Assessment of Pandemic and Seasonal Influenza A (H1N1) Virus Susceptibility to Neuraminidase Inhibitors in Three Enzyme Activity Inhibition Assays

The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC₅₀s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC₅₀s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC₅₀s between the wild type and the variants, whereas the IC₅₀s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.For the treatment and chemoprophylaxis of infections caused by influenza A viruses, the U.S. Food and Drug Administration (FDA) has approved four drugs: amantadine and rimantadine as well as zanamivir and oseltamivir. These drugs belong to two classes, adamantanes (i.e., M2 ion-channel blockers) and neuraminidase (NA) inhibitors (NAIs), respectively. In recent years, the effectiveness of M2 blockers has been greatly compromised, which limits their usefulness in clinical practice. This is largely due to the rapid emergence and widespread circulation of adamantane-resistant influenza viruses (1, 5, 6, 7, 14, 17). More recently, the emergence and worldwide spread of seasonal H1N1 viruses resistant to oseltamivir, currently the most widely used drug against influenza infections, became a considerable public health concern (15, 21, 25, 32). Monitoring the NAI resistance of influenza viruses is an ongoing public health issue since the emergence in 2009 of pandemic viruses that are resistant to M2 blockers.Cell culture-based assays are typically not used for assessment of virus sensitivity to NAIs because of the unpredictable effect of hemagglutinin (HA) receptor binding (2, 34). Instead, drug susceptibility can be monitored by functional (biochemical) NA inhibition (NI) assays, and subsequent genotypic methods are generally required to identify the molecular marker(s) of resistance in the NA. The principle underlying the functional methods relies on the enzymatic nature of the NA, a viral surface glycoprotein and antigen. NA acts by cleaving the terminal neuraminic acid (also called sialic acid) from receptors recognized by influenza viral HA, thus facilitating the release of progeny virions from infected cells and preventing self-aggregation (29). Structurally, NAIs mimic the natural substrate, neuraminic acid, and produce tight interactions, with conserved residues of the NA active site competing with neuraminic acid for binding (11, 23). Preincubation of virus with NAIs leads to the inhibition of enzyme activity, which is detected after the addition of enzyme substrate. Most NI assays commonly used for virus surveillance utilize as substrates small synthetic conjugates that produce either a luminescent or a fluorescent signal upon cleavage by the NA enzyme. The chemiluminescent (CL) assay uses the 1,2-dioxetane derivative of neuraminic acid substrate in the influenza neuraminidase inhibitor resistance detection (NA-Star) kit (8), while the fluorescent (FL) assay employs 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid substrate (MUNANA) (30). The results of the NI assays are expressed as the 50% inhibitory concentration (IC₅₀), which represents the NAI concentration that inhibits 50% of the enzyme activity of the virus. As the NA activity of clinical specimens is usually insufficient for determining the IC₅₀ due to a low viral content, NI assays, using either the substrate provided with the NA-Star kit or the MUNANA substrate, require virus propagation in cell cultures or embryonated chicken eggs. It is noteworthy that IC₅₀s are specific to the virus type/subtype and to the individual NAI tested (8, 19, 20, 24, 32, 37). The IC₅₀s obtained can be used for assessment of virus susceptibility to NAIs, including detection of resistant viruses, as well as for comparing the potencies of antiviral drugs belonging to the NAI class. Although both the CL and FL assays allow reliable detection of NAI resistance, the more recently developed CL assay was reported to be about 70 times more sensitive in detecting NA activity and has a greater linear range than the FL assay (8). The CL assay was also selected for use in the global drug susceptibility surveillance program by the Neuraminidase Inhibitor Susceptibility Network (NISN) (37, 39) and by other surveillance laboratories (28, 32). It should also be noted that IC₅₀s