Standardized Extract of Valeriana officinalis Improves Overall Sleep Quality in Human Subjects with Sleep Complaints: A Randomized, Double-Blind, Placebo-Controlled, Clinical Study.

INTRODUCTION: Sleep deficit or poor sleep leads to ill-health, whereas sleep deprivation for longer periods of time increases the risk of developing adverse conditions associated with poor quality of life, and high socioeconomic impact. The treatments for sleep disturbances include melatonin and over-the-counter medicines like diphenhydramine and doxylamine, all of which have negative side effects. Valerian (Valeriana officinalis L.) is a traditional herb and the most preferred alternate sleep solution to manage sleep complaints. METHODS: Eighty adult subjects with sleep complaints were randomized in 1:1 ratio to receive either V. officinalis extract (VE) or placebo for 8 weeks in a double-blind, placebo-controlled, parallel, clinical study. Primary efficacy endpoints included the Pittsburgh Sleep Quality Index (PSQI) and sleep latency using wrist actigraphy (WA), as well as a number of secondary endpoints, including sleep parameters such as actual sleep time and sleep efficiency using WA, the Epworth Sleepiness Scale (ESS), the Beck Anxiety Inventory (BAI), the Visual Analogue Scale (VAS) for the feeling of waking up refreshed, and a tertiary endpoint of sleep parameters using polysomnography (PSG) in a subset of 20 subjects per group. Safety parameters included physical examination, vital sign measurements, hematology, and clinical chemistry tests. Adverse events and serious adverse events were monitored throughout the study period. RESULTS: Seventy-two subjects (35 and 37 subjects in the placebo and VE groups, respectively) completed the study and were included in the efficacy assessments. On Days 14, 28, and 56, the PSQI Total Score in the VE group decreased significantly (p < 0.05) compared to the placebo group. Further, the VE group showed significant improvements (p < 0.05) in sleep latency and actual sleep time on Days 3, 14, 28, and 56, and sleep efficiency on Days 14, 28, and 56, as evaluated by WA. There was a decrease (p < 0.05) in anxiety (BAI) on Days 14, 28, and 56, daytime drowsiness (ESS) on Days 28 and 56, and an increased feeling of waking up refreshed (VAS) on Days 28 and 56 compared to placebo. PSG results carried out in subset of subjects revealed significant improvements (p < 0.05) in total sleep time, sleep latency, and sleep efficiency on Day 56 in the VE group compared to the placebo group. No safety concerns were observed throughout the study. CONCLUSION: VE supplementation significantly improved various subjective and objective parameters of sleep in young subjects with mild insomnia symptoms, such as overall sleep quality, sleep latency, sleep efficiency, and total sleep time. We also observed decreased anxiety and daytime sleepiness, and improved feeling of being refreshed after waking up with VE supplementation. VE was found to be safe and well tolerated throughout the study. TRIAL REGISTRATION: Clinical Trials Registry of India: CTRI/2022/05/042818.

A proprietary black cumin oil extract (Nigella sativa) (BlaQmax®) modulates stress-sleep-immunity axis safely: Randomized double-blind placebo-controlled study.

Objective: Stress, sleep, and immunity are important interdependent factors that play critical roles in the maintenance of health. It has been established that stress can affect sleep, and the quality and duration of sleep significantly impact immunity. However, single drugs capable of targeting these factors are limited because of their multi-targeting mechanisms. The present study investigated the influence of a proprietary thymoquinone-rich black cumin oil extract (BCO-5) in modulating stress, sleep, and immunity. Methods: A randomized double-blinded placebo-controlled study was carried out on healthy volunteers with self-reported non-refreshing sleep issues (n = 72), followed by supplementation with BCO-5/placebo at 200 mg/day for 90 days. Validated questionnaires, PSQI and PSS, were employed for monitoring sleep and stress respectively, along with the measurement of cortisol and melatonin levels. Immunity markers were analyzed at the end of the study. Results: In the BCO-5 group, 70% of the participants reported satisfaction with their sleep pattern on day 7 and 79% on day 14. Additionally, both inter- and intra- group analyses of the total PSQI scores and component scores (sleep latency, duration, efficiency, quality, and daytime dysfunction) on days 45 and 90 showed the effectiveness of BCO-5 in the improvement of sleep (p < 0.05). PSS-14 analysis revealed a significant reduction in stress, upon both intra (p < 0.001) and inter-group (p < 0.001) comparisons. The observed reduction in stress among the BCO-5 group, with respect to the placebo, was significant with an effect size of 1.19 by the end of the study (p < 0.001). A significant correlation was also observed between improved sleep and reduced stress as evident from PSQI and PSS. Furthermore, there was a significant modulation in melatonin, cortisol, and orexin levels. Hematological/immunological parameters further revealed the immunomodulatory effects of BCO-5. Conclusion: BCO-5 significantly modulated the stress-sleep-immunity axis with no side effects and restored restful sleep.

