In contrast, higher neutralising capacity for the yellow fever vi

In contrast, higher neutralising capacity for the yellow fever virus in subjects with anti-dengue IgG antibodies has been reported, and hypothesised that subgroups with positive serology for dengue could develop cross-reactions with anti-yellow fever antibodies [16].

In 2013, the WHO Strategic Advisory Group of Experts (SAGE) announced that a single dose of the yellow fever vaccine provides life-long immunity and that revaccination every 10 years is not necessary for people who live in or travel to risk areas [4]. This new guideline was based on surveillance data indicating that vaccination failures are extremely rare and do not cluster as time increases after immunisations [4]. However, the known limitations in the surveillance of yellow fever cases and in the management of vaccination records, particularly in adults, suggest that data on vaccination

selleck chemicals llc failure are underestimated [14]. The rarity of vaccination failure could also be partly explained by the revaccination requirement in immunisation programmes and prior to travel to endemic areas. However, the absence of yellow fever cases in vaccinated travellers Selleckchem PF01367338 does not appear to be a good indicator of the duration of immunity, considering that potential natural exposures, which warrant recommendation for vaccination, can impair the assessment of the long-term effects of vaccination. WHO’s recent recommendations have also generated controversies because the serological methods used have varied over the many decades during because which the studies that served as the basis for the recommendations

were performed [14]. In addition, the PRNT method that determines neutralising antibody titres, which is considered the best available measure of seroprotection following vaccination, has exhibited considerable heterogeneity and allows only limited comparability between results [14]. A review exploring the scientific evidence for a change in the vaccination recommendation proposed by the WHO [7] appears to disregard the possibility that seronegative subjects may have been a result of primary or secondary failures of the vaccine. In fact, the high levels of vaccine immunogenicity in clinical studies under controlled immunisation conditions in selected groups may not be reproduced in routine immunisation programmes. These are generally affected by problems related to vaccine conservation and application, and may include subjects with clinical complications that could compromise their immune response. Accordingly, the rate of seroconversion following routine vaccination in military personnel in this study has been reported to be slightly lower than that in healthy volunteers in controlled studies [15]. In addition, a weaker immune response can result in shorter immunity duration. Cut-off values correlating with protection are not available for antibody titres measured by serum-dilution plaque-reduction tests.

gondii In the present work, we constructed recombinant Influenza

gondii. In the present work, we constructed recombinant Influenza A viruses harboring a dicistronic neuraminidase segment encoding T. gondii tachizoyte surface antigen SAG2 under control of a duplicated internally located 3′ promoter. Recombinant FLU-SAG2 viruses were genetically stable and induced expression of SAG2 in cell culture as well as in lungs of infected mice. We also observed that FLU-SAG2 was immunogenic

and, when associated with recombinant adenoviruses expressing SAG2 in vaccination protocols, elicited humoral and cellular anti-SAG2 immune responses, conferring a high degree of protection against challenge infection this website with the P-Br strain of T. gondii. Previous studies demonstrated that sequence of vector administration has pivotal importance in induction of heterospecific immune response in heterologous vaccination protocols [13], [14], [47] and [50]. Indeed, Li and co-workers showed that mice primed with a recombinant influenza and boosted with recombinant

Vaccinia encoding CS antigen, were protected after challenge with Plasmodium. However, no protection was observed in mice primed with Vaccinia and boosted with influenza. According to the authors, the systemic boost with Vaccinia could induce CTL migration to the liver, where Plasmodium circumsporozoyte replication occurs, while the intranasal boost with influenza viruses SCH772984 cost would favor CTL migration to lungs [13]. Based on these previous observations, we have chosen to prime the animals with FLU-SAG2 and to boost with Ad-SAG2. We speculate that the influenza vector would elicit anti-SAG2 immune response mainly at the respiratory level, priming both B and T cells, whereas a boost with adenovirus would reinforce the response at systemic level. Indeed, detection of IFN-γ producing

T cells specific for SAG2 in spleen and protection after challenge infection were only demonstrated in mice that received Ad-SAG2 boost by subcutaneous route. Although we did not evaluate the cellular immune response in respiratory tract, we speculate that boosting by intranasal route could detour T lymphocytes to respiratory tract and to abrogate the systemic cellular immune response. In our experiments, mice primed ADP ribosylation factor with FLU-SAG2 and boosted with recombinant Ad-SAG2 displayed significant reduction of parasite burden after challenge with the P-Br strain of T. gondii. On the other hand, mice vaccinated with a single dose of Ad-SAG2 showed parasite burden similar to that found in animals vaccinated with control vectors. These results support previous studies showing that often significant protection cannot be achieved after a single immunization [3] and [51]. In addition, our results showed that innate immune response triggered by influenza inoculation was not sufficient to explain protection observed after the boost with Ad-SAG2.

