2 field isolate (A/chicken/Uttar Pradesh/2543/2004) for generation of the rgH5N2 virus. A single dose (0.5 ml/bird) of the inactivated rgH5N2 vaccine protected 100% of the vaccinated chickens (n = 10) on 28(th) dpv (early challenge) and 90% of the vaccinated chickens (n = 10) on 200(th) dpv (late challenge) against high dose challenge with HPAI virus (10(9) EID50/bird). Challenge virus shedding via oropharynx and cloaca of the vaccinated chickens was detectable by realtime RT-PCR during 1-5 dpc and 1-9 days dpc in the early and the late challenge, respectively. The protective level of antibodies (mean HI titre > 128) was maintained without booster vaccination for 200 days. The present study provides the experimental evidence about the extent of protection provided by a reverse genetics based vaccine for clade 2.2 H5N1 viruses against challenge with high dose of field virus at two different time points (28 dpv and 200 dpv). The challenge study is uniquely different from the previous similar experiments on account of 1000 times higher dose of challenge and protection at 200 dpv. The protection and virus shedding data of the study may be useful for countries planning to use H5 vaccine in poultry especially against the clade 2.2 H5N1 viruses.

Identification and molecular characterization of border disease virus (BDV) from sheep in India.

Pestiviruses isolated from sheep and goats in India thus far have been bovine viral diarrhoea virus 1 (BVDV-1) or BVDV-2. During routine genetic typing of pestiviruses in the years 2009-10, border disease virus (BDV) was detected in eight Indian sheep of a flock showing clinical signs of BD by real time RT-PCR. All the samples yielded positive virus isolates in cell culture but were found negative by a BVDV antigen ELISA. A representative BDV isolate was characterized at genetic and antigenic level. Phylogenetic analysis carried out in 5'-UTR, N(pro) and E2 regions of genome typed the Indian BDV isolate as BDV-3. A more detailed analysis in N(pro) and entire region coding structural proteins showed that the N(pro) (168), C (100 aa), E(rns) (227 aa), E1 (195 aa) and E2 (373 aa) proteins were of size characteristic for BDV reference strain X818. Antigenic differences were evident between the BDV-3 isolate and previously reported BDV-1, BDV-5 and BDV-7 strains. Although origin of BDV-3 in India is not clear, the results reflect probable introduction through trade in sheep between India and other countries or BDV-3 may be more widely distributed. Additionally, this study suggests that for diagnosis of BDV infection, the commercial BVDV Ag-ELISA should be used with caution. This is the first identification of BDV in sheep in India which highlights the need for continued pestivirus surveillance and assessing its impact on sheep and goat production.

Expression and purification of recombinant H5HA1 protein of H5N1 avian influenza virus in E. coli and its application in indirect ELISA.

The PCR amplified HA1 fragment of H5N1 (H5HA1) avian influenza virus (AIV) hemagglutinin gene was cloned into pET28a (+) expression vector and expressed in Rosetta Blue (DE3) pLysS cells. The recombinant H5HA1 (rH5HA1) protein purified by passive gel elution after SDS-PAGE of the inclusion bodies reacted specifically with H5N1 serum in Western blot analysis. A subtype specific indirect enzyme linked immunosorbent assay (iELISA) using the rH5HA1 protein as the coating antigen was developed for detecting antibodies to H5 subtype of AIV. The assay had 89.04% sensitivity and 95.95% specificity when compared with haemagglutination inhibition test. The Kappa value of 0.842 indicated a perfect agreement between the tests. The iELISA developed can be used for serosurveillance of avian influenza in chickens.