Influence of CurQfen®-curcumin on cognitive impairment: a randomized, double-blinded, placebo-controlled, 3-arm, 3-sequence comparative study.

Background: Although curcumin is a blood-brain-barrier permeable molecule with the ability to bind and segregate ß-amyloid plaques and neurofibrillary tangles of hyperphosphorylated tau proteins, its poor oral bioavailability, rapid biotransformation to inactive metabolites, fast elimination from the systemic circulation, and hence the poor neuronal uptake has been limiting its clinical efficacy under neurodegenerative conditions. Objective: We hypothesized that the highly bioavailable CurQfen-curcumin (CGM), which has been shown to possess significant blood-brain-barrier permeability and brain bioavailability, would ameliorate dementia in neurodegenerative conditions. Methods: In the present double-blinded placebo-controlled 3-arm 3-sequence comparative study, 48 subjects characterized with moderate dementia due to the onset of Alzheimer's disease were randomized into three groups (N = 16/group) and supplemented with 400 mg × 2/day of either placebo (MCC), unformulated standard curcumin complex with 95% purity (USC), or CGM as a sachet for six months. The relative changes in cognitive and locomotor functions and biochemical markers were compared. Results: Supplementation with CGM produced significant (P < 0.05) improvement in the Mini-Mental State Examination (MMSE) and the Geriatric Locomotive Function Scale (GLFS) scores in both intra- and inter-group comparison by 2 × 2 repeated measures (RM) ANOVA. Further, analysis of the serum levels of specific biomarkers (BDNF, Aß42, tau protein, IL-6, and TNF-α) also revealed a significant (P < 0.05) improvement among CGM subjects as compared to placebo and the USC groups. Conclusion: Supplementation with CGM as sachet was found to offer significant delay in the progress of Alzheimer's disease, as evident from the improvements in locomotive and cognitive functions related to dementia. Clinical trial registration: http://ctri.nic.in, identifier: CTRI/2018/03/012410.

A phase I clinical trial to evaluate the safety of thymoquinone-rich black cumin oil (BlaQmax®) on healthy subjects: Randomized, double-blinded, placebo-controlled prospective study.

Black cumin or black seed (Nigella sativa L.) is a popular medicinal herb and culinary spice belonging to Ranunculacea family. Thymoquinone (TQ) is the major active phytoconstituent in black cumin and is abundant in the volatile oil fraction. Though black cumin oil containing low TQ content (less than 1%) has been clinically investigated, clinical efficacy and safety data of TQ-rich oil is limited. A recent study with black cumin oil formulation containing 5% TQ (BCO-5) exhibited significant clinical efficacy to alleviate sleep disorders and stress. So, the present phase 1 randomized, double-blinded, placebo-controlled trial evaluated the safety of BCO-5 at a dose of 200 mg/adult/day for 90 days on healthy subjects (n = 70). Both the biochemical and hematological parameters were analysed along with the adverse events or side effects to establish the clinical safety of BCO-5. The study reported neither serious adverse side effects nor any significant alterations in the hematological parameters. The absence of significant changes in the biochemical parameters related to liver function (ALT, AST, ALP), renal function (serum creatinine and urea) were also observed. However, analysis of lipid profile showed a significant (P < 0.05) reduction in total cholesterol, LDL, VLDL and triglycerides, but within the normal range. In conclusion, BCO-5 is safe at 200 mg/adult/day for human consumption and may be clinically evaluated for various health beneficial pharmacological activities where black cumin oil has been shown to have positive effects.