Older patients, those with back pain, and those who had previousl

Older patients, those with back pain, and those who had previously

taken sick leave for neck pain were more likely to report activity due to neck pain at the 3-month follow-up. Ethics: The University of Sydney Human Research Ethics Committee(s) approved this study. All participants gave written informed consent before data collection began. Support: This study was supported by the Australian National Health and Medical Research Council (Grant Docetaxel no. 402686) and The University of Sydney. “
“Regular physical activity is directly related to positive health outcomes (Schnohr et al 2003, Wen et al 2011). To achieve positive health outcomes guidelines recommend that adults should accumulate 30 minutes of moderate intensity aerobic activity on most days of the week (Pate et al 1995). Updated versions of these guidelines, which also consider older adults (≥ 65 years) and people with chronic health conditions, state that the activity must be completed in bouts of 10 minutes or more, on at least 5 days of the week (Haskell et al 2007, Nelson et al 2007, WHO 2011). There is emerging evidence to suggest that as little as 15 minutes of moderate intensity physical activity may be beneficial to health for community-dwelling adults and older adults (Wen et al 2011). Furthermore, GSK J4 nmr it is recommended that older adults

who are limited by health conditions be ‘as physically active as their abilities and conditions allow’ (WHO 2011). Orthopaedic rehabilitation aims to promote independence and improve function to prepare patients to return to living independently in the community. Therefore, it could be expected that patients are trained while in rehabilitation to have levels of physical activity that are recommended for maintenance of health, in preparation for living Montelukast Sodium independently in the community. However, adults with lower limb orthopaedic conditions in inpatient rehabilitation

may find it difficult to be sufficiently active to meet physical activity guidelines because of the difficulty in restoring mobility after injury and/or surgery (Beringer et al 2006, Groen et al 2012, Koval and Zuckerman 1994, Resnick et al 2011, Schmalzried et al 1998, Silva et al 2005). Following hip fracture, inpatients who were more active during therapy sessions had better functional outcomes than those who were less active (Talkowski et al 2009), suggesting a positive relationship between physical activity and functional outcome. However, we were unable to locate any research that quantifies the physical activity levels of adults with lower limb orthopaedic conditions during inpatient rehabilitation in relation to physical activity guidelines. Therefore, the research questions for this study were: 1.

Moreover, studies of mainly adults have shown that PCV7 is immuno

Moreover, studies of mainly adults have shown that PCV7 is immunogenic in patients with leukemia, especially if it

is administered at an early stage of the disease (i.e. before the start of chemotherapy and the development of hypogammaglobulinemia) [54] and [55]. None of these few studies reported any safety or tolerability problems [53], [54] and [55]. Although meningococcal vaccine is recommended by health authorities for all high-risk subjects, find more there are very few studies of its use in children with cancer receiving standard chemotherapy [24] and [56]. One of the main study was performed by Yu et al., who administered meningococcal C CRM197 conjugate vaccine to 35 children aged 2.1–17.8 years, most of whom had ALL [56]. The children were on maintenance therapy or had completed chemotherapy between three and 18 months earlier. Fifty percent of the children showed a positive serological response, defined as a four-fold increase in meningococcal-specific

IgG, and a complement-mediated bactericidal response was demonstrated in 44%; however, only 39% of children showed a simultaneous serological and bactericidal response. The response was strictly related to total B cell counts and the proximity of chemotherapy. The vaccine was safe and well tolerated by all of the children [56]. Children with cancer are considered to be at risk of influenza-related complications because they often require intensive care and prolonged hospitalisation during the course Carnitine palmitoyltransferase II of influenza, and influenza can considerably

