Characterization of a broadly reactive monoclonal antibody against norovirus genogroups I and II: recognition of a novel conformational epitope

Norovirus, which belongs to the family Caliciviridae, is one of the major causes of nonbacterial acute gastroenteritis in the world. The main human noroviruses are of genogroup I (GI) and genogroup II (GII), which were subdivided further into at least 15 and 18 genotypes (GI/1 to GI/15 and GII/1 to GII/18), respectively. The development of immunological diagnosis for norovirus had been hindered by the antigen specificity of the polyclonal antibody. Therefore, several laboratories have produced broadly reactive monoclonal antibodies, which recognize the linear GI and GII cross-reactive epitopes or the conformational GI-specific epitope. In this study, we characterized the novel monoclonal antibody 14-1 (MAb14-1) for further development of the rapid immunochromatography test. Our results demonstrated that MAb14-1 could recognize 15 recombinant virus-like particles (GI/1, 4, 8, and 11 and GII/1 to 7 and 12 to 15) and showed weak affinity to the virus-like particle of GI/3. This recognition range is the broadest of the existing monoclonal antibodies. The epitope for MAb14-1 was identified by fragment, sequence, structural, and mutational analyses. Both terminal antigenic regions (amino acid positions 418 to 426 and 526 to 534) on the C-terminal P1 domain formed the conformational epitope and were in the proximity of the insertion region (positions 427 to 525). These regions contained six amino acids responsible for antigenicity that were conserved among genogroup(s), genus, and Caliciviridae. This epitope mapping explained the broad reactivity and different titers among GI and GII. To our knowledge, we are the first group to identify the GI and GII cross-reactive monoclonal antibody, which recognizes the novel conformational epitope. From these data, MAb14-1 could be used further to develop immunochromatography.     

An Economic Evaluation of Neonatal Screening for Inborn Errors of Metabolism Using Tandem Mass Spectrometry in Thailand

Background

Inborn errors of metabolism (IEM) are a rare group of genetic diseases which can lead to several serious long-term complications in newborns. In order to address these issues as early as possible, a process called tandem mass spectrometry (MS/MS) can be used as it allows for rapid and simultaneous detection of the diseases. This analysis was performed to determine whether newborn screening by MS/MS is cost-effective in Thailand.

Method

A cost-utility analysis comprising a decision-tree and Markov model was used to estimate the cost in Thai baht (THB) and health outcomes in life-years (LYs) and quality-adjusted life year (QALYs) presented as an incremental cost-effectiveness ratio (ICER). The results were also adjusted to international dollars (I$) using purchasing power parities (PPP) (1 I$ = 17.79 THB for the year 2013). The comparisons were between 1) an expanded neonatal screening programme using MS/MS screening for six prioritised diseases: phenylketonuria (PKU); isovaleric acidemia (IVA); methylmalonic acidemia (MMA); propionic acidemia (PA); maple syrup urine disease (MSUD); and multiple carboxylase deficiency (MCD); and 2) the current practice that is existing PKU screening. A comparison of the outcome and cost of treatment before and after clinical presentations were also analysed to illustrate the potential benefit of early treatment for affected children. A budget impact analysis was conducted to illustrate the cost of implementing the programme for 10 years.

Results

The ICER of neonatal screening using MS/MS amounted to 1,043,331 THB per QALY gained (58,647 I$ per QALY gained). The potential benefits of early detection compared with late detection yielded significant results for PKU, IVA, MSUD, and MCD patients. The budget impact analysis indicated that the implementation cost of the programme was expected at approximately 2,700 million THB (152 million I$) over 10 years.

Conclusion

At the current ceiling threshold, neonatal screening using MS/MS in the Thai context is not cost-effective. However, the treatment of patients who were detected early for PKU, IVA, MSUD, and MCD, are considered favourable. The budget impact analysis suggests that the implementation of the programme will incur considerable expenses under limited resources. A long-term epidemiological study on the incidence of IEM in Thailand is strongly recommended to ascertain the magnitude of problem.     

Genome-wide analyses of human noroviruses provide insights on evolutionary dynamics and evidence of coexisting viral populations evolving under recombination constraints

Norovirus is a major cause of acute gastroenteritis worldwide. Over 30 different genotypes, mostly from genogroup I (GI) and II (GII), have been shown to infect humans. Despite three decades of genome sequencing, our understanding of the role of genomic diversification across continents and time is incomplete. To close the spatiotemporal gap of genomic information of human noroviruses, we conducted a large-scale genome-wide analyses that included the nearly full-length sequencing of 281 archival viruses circulating since the 1970s in over 10 countries from four continents, with a major emphasis on norovirus genotypes that are currently underrepresented in public genome databases. We provided new genome information for 24 distinct genotypes, including the oldest genome information from 12 norovirus genotypes. Analyses of this new genomic information, together with those publicly available, showed that (i) noroviruses evolve at similar rates across genomic regions and genotypes; (ii) emerging viruses evolved from transiently-circulating intermediate viruses; (iii) diversifying selection on the VP1 protein was recorded in genotypes with multiple variants; (iv) non-structural proteins showed a similar branching on their phylogenetic trees; and (v) contrary to the current understanding, there are restrictions on the ability to recombine different genomic regions, which results in co-circulating populations of viruses evolving independently in human communities. This study provides a comprehensive genetic analysis of diverse norovirus genotypes and the role of non-structural proteins on viral diversification, shedding new light on the mechanisms of norovirus evolution and transmission.     