may vary even for the same virus when the NI assay is done using the NA-Star substrate (CL assay) and the MUNANA substrate (FL assay), according to reports on seasonal viruses (37). Whether one of the two assays, the CL or FL assay, more reliably predicts the level of resistance and the drug concentration required for the NA activity inhibition in vivo are key points of interest and remain to be elucidated.A third assay, the colorimetric (CM) assay, which utilizes fetuin as the substrate of the NA, is typically used to determine the titer of anti-NA antibodies because small substrates do not effectively compete with antibodies (3, 31). This assay is not widely used for antiviral susceptibility testing. Unlike the NA-Star and MUNANA synthetic substrates, fetuin is a large, natural, and soluble bovine glycoprotein that contains abundant neuraminic acids at the ends of its oligosaccharide moiety (which include the presence of two residues of α2,3-linked sialic acid and one residue of α2,6-linked sialic acid) (4, 33) and has been used as a substrate in NA-catalyzed reactions (3). Given that NAIs compete with the enzyme substrate for binding to the active site, the structure of the substrate can potentially influence the outcome of the competition and, as a result, the IC₅₀. In this respect, fetuin may represent a better natural substrate for the enzyme-neuraminic acid attached via an α2,3 or α2,6 linkage to oligosaccharide chains on the cell surface. Furthermore, since the cleavage of each neuraminic acid is chemically converted, the CM assay can be a quantifiable method from which the resulting IC₅₀s would correlate more closely to the NA activity of the virus tested. Despite these apparent advantages to the use of fetuin, the CM method relies on chemical reactions that are time-consuming, cumbersome, and impractical for high-throughput use. In addition, the assay requires concentrated virus stocks for testing. Thus, fetuin is still considered an undefined substrate that does not confer sufficient sensitivity or specificity for use in routine NAI susceptibility assays (34). The potential usefulness of a large substrate such as fetuin for assessment of the NAI susceptibilities of novel H1N1 viruses or novel inhibitors remains largely unexplored.Resistance to NAIs is not defined as clearly as that to adamantanes. In NI assays, a drug-resistant virus should have IC₅₀s consistently greater than the threshold value that is determined for each viral type/subtype and drug tested (27, 32, 37). Since the 2007-2008 influenza season, about a decade after the introduction of NAIs into clinical use, an NA framework mutation, H275Y (H274Y in N2 numbering), was consistently and most commonly detected in oseltamivir-resistant H1N1 viruses isolated worldwide (15, 21, 25, 32). Although the H275Y substitution represents the most-defined oseltamivir resistance marker of influenza viruses carrying the NA of the N1 subtype (35), novel NAI resistance-associated mutations—determined by elevated IC₅₀s in NI assays—continue to be revealed (21, 22, 32). Importantly, oseltamivir-resistant viruses from the ongoing H1N1 pandemic have been detected and reported around the world (9, 10, 26, 38). Seasonal and 2009 pandemic H1N1 viruses have the same phylogenetically distant NA gene ancestors (16), which necessitates the comprehensive assessment of the drug susceptibilities of the new pandemic viruses. Therefore, it is necessary to evaluate existing NI assays in order to better understand which assay may be the most sensitive for the detection of NAI resistance and/or the most predictive of virus susceptibility to NAIs in vivo.In the present study, we assessed the susceptibilities of a panel of seasonal and pandemic H1N1 influenza viruses, including virus variants bearing the established oseltamivir resistance mutation, H275Y in the NA, against five NAIs: two FDA-approved NAIs, zanamivir and oseltamivir, and three investigational NAIs, peramivir, R-125489 (the bioactive metabolite of the prodrug CS-8958 [laninamivir]), and A-315675 (a bioactive form of the prodrug A-322278). In order to better characterize and assess the consistency of IC₅₀s and levels of susceptibility, these viruses were tested in the widely used CL and FL assays, as well as with the CM method.     