delay the start of chemotherapy drug administration [57], [58] and [59]. A number of studies investigating the use of trivalent influenza vaccine in children with ALL and solid tumours have been carried out, but most of them were published some years ago, and only a few have made use of newer vaccines [60], [61], [62], [63], [64], [65], [66], [67], [68] and [69]. Furthermore, although all of these studies evaluated immunity, safety and tolerability, there are no published data concerning vaccine efficacy in laboratory-confirmed cases, hospitalisation, chemotherapy delays or mortality. Nevertheless a global evaluation indicates that inactivated influenza vaccines are safe and well tolerated: no serious adverse events have been observed and the proportion of mild adverse events is no different from that observed in healthy subjects [60], [61], [62], [63], [64], [65], [66], [67], [68] and [69]. Children with cancer seem to be able to generate a sufficient immune response to the influenza antigens contained in the vaccines when receiving chemotherapy, but this response is weaker than that of healthy children or children with cancer who have discontinued chemotherapy for more than 1 month (both groups show similar antibody production) [61], [62], [63] and [64].

AdGFP served as a positive control and showed that 90% of the cel

AdGFP served as a positive control and showed that 90% of the cells were transduced by this adenovector and expressed the GFP transgene following infection at an MOI of 200 pu/cell. These data support the previous finding that DAF anchor is more efficient to attach antigen to the cell membrane than the native MSP142 anchor in mammalian cells. We next evaluated the immunogenicity of adenovectors expressing the various forms of MSP142 in mice. We observed robust T cell responses 2 weeks after a single immunization for each of the MSP142 expressing adenovectors (Fig. 7a). A second immunization of MSP142 adenovector 6 weeks later did not increase MSP142-specific IFN-γ

responses relative to the 2-week time point (although responses induced by DSA and DS-GM constructs appear MS-275 to be sustained longer in animals that received two doses). The various MSP142 adenovectors differed in their capacity to induce BI6727 MSP142-specific antibody responses in mice. MSP142-specific antibodies were observed in mice after immunization

with a single administration of either AdMSP142-DS or AdMSP142-DSA, but not after one or two doses of AdMSP142-DS-GM or AdMSP142-IC. Interestingly, a second dose of either AdMSP142-DS or AdMSP142-DSA boosted MSP142-specific antibody responses by about 10-fold relative to a single administration of adenovector (Fig. 7b). Adenovector-induced antibody responses were also evaluated in rabbits following two immunizations at an 8-week interval. MSP142-IC was not included in this analysis as it was a poor inducer of antibody responses in the murine studies. The ELISA data with rabbit sera were similar to those from the murine studies. Specifically, the DS and DSA constructs induced the highest responses and

the glycosylation mutant DS-GM induced weak MSP1-specific serum antibody (Fig. 7c). The ability of the MSP142 adenovectors to induce functional antibodies, capable of inhibiting the invasion of erythrocytes by blood stage forms of P. falciparum, was evaluated using GIA. High titers of functional antibodies were induced in rabbits by the adenovectors expressing MSP142. Approximately 60% inhibition was achieved in the standard assay using 2.5 mg/ml of purified IgG from Endonuclease immunized rabbits. The DS and DSA versions of MSP142 induced equally high levels of functional antibodies by GIA ( Fig. 8a) and total antibody by ELISA ( Fig. 8b). We observed similar results using diluted antibody ( Fig. 8c and d). AdMSP142-DS and AdMSP142-DSA performed comparably inducing statistically significant increases in GIA relative to AdNull and AdMSP142-GM (p = 0.0005). There is considerable enthusiasm for the evaluation of adenovirus-based vectors as a gene delivery platform for vaccines. This is driven by findings from different laboratories that adenovectors induce robust and protective T cell responses in multiple animal models of infectious diseases [20], [21], [22], [23] and [24].

In other systems, however, EMS transport to hospital may not alwa

In other systems, however, EMS transport to hospital may not always be quicker than self-transport. [15] Moreover, other patient-related factors, such as atypical symptoms, diabetes, race, gender, as well as psychosocial factors, have been shown to impact pre-hospital Selleckchem LY2835219 delays [16], [17], [18], [19], [20] and [21]. Among the known factors associated with delays in DTB, our study found that self-transport (versus EMS-transport) and off-hours presentation (versus on-hours) correlate independently with DTB > 90 minutes. The impact of off-hours presentation causing

delay was also demonstrated in recent studies [22] and [23]. However, other known patient-related factors did not correlate with delays in DTB in our study [24], [25] and [26]. Our study identifies a practical approach to help expedite in-hospital processing