Influence of a Novel Food-Grade Formulation of Red Chili Extract (Capsicum annum) on Overweight Subjects: Randomized, Double-Blinded, Placebo-Controlled Study.

Capsaicinoids from pungent red chilies (Capsicum annum and Capsicum frutescens) have received significant attention as a natural supplement for the management of obesity. However, the consumption of chili extract at physiologically relevant dosage of capsaicinoids is a challenge owing to its pungency and gastrointestinal discomforts. The present study reports the systemic absorption, safety and influence of a novel, food-grade, and sustained-release formulation of capsaicinoids-rich red chili extract using fenugreek dietary fiber (Capsifen®). Twenty-four healthy overweight subjects were randomized into placebo (n = 12) and Capsifen (n = 12) groups and supplemented with 200 mg × 1/day of Capsifen (4 mg capsaicinoids/day) for 28 days. Influence of Capsifen on eating behavior and appetite was followed by Three-Factor Eating Questionnaire (TFEQ) and Council of Nutrition Appetite Questionnaire (CNAQ), respectively. Consumption of Capsifen did not reveal any adverse events or deviations in hematology and biochemical parameters related to safety. However, a significant decrease in body weight (2.1%), w/h ratio (4%) and body mass index (BMI) (2.2%) were observed among Capsifen group when compared to placebo. The TFEQ and appetite analysis revealed a significant improvement in uncontrolled eating and reduction in appetite among Capsifen subjects. The UPLC-ESI-MS/MS analysis confirmed the absorption of capsaicinoids from CAP supplementation. The study further demonstrated the safety and tolerability of Capsifen at the investigational dosage. Thus, the significant reduction in anthropometric parameters such as body weight, w/h ratio, and BMI along with the improvement in eating behaviour as well as appetite, indicated the potential body weight management effect of Capsifen.

Influence of a low-dose supplementation of curcumagalactomannoside complex (CurQfen) in knee osteoarthritis: A randomized, open-labeled, active-controlled clinical trial.

A 6-week, randomized, open-label, active-controlled clinical trial was conducted to evaluate the influence of a low-dose curcumagalactomannosides (CGM) (400 mg once daily) in OA subjects. The treatment was compared with a standard combination of 500 mg glucosamine hydrochloride (GLN) and 415 mg chondroitin sulphate (CHN), supplied as a single oral dose twice a day. Out of 84 subjects randomized, 72 subjects who have completed the study were evaluated for the safety and efficacy of the treatments at baseline and subsequent visits (day 28 and 42), by measuring walking performance, VAS, KPS, and WOMAC scores. CGM exhibited 47.02, 21.43, and 206% improvement in VAS, KPS, and walking performance, respectively, compared to the baseline. Similarly, there was 31.17, 32.93, 36.44, and 35% improvement in the pain, stiffness, physical function, and total WOMAC scores. CGM also caused a substantial reduction in the serum inflammatory marker levels. The results indicate that a short-term supplementation of a low dosage CGM exerted superior beneficial effects than a high-dosage CHN-GLN combination in alleviating the pain and symptoms of OA subjects. Further clinical trials of extended duration in a larger population is required to substantiate the efficacy of CGM in the long-term management of OA.

Curcumagalactomannoside/Glucosamine Combination Improved Joint Health Among Osteoarthritic Subjects as Compared to Chondroitin Sulfate/Glucosamine: Double-Blinded, Randomized Controlled Study.