Effect of rifampicin in the treatment of pruritus in hepatic cholestasis

Pruritus in hepatic cholestasis has been suggested to be secondary to a high concentration of serum bile acids. Rifampicin, which inhibits the uptake of bile acids by hepatocytes, has been used to treat pruritus. To determine the efficacy of rifampicin as a treatment for refractory pruritus, the medical records of 33 children (median age 25 months, range 4-135; 19 boys) with chronic cholestasis liver disease (21 with Alagille's syndrome, eight with progressive intrahepatic cholestasis, one with extrahepatic biliary atresia, one with an inborn error of bile acid metabolism, and one with cryptogenic cirrhosis) were reviewed retrospectively. The median dose of rifampicin was 5(4-10) mg/kg/day. The median duration of intake was 36(4-120) weeks. Complete relief of pruritus was noted in five (15%) patients and a partial response in 12 (36%). Overall, no significant difference was noted in the laboratory parameters before and after treatment with rifampicin. In the 21 patients with Alagille's syndrome, however, a significant decrease in alkaline phosphatase was seen before and after one and six months of starting treatment. No adverse side effects were seen. Rifampicin appears to be effective in the treatment of refractory pruritus. A prospective study is warranted to assess further the effect of rifampicin treatment in children with hepatic cholestasis.     

Leukocyte-technetium-99m uptake in Crohn’s disease: Does it show subclinical disease?

AIM: To evaluate inflammatory activity in patients with Crohn’s disease (CD) using technetium-99m-hexamethylpropyleneamine oxime (99mTc-HMPAO) granulocyte scintigraphy.METHODS: Twenty patients (7 male and 13 female) with CD and five healthy volunteers were selected for 99mTc-HMPAO granulocyte scintigraphy. The Crohn’s Disease Activity Index (CDAI), blood tests and C-reactive protein (CRP) of each patient were performed 7 d before the scintigraphic images. The leukocytes were labeled according to the International Society of Radiolabeled Blood Elements (ISORBE) consensus protocol and the scintigraphic images, including single photon emission computed tomography, were obtained 30 min and 2 h after injection of the radiolabeled leukocytes.RESULTS: The labeling yield of the leukocytes with the lipophilic complex 99mTc-HMPAO was 55.0% ± 10%. Six of the 20 patients (30%) presented congruent results for the three parameters investigated (CDAI, Scintigraphic Index and CRP). On the other hand, 14 patients (70%) did not show congruent results. There was no significant correlation between the indices analyzed according to the Spearman test (P > 0.05, n = 20).CONCLUSION: The results suggest that 99mTc-HMPAO-labeled leukocyte scintigraphy could be important for determining inflammatory activity in CD even in the absence of clinical symptoms.     

Transforming growth factor beta 1 directly and reversibly inhibits the initial cell divisions of long-term repopulating hematopoietic stem cells

Hematopoiesis appears to be regulated, in part, by a balance between extracellular positive and negative growth signals. Transforming growth factor beta-1 (TGF-beta 1) has been shown to be a negative regulator of primitive hematopoietic cells. This study examined the direct effect of TGF-beta 1 on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC) in vitro. We previously reported a cell fractionation approach that includes the selection of low Hoescht 33342/low Rhodamine 123 (low Ho/Rh) cell fractions that are highly enriched for long-term repopulating cells (LTR-HSC) and also clone to a very high efficiency in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6: 90% to 100% of individually cultured low Ho/Rh cells formed high proliferative potential clones. This high cloning efficiency of an LTR-HSC enriched cell population enabled proliferation inhibition studies to be more easily interpreted. In this report, we show that the continuous presence of TGF-beta 1 directly inhibits the cell division of essentially all low Ho/Rh cells (in a dose-dependent manner) during their 0 to 5th cell division in vitro. Therefore, it follows that TGF-beta 1 must directly inhibit the proliferation of LTR-HSC contained within these low Ho/Rh cells. The time required for some low Ho/Rh cells to undergo their first cell division in vitro was also prolonged in the presence of TGF-beta 1. Furthermore, when low Ho/Rh cells were exposed to TFG-beta 1 for varying lengths of time before neutralization of the TGF-beta 1 by monoclonal antibody, the ability to form macroclones was markedly decreased after approximately 4 days of TGF-beta 1 exposure. In addition, 1 to 10 ng/mL of TGF-beta 1 resulted in a maintenance of high proliferative potential-colony-forming cell (HPP-CFC) during 8 days of culture compared with loss of HPP-CFC in cultures with no added TGF- beta 1. In conclusion, this study shows that TGF-beta 1 directly inhibits the initial stages of proliferation of LTR-HSC and appears to slow the differentiation of daughter cells of low Ho/Rh cells.     