A 5,7-Dimethoxyflavone/Hydroxypropyl-β-Cyclodextrin Inclusion Complex with Anti-Butyrylcholinesterase Activity

This study aimed to improve the water solubility of 5,7-dimethoxyflavone (5,7-DMF) isolated from Kaempferia parviflora by complexation with 2-hydroxypropyl-β-cyclodextrin (HPβ-CD). The phase solubility profile of 5,7-DMF in the presence of HPβ-CD was classified as A_L-type and indicated a 1:1 mole ratio. Differential scanning colorimetry, X-ray diffraction, NMR and SEM analyses supported the formation of a 5,7-DMF/HPβ-CD inclusion complex involving the A ring of 5,7-DMF inside the HPβ-CD cavity. This is the first example of CD inclusion with the A ring of non-hydroxyl flavones. The stability and binding constants of the complexes were determined using the phase solubility and UV-vis absorption spectroscopy, respectively. The water solubility of 5,7-DMF was increased 361.8-fold by complexation with HPβ-CD and overcame the precipitation problem observed in aqueous buffers, such as during in vitro anti-butyrylcholinesterase activity assays. The 1:1 mole ratio of the 5,7-DMF/HPβ-CD complex showed a 2.7-fold higher butyrylcholinesterase inhibitory activity (in terms of the IC₅₀ value) compared to the non-complexed compound.     

Coumarins from the heartwoods of Mansonia gagei Drumm

Three coumarins and three known mansonones were isolated from the heartwood of Mansonia gagei Drumm. The structures of the three coumarins were elucidated as 3,8-dimethyl-5-isopropyl-6-methoxycoumarin (mansonrin A) 3,8-dimethyl-5-isopropyl-6-hydroxycoumarin (mansorin B) and 2,3-dihydro-3,6,9-trimethyl naphtho[1,8-bc]pyran-7-oxa-8-one (mansorin C) by analyses of physical properties and spectroscopic data. The cytotoxicity of the isolated compounds against brine shrimp Artemia salina Linn. was also evaluated.     

Calicivirus

Caliciviruses represented by norovirus and sapovirus exist not only in human but also in other animal species. Clinical manifestations are gastroenteritis, respiratory infections, vesicles and hemorrhagic skin diseases and others symptoms depended on the viruses. Inapparent symptom of calicivirus infection is also recognized. Calicivirus is stable in the environment and found sometimes in contaminated food or water sources. In addition to intragenomic mutation, intragenomic recombination is the common phenomenon that usually found in calicivirus genome. The genomic recombinations have been reported among the strains within the same animal species. For diagnosis and molecular epidemiological study, several laboratory methods are available, such as genetic molecular analysis, enzyme immunoassay and immunochromatography, which developed by using the antibody against virus-like particles. The reactivity between virus and host immunity is type specific and the titer of cross reaction is not so high. There are evidences that the new variant strains are emerged and spread quickly year by year. Histo-blood group antigen (HBGA) is one of the specific host cells receptor for calicivirus. Infectivity of the virus depends on specificity of the HBGA on the host cells. Because of the inability to culture human norovirus and sapovirus, pathogenesis and immunological data are limited. So far, only feline calicivirus and mouse norovirus are cultivable. Animal model studies for calicivirus by gnotobiotic pigs with human calicivirus and mouse with mouse norovirus are mainly used for experiments of pathobiological study, treatment and vaccine development.     

Coumarins from the heartwoods of Mansonia gagei Drumm.

Three coumarins and three known mansonones were isolated from the heartwood of Mansonia gagei Drumm. The structures of the three coumarins were elucidated as 3,8-dimethyl-5-isopropyl-6-methoxycoumarin (mansonrin A) 3,8-dimethyl-5-isopropyl-6-hydroxycoumarin (mansorin B) and 2,3-dihydro-3,6,9-trimethyl naphtho[1,8-bc]pyran-7-oxa-8-one (mansorin C) by analyses of physical properties and spectroscopic data. The cytotoxicity of the isolated compounds against brine shrimp Artemia salina Linn. was also evaluated.     

Screening of plants containing Naja naja siamensis cobra venom inhibitory activity using modified ELISA technique

Enzyme-linked immunosorbent assay (ELISA) has been modified for screening plants with antagonistic activity to Naja naja siamensis cobra venom. Aqueous extracts from plants were investigated for their inhibitory effects on the binding of anti-cobra venom antibody to antigen, cobra venom, fixed onto 96-well microtiter plates. Ingredients in extracts were allowed to react with immobilized venom before the subsequent addition of antivenom antibody. Venom components affected by exposure to the extracts, unable to interact with their specific antibody, were predicted to be unable to bind to their native destinations or natural receptors. Curcuma cf. zedoaria, an old Thai medicinal plant, showed clear inhibitory activity in the ELISA test. Neurotoxin and protein degradative enzymes, major components in venom, were identified as targets of this extract in Western immunoblotting analysis. Ingredients in the extract showed high affinity to the toxin in competition assay by immunoprecipitation. The extract attenuated toxin activity by extending contraction time of diaphragm muscle after envenomation and had a potency to protect cellular proteins from venom degradative enzymes. Curcuma parviflora, with less activity in ELISA, exhibited acceptable results in two experiments but negative results in two experiments, whereas Curcuma longa, having low activity in the ELISA test, never showed any favorable results. Screening of 36 samples could classify plants into an inhibition range of 0 to 86%. This modified ELISA is recommended as a preliminary screening method for inhibitors with a large number of samples.