of STEMI patients – use of EMS will actually facilitate more efficient ED processes leading to catheterization laboratory activation. The availability of pre-hospital ECGs may have helped in the ED triage process leading to catheterization laboratory activation AZD8055 purchase [27], and door-to-activation time is a key determinant of DTB times [12]. At present, EMS is still underutilized based on large national registries [11], and for reasons unclear, this has not changed Isotretinoin since a decade back [10], although the median DTB times have improved due to improvements in hospital best practice strategies [28]. Increasing

the use of EMS would certainly provide further opportunities to improve DTB times in most systems similar to ours. Other strategies may include pre-hospital activation of the catheterization laboratory and bypassing the ED altogether for patients with a clear STEMI diagnosis [29]. This approach has its pitfalls, however, the least of which include erroneous diagnosis, incomplete assessment of patient’s condition, and false activations [30], [31] and [32]. In addition, many systems in the United States do not practice pre-hospital activation. In line with Mission: Lifeline, a nationwide initiative for STEMI care launched by the American Heart Association [33], community education efforts should be directed not only at recognizing symptoms of myocardial infarction, but also at the exigency and benefit of EMS activation. The key message to the community is to call EMS early in order to avoid delays. Moreover, efforts should be made to identify major barriers to EMS use (e.g. denial, lack of awareness, fear of costs, trustworthiness of others to provide care, as well as other psychosocial and educational factors) [19], [20] and [21], to enhance the effectiveness of community outreach. This study has several limitations.

It may be beneficial to select a discrete dengue outbreak, such a

It may be beneficial to select a discrete dengue outbreak, such as the recent outbreak in Martinique, and examine all the associated costs. This could then be more broadly applied to better understand the total costs of dengue. The indirect costs PD-0332991 cell line that are typically unaccounted for include the cost of disruption to health care services (caused by the influx of dengue cases), and the cost of decreased tourism, shipping, transport, and commerce due to fears of the disease spreading. The impact of dengue on patients and their families is significant, both

economically and in terms of quality of life. The economic cost disproportionately falls on the poor, particularly in countries where most costs are covered by the patient. A study in Cambodia showed that patients with dengue cover, on average, 78% of the total cost and 63% of the direct medical cost [28]. In a study in Thailand, 47% of patients with dengue could not afford to visit a reputable medical provider, 14% could not afford treatment, and 17% had to borrow money to cover the cost of illness [29]. Other studies in Cambodia show how these costs are a continuing burden to the

poor [30], with the majority (62%) still unable to repay their debts up to one year later [31]. There is also a significant drop (>50%) in the quality of life of both children and adults with dengue, which does not return to baseline until 12–16 days after onset of illness which is almost twice the duration of fever [32]. To raise the profile of dengue among governments and global decision-makers, which will be essential to secure funding for vaccine from introduction, it will be necessary to publicise the full

extent of the human burden of dengue. The morbidity caused by dengue should be highlighted and attempts made to move the global focus away from simply considering mortality statistics. While the mortality statistics for dengue are lower than for some other diseases considered a global health priority, the human impact of dengue morbidity is profound and, if better conveyed, persuasive. In particular, the impact of dengue on communities and its psychological impact on patients and families are often ignored. Computational modelling is an additional tool to support the decision-making process. It has proven to be highly advantageous in dengue research, for example in mapping the movement of the dengue virus from urban centres [33] and identifying the causes of the upwards shift in the average age of patients with dengue in some countries [34]. Each dengue-endemic country should have the opportunity to run its own modelling programs, however both human (skilled technicians/programmers) and material (sufficient computational power) resources are currently lacking.

1 mM EDTA, pH 7 4) After

centrifugation through a Spin-X

1 mM EDTA, pH 7.4). After

centrifugation through a Spin-X centrifuge tube filter (Corning, U.S.A.), the sterile stock solution was stored at 4 °C for use within one month. A stock of A/PR8 (H1N1) influenza virus propagated on Madin–Darby canine kidney cells (MDCK) was kindly provided by Solvay Biologicals (Weesp, The Netherlands). The virus titer was determined by measuring the tissue culture infectious dose 50 (TCID50). To this end serial twofold dilutions of virus suspension were inoculated on MDCK cells grown in serum-free medium. 1 h later TPCK trypsin (Sigma, Zwijdrecht, Netherlands) was added to a final concentration of 7.5 μg/ml. After 72 h, supernatants were collected and transferred to a round-bottom 96-well plate followed by the addition of 50 μl 1% guinea pig erythrocytes to each well. The plate