Objective: A combination of curcumagalactomannosides (CGM) (400 mg) with glucosamine hydrochloride (GLN) (500 mg) was evaluated against a standard dietary supplement combination chondroitin sulfate (CHN) (415 mg)/GLN (500 mg) for their effectiveness in alleviating the pain and symptoms among osteoarthritic subjects. Design: Randomized, double-blinded and active-controlled study. Settings/Location: The study was conducted in a hospital-based research center in Vadodara, Gujarat, India. Subjects: Eighty subjects (38 males and 42 females), with confirmed osteoarthritis (OA) (Class I-III), were randomized into two parallel groups designated as Group I (CGM-GLN) and Group II (CHN-GLN). Interventions: All the study subjects were supplemented with their corresponding intervention capsules (ether CGM along with GLN or CHN along with GLN), as a single oral dose twice a day, once in the morning 10-15 min before breakfast and again in the evening before dinner, for 84 days. Outcome measures: A validated treadmill uphill walking protocol was used for the study, and the efficiency of supplementation was evaluated using visual analogue scale (VAS) score, Karnofsky Performance Scale (KPS) score, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaire at the baseline, 28th, and 84th day following the treatment. Mechanism of action of CGM-GLN combination was analyzed by measuring the levels of serum inflammatory markers interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), and soluble vascular cell adhesion molecule-1 (sVCAM) at the baseline and 84th day. Results: CGM-GLN was found to offer significant beneficial effects to pain, stiffness, and physical function of OA subjects compared with CHN-GLN, which was evident from the improvement in walking performance, VAS score, KPS score, and WOMAC score. The efficiency of CGM-GLN was almost double compared with the CHN-GLN by the end of the study (84th day). A significant reduction of inflammatory serum marker levels was observed among CGM-GLN subjects compared with CHN-GLN subjects. Compared with the baseline, CGM-GLN produced 54.52%, 59.08%, and 22.03% reduction in IL-1ß, IL-6, and sVCAM levels, respectively. Whereas CHN-GLN group of subjects expressed only 23.17%, 21.38%, and 6.82% reduction in IL-1ß, IL-6, and sVCAM levels, respectively. Conclusions: In conclusion, the present study demonstrated the potential benefits of CGM-GLN supplements in alleviating the symptoms and function of OA subjects compared with the standard CHN-GLN treatment. The augmented efficacy of CGM-GLN combination could be attributed to the enhanced anti-inflammatory effect of CGM.

A double blind, placebo-controlled randomized comparative study on the efficacy of phytosterol-enriched and conventional saw palmetto oil in mitigating benign prostate hyperplasia and androgen deficiency.

BACKGROUND: The present clinical trial was conducted to evaluate the efficacy and tolerability of a standardized saw palmetto oil containing 3% ß-sitosterol in the treatment of benign prostate hyperplasia (BPH) and androgen deficiency. METHODS: Subjects aged 40-65 years with symptomatic BPH were randomized to 12-week double-blind treatment with 500 mg doses of ß-sitosterol enriched saw palmetto oil, conventional saw palmetto oil and placebo orally in the form of capsules (n = 33 in each group). BPH severity was determined using the International Prostate Symptom Score (IPSS), uroflowmetry, serum measurement of prostate specific antigen (PSA), testosterone and 5α-reductase. During the trial, the androgen deficiency was evaluated using Aging Male Symptoms (AMS) scale, the Androgen Deficiency in the Aging Male (ADAM) questionnaire, serum levels of free testosterone. RESULTS: Subjects treated with ß-sitosterol enriched saw palmetto oil showed significant decrease in IPSS, AMS and ADAM scores along with reduced postvoiding residual volume (p < 0.001), PSA (p < 0.01) and 5α-reductase from baseline to end of 12-week treatment as compared to placebo. There was also a significant increment in the maximum and average urine flow rate (p < 0.001), and serum free testosterone level of subjects treated with enriched saw palmetto oil as compared to placebo. CONCLUSION: This study demonstrates the efficacy of ß-sitosterol enriched saw palmetto oil superior to conventional oil thus extending the scope of effective BPH and androgen deficiency treatment with improved quality of life through the intake of functional ingredients. TRIAL REGISTRATION: CTRI/2018/12/016724 dated 19/12/2018 prospectively registered. URL: http://ctri.nic.in/Clinicaltrials/advsearch.php.

A Novel Curcumin-Galactomannoside Complex Delivery System Improves Hepatic Function Markers in Chronic Alcoholics: A Double-Blinded, randomized, Placebo-Controlled Study.