The incidence,spectrum and outcome of paediatric trauma managed by the Pietermaritzburg Metropolitan Trauma Service

Introduction

The Pietermaritzburg Metropolitan Trauma Service (PMTS) has run a systematic quality improvement programme since 2006. A key component included the development and implementation of an effective surveillance system in the form of an electronic surgical registry (ESR). This study used data from the ESR to review the incidence, spectrum and outcome of paediatric trauma in Pietermaritzburg, South Africa.

Methods

The ESR was reviewed, and all cases of paediatric trauma managed between 1 January 2012 and 30 July 2014 were retrieved for analysis.

Results

During the study period, 1,041 paediatric trauma patients (724 male, 69.5%) were managed by the PMTS, averaging a monthly admission of 36. The mean age was 10.9 years (standard deviation: 5.4 years). The mechanism of injury (MOI) was blunt trauma in 753 patients (72.3%) and penetrating trauma in 170 (16.3%). Pedestrian vehicle collisions accounted for 21% of cases and motor vehicle collisions for a further 11%. Intentional trauma accounted for 282 patients (27.1%) and self-inflicted trauma for 14 cases (1.3%). Ninety patients admitted to the intensive care unit and fifty-one required high dependency unit admission. There were 17 deaths, equating to an in-hospital mortality rate of 1.7%. A total of 172 children died on the scene of an incident. There were 35 road traffic related deaths, 26 suicides by hanging, 27 deaths from blunt assault and 23 deaths from penetrating assault. The overall mortality rate for paediatric trauma was 18.2%.

Conclusions

The ESR has proved to be an effective surveillance system and has enabled the accurate quantification of the burden of paediatric trauma in Pietermaritzburg. This has improved our understanding of the mechanisms and patterns of injury, and has identified a high incidence of intentional and penetrating trauma as well as road traffic collisions. These data can be used to guide strategies to reduce the burden of paediatric trauma in our environment.     

Release of galactosyltransferase from human platelets and a subset of monocytes in culture

We show that human monocytes and platelets release considerable amounts of galactosyltransferase (GT) in serum-free culture as measured by the amount of incorporation of 3H-galactose into ovalbumin. Enzyme production was the greatest among medium-sized mononuclear cells separated by counter-current elutriation. The cells were adherent and positive for the monocyte-specific monoclonal antibody FMC-32. The activity in the monocyte fractions was not due to platelet contamination as shown from experiments in which platelets or platelet antigens were eliminated. Cell viability decreased by less than 3% during the overnight culture, and results from cell disruption experiments showed that the enzyme was not released from dead or dying cells. Cycloheximide inhibited release during 20 hours culture. Approximately 50% of the enzyme in the cell culture supernatant was pelletable at 105,000 g. Platelets released the enzyme more rapidly than did monocytes and were readily stimulated by thrombin to release more GT. Thrombin also increased monocyte GT activity after overnight incubation, but other stimulants, zymosan and lipopolysaccharide (LPS), decreased release. We conclude that GT is released into culture supernatants by platelets and by a subset of peripheral blood monocytes. These sources may account for a significant proportion of the serum enzyme and may be important in modification of extracellular carbohydrates during inflammation and coagulation.     

Structure of over lapping region of ICP18.5 and gB genes of type 1 bovine herpesvirus strain TK-A]

Nucleotide sequence of DNA fragment coding 3'-terminal of ICO 18.5 gene that overlaps the regulatory region and 5'-terminal of open reading frame of gB gene of bovine herpesvirus (BHV-1, subtype 1.3), strain TK-A, was determined. Comparative analysis of the sequence with the corresponding DNA regions of BHV-1, subtype 1.1, equine herpesviruses of the first and fourth types, and porcine pseudorabies virus was performed.