was incubated for 2 h before reading. The titer was determined selleck kinase inhibitor as the highest virus dilution at which hemagglutination was visible and the TCID50 was calculated by the method of Reed and Muench [19]. For inactivation, the virus was incubated with freshly prepared 10% β-propiolactone in citrate buffer (125 mM sodium citrate, 150 mM sodium chloride, pH 8.2) at a final concentration of 0.1% β-propiolactone. Inactivation was carried out for 24 h at 4 °C under continuous stirring. After inactivation, the virus was dialyzed against phosphate-buffered saline (PBS) overnight at 4 c. Subunit vaccine was prepared by solubilizing the inactivated virus (0.8 mg virus protein/ml) in PBS

containing Tween 80 (0.3 mg/ml) and hexadecyltrimethylammonium Regorafenib price bromide (CTAB, 1.5 mg/ml) for 3 h at 4 °C under continuous stirring, and oxyclozanide removal of the viral nucleocapsid from the preparation by ultracentrifugation for 30 min at 50,000 rpm in a TLA100.3 rotor at 4 c. Detergents were then removed by overnight absorption onto Biobeads SM2 (634 mg/ml, Bio-Rad, Hercules, CA) washed with methanol prior to use. Protein content of the inactivated virus and subunit material was determined by a modified Lowry assay [20]. Hemagglutinin (HA) content was assumed to be one third of the total protein for whole inactivated virus (based on the known protein composition of influenza virus and the molecular weight of the viral proteins) and to be equal to the total protein for subunit material (based on silver-stained SDS polyacrylamide gels run under reducing and non-reducing condition) [21]. Vaccines were mixed at the indicated amounts of subunit and GPI-0100 just before immunization. The protocol for the animal experiment described here was approved by the Ethics Committee on Animal Research of the University of Groningen. Female Balb/c mice (Harlan, The Netherlands) aged 8–10 weeks were grouped (n = 6 per group) and immunized intramuscularly (i.m.) with A/PR/8 subunit vaccine with or without GPI-0100 adjuvant in a two-dose immunization regimen (day 0 and day 20). Control mice were injected with HNE buffer.

Règle 5 : « Je m’hydrate régulièrement à l’entraînement comme en

Règle 5 : « Je m’hydrate régulièrement à l’entraînement comme en compétition ». La déshydratation, même modeste, diminue la performance et, associée à l’ambiance hypercatécholergique de l’effort intense, augmente le risque d’accident cardiovasculaire. Règle 6 : « J’évite les activités intenses en cas de changement brutal et marqué de la température extérieure (< −5 °C ou > 30 °C) et lors des pics de pollution ». Chez le sujet peu entraîné et/ou à risque, ces deux éléments majorent le risque d’angor et de troubles du rythme. Des efforts intenses peuvent cependant être réalisés par le sportif entraîné, acclimaté et bien équipé. Règle 7 : « Je ne fume

pas et en tout cas jamais 2 heures avant ou après une pratique sportive ». Les sportifs fumeurs sont trop nombreux. L’association activité physique intense et tabac majore fortement la survenue NVP-AUY922 concentration Selleckchem STI571 d’un thrombus occlusif en particulier coronaire. Règle 8 : « Je ne consomme jamais de substances dopantes et j’évite l’automédication en général ». Les effets cardiovasculaires délétères des produits dopants sont bien démontrés. L’automédication comporte aussi des risques tels que thrombi-vasculaires, hémorragies, troubles du rythme, insuffisance rénale. Règle 9 : « Je ne fais pas de sport intense en cas de fièvre, ni dans les 8 jours qui suivent un épisode grippal (fièvre + courbatures) ». until L’inflammation peut toucher

le myocarde au même titre que les autres muscles « courbaturés ». Elle favorise la survenue d’arythmies à l’effort. Règle 10 : « Je pratique un bilan médical avant de démarrer ou reprendre une activité sportive intense si j’ai plus de 35 ans pour les hommes et plus de 45 ans pour les femmes ». Le risque d’accident cardiovasculaire est transitoirement majoré lors d’une activité sportive intense surtout chez le sédentaire. Ces règles ne permettront malheureusement pas de prévenir tous les accidents. La mort subite