Considering the recent interest in free (unconjugated) curcuminoids delivery, the present study investigated the efficacy of a novel food-grade free-curcuminoids delivery system (curcumin-galactomannoside complex; CGM) in improving the hepatic function markers (inflammation and oxidative stress) in chronic alcoholics. The double-blinded, placebo-controlled study randomized 48 subjects with elevated serum transaminases and gamma-glutamyl transferase (GGT) levels, who were allocated to two groups (n=24) and to receive either placebo or CGM at (250 mg × 2)/day for 8 weeks. While liver function markers (transaminases and GGT) in the placebo group showed an increase (~ 9.5%), CGM group indicated a significant decrease in transaminases (31%) and GGT (29%) from the baseline levels. The beneficial effect of CGM was also clear from the significant increase (p <0.001) in endogenous antioxidants (GSH, SOD, and GPx) and decrease in inflammatory markers (IL-6 and CRP) levels (p <0.001) as compared to both the baseline and placebo group. To summarize, the nutritional intervention of CGM-curcumin was found to offer a significant hepatoprotective effect to attenuate the alcohol induced alterations to hepatic function markers. The Indian Medical Council and Drug Controller General of India approved Clinical Trial Registry No. CTRI/2018/03/012385.

Phylogeny and genotyping of recent avian low-pathogenic H5 subtype influenza viruses from French ducks.

H5 low-pathogenic avian influenza virus (LPAIV) has the potential to become highly pathogenic and to cause serious problems in animal and public health. AIV surveillance and characterization in both wild and domestic species is therefore necessary. In order to acquire molecular information and to identify possible reassortments in French viruses, we analysed the entire genome of five H5N3, three H5N2 and two H5N1 LPAIV, isolated in France between 2002 and 2008 mostly from captive ducks (free-range commercial poultry or decoy ducks). Some of the genome sequences showed atypical characteristics, such as an insertion of 1 aa in the PB1 protein of one H5N3, a highly truncated PB1-F2 protein (11 aa in length instead of 90 aa) in one H5N2, and an insertion of 8 aa in the NS1 protein of H5N1. These two last molecular characteristics have not been described previously. Phylogenetic analysis demonstrated that all genes of French LPAIV, except the closely related matrix protein genes, clustered within the Eurasian avian influenzavirus lineage and fell into at least two phylogenetic subgroups. In addition, the French H5 LPAIV were segregated into eight genotypes, suggesting that many reassortment events have occurred in H5 LPAIV in Europe. However, it is not known whether the reassortment events have occurred in wild waterfowl and/or in captive birds in direct or indirect contact with wild birds.

Vaccination as a tool to combat introductions of notifiable avian influenza viruses in Europe, 2000 to 2006.

In late 2000, Italy was the first country of the European Union (EU) to implement an emergency vaccination programme against notifiable avian influenza. Vaccination with a conventional vaccine containing a seed strain with a different neuraminidase subtype from that of the field virus was used to complement biosecurity and restriction measures as part of an overall eradication strategy. This vaccination technique, in line with the Differentiating Infected from Vaccinated Animals system (DIVA), was applied several times until March 2008. This strategy enabled the identification of field exposed flocks and ultimately the eradication of low pathogenic H7N1, H7N3 and H5N2 infections. Italy was also the first country to implement a bivalent H5/H7 prophylactic vaccination programme of defined poultry populations, which was discontinued in December 2006. Following the incursion of highly pathogenic H5N1 into Europe, in 2005 and 2006, two other EU Member States, namely France and the Netherlands, implemented preventive vaccination programmes in 2006 but they targeted selected poultry populations different from those targeted in Italy and were implemented for short periods of time. Data generated during six years of experience with vaccination against avian influenza in Italy indicate that it is a useful tool to limit secondary spread and possibly prevent the introduction of low pathogenic avian influenza viruses in a susceptible population. The experience of France and the Netherlands provides data on vaccination of ducks and hobby poultry respectively and monitoring programmes associated with vaccination and difficulties related to their application. The advantages and disadvantages of vaccination need to be considered in the decision-making process, including the financial aspects of vaccination.

Identification of sensitive and specific avian influenza polymerase chain reaction methods through blind ring trials organized in the European Union.