liée au sport survient presque toujours en présence de témoins. Il est prouvé qu’en France ceux-ci interviennent très peu. La rapidité de la mise en œuvre du massage cardiaque est pourtant un facteur majeur de survie [25]. Il faut donc insister auprès de l’environnement sportif et de la population générale pour qu’elle se forme aux gestes d’urgence qui se résument à appeler, masser, défibriller (Fédération française de cardiologie). Nous avons vu que la pratique d’un sport en compétition aggravait le risque de mort subite en révélant une cardiopathie méconnue. Éthiquement, médicalement et légalement, il est justifié de proposer une prévention la plus efficace possible de ces accidents. Elle repose sur une visite médicale de non-contre-indication (VNCI) efficace, complétée si besoin d’examens complémentaires ciblés. Le terme de compétition mérite d’être précisé.

Two viruses, A− with a 13 amino acid deletion within the VP1 G-H

Two viruses, A− with a 13 amino acid deletion within the VP1 G-H loop and A+ with the native VP1 G-H loop, were derived from a Middle Eastern serotype A vaccine strain of FMDV by three rounds of plaque purification in BHK-21 cell cultures. Field isolates of FMDV serotype A, namely, A22/IRQ/24/64, A/IRN/2/87, A/IRN/41/2003, A/IRN/4/2005, A/IRN/5/2005, A/IRN/31/2001, A/IRN/6/2002, A/IRN/32/2004, A/KEN/2/2003, A/LAO/36/2003, A/MAY/2/2002,

A/PAK/9/2003, A/PAK/11/2003, click here A/TAI/10/2003, and A/TUR/5/2003, were received as primary bovine thyroid cell culture supernatants from the World Reference Laboratory for FMD (WRLFMD) at Pirbright. These viruses were subsequently passaged once in BHK-21 monolayer cells in 175 cm2 flasks in order to increase the virus titre and volume. The sequence of the capsid coding regions which encode the VP1 G-H loops of the A+ and A− viruses were determined to confirm that the VP1 G-H loop was retained in the A+ and that the loop deletion remained in the A− following one passage on BHK-21 cells. Sequencing and comparison between the entire capsid coding regions of A+ and A− were Lapatinib in vivo also performed to resolve any other amino acid changes. Total RNA was obtained using RNeasy Kit (Qiagen) following the manufacturer’s guidelines. The capsid coding region was obtained using Ready-To-Go™ RT-PCR

beads (GE Healthcare) in seven overlapping fragments using a one-step reverse transcription polymerase

chain reaction (RT-PCR), 14 primers (LF, ACCCCTGGACACCGGACCCGTC, 516R, TGTTCGGTGGGGAGTTCCAAC, 252F, CGCCGACAAAAAGACAGAGG, 875R, TGGGTTGGGGCGATGTTGGCGT, 552F, CGCGTACATGAGAAATGGCTG, 1159R, TTGCAGCCAGGGAAACATCAAAC, 854F, ACGCCAACATCGCCCCAACCCA, 1438R, CTGCCACGTCAGACGCGGTGT, 1137F, GTTTGATGTTTCCCTGGCTGCAA, 1743R, GTGGGTCTGCATGAGGTCAATG, 1420F, ACCGCGTCTGACGTGGCAGA, 2088R, GTGGATGGTCGTGGCCCGAATT, 1728F, CCTCATGCAGACCCACCAACAC, NK61, GACATGTCCTCCTGCATCTG) and cycle parameters (50 °C for 30 min, 95 °C for 15 min, [94 °C – 1 min, 55 °C – 1 min, 72 °C – 1 min] × 35 cycles, Carnitine palmitoyltransferase II 72 °C – 10 min). All thermal treatments were performed on an Eppendorf mastercycler (Eppendorf). RT-PCR reactions were separated on an appropriate percentage agarose gel and the products visualised by ethidium bromide staining. RT-PCR products were purified using a GFX DNA purification kit (GE Healthcare) following manufacturer’s guidelines. PCR products were sequenced using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) as per the manufacturer’s guidelines. The same 14 primers detailed for the RT-PCR were used for the sequencing reactions in conjunction with cycle parameters of 96 °C for 1 min, [96 °C for 10 s, 5 °C – 5 s, 60 °C – 4 min] × 25 cycles provided by an Eppendorf Mastercycler (Eppendorf). Subsequent sequencing was performed on an Applied Biosystems (ABI) 3730 DNA analyser.