Many different polymerase chain reaction (PCR) protocols have been used for detection and characterization of avian influenza (AI) virus isolates, mainly in research settings. Blind ring trials were conducted to determine the most sensitive and specific AI PCR protocols from a group of six European Union (EU) laboratories. In part 1 of the ring trial the laboratories used their own methods to test a panel of 10 reconstituted anonymized clinical specimens, and the best methods were selected as recommended protocols for part 2, in which 16 RNA specimens were tested. Both panels contained H5, H7, other AI subtypes, and non-AI avian pathogens. Outcomes included verification of 1) generic AI identification by highly sensitive and specific M-gene real-time PCR, and 2) conventional PCRs that were effective for detection and identification of H5 and H7 viruses. The latter included virus pathotyping by amplicon sequencing. The use of recommended protocols resulted in improved results among all six laboratories in part 2, reflecting increased sensitivity and specificity. This included improved H5/H7 identification and pathotyping observed among all laboratories in part 2. Details of these PCR methods are provided. In summary, this study has contributed to the harmonization of AI PCR protocols in EU laboratories and influenced AI laboratory contingency planning following the first European reports of H5N1 highly pathogenic AI during autumn 2005.

Virologic findings in selected free-range mule duck farms at high risk for avian influenza infection.

Prevalence of avian influenza infection in free-range mule ducks (a cross between Muscovy [Cairina moschata domesticus] and Pekin ducks [Anas platyrhychos domesticus]) is a matter of concern and deserves particular attention. Thus, cloacal swabs were collected blindly from 30 targeted mule flocks at 4, 8, and 12 wk of age between October 2004 and January 2005. They were stored until selection. On the basis of a positive H5 antibody detection at 12 wk of age with the use of four H5 antigens, the samples from eight flocks were selectively analyzed. Positive samples were first screened with a matrix gene-based real-time reverse transcriptase-polymerase chain reaction assay before virus isolation. Eight avian influenza subtypes (H5N1, H5N2, H5N3, H6N2, H6N8, and H11N9) and three avian paramyxovirus type 1 viruses were isolated. All 11 are characterized as low pathogenicity (LP) and avirulent, respectively, by in vivo tests and, when relevant, nucleotide sequencing of the hemagglutinin (or fusion [F]) protein cleavage site. Regarding H5 isolates, all of their eight genes belong to the avian lineage and some particular genetic traits were determined. H5 genes were fully sequenced and phylogenetically analyzed; they all belong to the Eurasian lineage, lack additional glycosylation sites, and do not cluster, suggesting separate introductions from the wild reservoir. None were grouped with the Asian isolates. The N1 gene (H5N1 isolate) was very close genetically to an Italian LP-H7N1 gene. Antigenic relationships between these H5 isolates and others were assessed comparatively by crossed hemagglutination inhibition tests. All these data are very useful to control the evolution of H5 viruses at the genetic and antigenic level to better understand the source of new outbreaks (new introductions from wild birds or the result of spread among poultry) and to assess the immunity afforded by available vaccines. These data are useful also to update antigens for avian influenza survey and to choose the most suitable vaccine in the case of preventive vaccination of ducks.

Importance of a prime-boost DNA/protein vaccination to protect chickens against low-pathogenic H7 avian influenza infection.

Control of H5/H7 low-pathogenic avian influenza (LPAI) virus circulation is a major issue regarding animal and public health consequences. To improve vaccines and to prevent vaccinated poultry from becoming infected and from shedding wild viruses, we initiated studies targeting prevention of H7 infection through DNA vaccines encoding H7 and M1 viral proteins from an Italian H7N1 LPAI virus isolated from poultry in 1999. More recently, we expressed recombinant H7 and M1 proteins in the baculovirus system to assess whether they might enhance immunity when given as a boost after DNA vaccination. The protection afforded by three vaccine combinations-DNA/DNA, DNA/protein, protein/protein-given 3 wk apart were experimentally compared in 20 specific-pathogen-free chickens per group. Ten days after the boost, chickens were challenged with a homologous (Italian H7N1 LPAI) or heterologous (French H7N1 LPAI isolated from mallards in 2001) virus. Tracheal and cloacal shedding was measured by a matrix gene (M)-based real-time reverse transcription polymerase chain reaction assay and compared with that displayed by unvaccinated infected controls. After the homologous challenge, chickens of every vaccinated group displayed a significant decrease in cloacal shedding, whereas tracheal shedding was not significantly reduced in the protein/protein group. After the heterologous challenge, only the DNA/DNA group showed a nonsignificant decrease in tracheal shedding. According to these two trials, prime-boost DNA/protein vaccination appeared be more advantageous. Further development could be aimed at improving protein expression, shifting subtype (H5), and assessing the interest of proteins as a boost of recombinant vaccines.

European and American subgroup C isolates of avian metapneumovirus belong to different genetic lineages.

The gene encoding the attachment glycoprotein (G) was sequenced in three French isolates of-subgroup C avian metapneumovirus (APV-C) from ducks. With 1771 nt, this gene proved as long as recently published for North-American APV-C isolates from turkeys. The nt sequences of the duck viruses shared 99% identity but proved only 75-83% identical with their North-American counterparts, viruses of both origins encoding 585 amino acid (aa)-long G proteins. Alignments revealed more homogeneity within the European and North-American groups (at least 98 and 79% aa identity, respectively) than between European and North-American viruses (at best 70% a identity), and confirmed the presence of an extracellular divergent domain (positions 302-484) in APV-C G. A phylogenetic analysis demonstrated that North-American and French isolates of APV-C belonged to significantly different genetic lineages, in agreement with the different geographical origin and host species of these viruses.

Molecular study of the quasispecies evolution of a typical pigeon paramyxovirus type 1 after serial passages in pigeons by contact.

The quasispecies nature of a typical pigeon paramyxovirus type 1 (pPMV-1) was, for the first time, studied under conditions close to the natural infectious environment. The virus was serially passaged in pigeons by successive contacts. Viral heterogeneity was analysed in the kidneys and brain of five pigeons from the last contact, by reverse transcriptase-polymerase chain reactions performed on RNA directly extracted from the organ and targeting the P and HN genes of the virus. The viral diversity following in vivo passage was found to be different from that in the inoculum, but demonstrated the reality of the quasispecies concept for pPMV-1 strains. Moreover, some aberrant genomic RNAs comprising insertions in the P gene editing site or deletions in the HN gene were also detected, with possible consequences for the pathogenicity and infectivity of the virus.

Strategies to improve protection against low-pathogenicity H7 avian influenza virus infection using DNA vaccines.

Eukaryotic expression plasmids encoding either the avian influenza hemagglutinin or matrix genes (pCMV-HA and pCMV-M, respectively) were constructed. The viral genes were derived from a low-pathogenicity H7N1 strain, A/Chicken/Italy/1067/99, isolated during the 1999-2001 epizootic in Italy. The plasmid was administered to 4-to-5-wk-old specific-pathogen-free chickens by several different injection methods. For the initial studies comparing methods of vaccine injection, results were compared based on hemagglutination inhibition (HI) response following immunization with pCMV-HA. Additional studies with coadministration of both pCMV-HA and pCMV-M was evaluated based on HI response and viral isolation after homologous challenge. Preliminary results indicate that a device intended to inject insulin in humans (Medijector) and the coadministration of both plasmids improved protection against H7 infection.

Monitoring of pigeon paramyxovirus type-1 in organs of pigeons naturally infected with Salmonella Typhimurium.

An experimental pigeon paramyxovirus (pPMV-1) infection was followed by reverse transcription-nested polymerase chain reaction for 31 days after infection, in 16 organs of inoculated or contact pigeons naturally infected with Salmonella Typhimurium. With two exceptions, both groups presented similar results. Typical nervous signs and a green diarrhoea were observed. The spread of pPMV-1 was relatively quick, all organs being largely positive at 4 days after inoculation or contact. The lung, spleen, caecal tonsils, kidneys and brain, for which almost all tested samples remained positive during most of the experiment, seemed to be the principal targets for virus persistence. However, the virus was significantly recovered later in the brain parts and for longer in the trachea of the contact pigeons than of the inoculated ones.

Development of a RT-nested PCR test detecting pigeon Paramyxovirus-1 directly from organs of infected animals.

A RT-nested PCR that amplifies part of the conserved nucleoprotein gene of avian Paramyxovirus type 1 is described. The technique allowed the detection of pigeon Paramyxovirus type 1 (pPMV-1) virus directly from a wide range of infected chicken and pigeon organs, and should be able to detect typical Newcastle disease viruses too. Compared with the reference method, the developed RT-nested PCR was found more sensitive, as it was able to detect virus genome in infected pigeon organs at late stage of infection, when virus isolation failed. Such a molecular technique represents an alternative method of diagnosis for research purposes on pPMV-1 variants, for example to study pathogenesis aspects of the infection or to assess the efficacy of vaccines.