N-glycosylation of the premembrane protein of Japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles.

Premembrane (prM) and envelope (E) proteins, the major structural proteins of Japanese encephalitis virus (JEV) each contain single potential N-glycosylation site. In this study, the role of N-glycosylation of these proteins on their folding and activity were investigated. Three mutant prM and/or E (prM-E) genes lacking N-glycosylation sites were generated by site-directed mutagenesis. The effects of the N-glycan on folding, secretion and cytotoxicity of mutant proteins were determined by comparison with their wild type (wt) counterparts. Removal of N-glycan from the prM protein resulted in a complete misfolding of the E protein and failure to form virus-like particles (VLPs). A similar removal of N-glycan from the E protein led to a low efficiency of its folding and VLPs formation. The secretion and cytotoxicity of the E protein was also markedly impaired in case the glycosylation sites in the prM or E or both proteins were removed. These results suggest that the N-glycosylation of the prM protein is critical to the folding of the E protein, which makes it pivotal in the cytotoxicity of JEV particles and their production.

The rabies situation in Far East Asia.

This study evaluated rabies epidemiology in Far EastAsia. Questionnaires were sent by the OIE to Far East Asian countries and eight questionnaires were returned. Data were collected from these returns, as well as from recent publications, to gather information regarding rabies epidemiology in these countries. More than 29,000 human deaths were reported in 2006 in Far East Asia, representing more than 50% of all human rabies cases around the globe. There are only a few countries or regions from which no human rabies was reported in 2006 such as Japan, Singapore, South Korea, Malaysia, Hong Kong, and Taiwan. In many of these rabies endemic countries, the number of human rabies cases has not changed much during the past decade. The only country with a steady decline is Thailand, where the number of cases has decreased from around 200 to about 20 cases per year. The most dramatic changes were observed in China. Human rabies cases declined from around 5,000 cases per year in the 1980s to about 160 in the mid-1990s. However, these trends have since been reversed. A steady increase has been reported over the past 10 years with more than 3,200 cases reported in 2006. Although there are many factors that contribute to the epidemic or endemic nature of rabies in these countries, the single most important factor is the failure to immunize domestic dogs, which transmit rabies to humans. Dog vaccination is at or below 5% in many of these countries, and cannot stop the transmission of rabies from dogs to dogs, thus to humans. It is thus most importantforthese countries to initiate mass vaccination campaigns in dog populations in order to stop the occurrence of human rabies in Far East Asia.

Pathogenic rabies virus alters host protein expression in the central nervous system: implications for neuronal dysfunction.

Proteomics technology was employed to profile host responses to rabies virus (RABV) infection in order to understand how RABV infection results in neuronal dysfunction. In mice infected with wild-type (wt) RABV, the expression of proteins involved in ion homeostasis was altered. H+ ATPase and Na+/K+ ATPase were up-regulated while Ca2+ ATPase was downregulated, which resulted in reduction of intracellular Na+ and Ca2+ concentrations. Furthermore, infection with wt RABV resulted in down-regulation of SNAREs such as alpha-SNAP, TRIM9, syntaxin, and pallidin, all of which are involved in docking and fusion of synaptic vesicles to and with the presynaptic membrane. As a consequence, the accumulation of synaptic vesicles was observed in the presynapses of mice infected with wt RABV. These data demonstrate that infection with wt RABV results in the alteration of host protein expression, particularly those involved in ion homeostasis and docking and the fusion of synaptic vesicles to the presynaptic membrane, which may lead to neuronal dysfunction.

[Planned neck dissection in the treatment of locally advanced head and neck squamous cell carcinoma].

Objective: To investigate the value of planned neck dissection combined with induction chemotherapy and concurrent chemoradiotherapy in regional control and the outcome of locally advanced head and neck squamous cell carcinoma. Methods: A prospective randomized controlled study totally enrolled sixty-four patients of head and neck squamous cell carcinomas(include oropharynx, hypopharynx, and larynx) in stages Ⅳa-Ⅳb with lymph node metastase was were N2-N3. All patients firstly received 2-3 cycles of induction chemotherapy(ICT), then divided into two groups randomly, according to the efficacy of ICT. Group A(the study group) received planned neck dissection(PND) and concurrent chemoradiotherapy(CCRT). Group B(the control group) received concurrent chemoradiotherapy(CCRT). The differences in clinicopathologic features, local recurrence(LR), regional recurrence(RR), disease-free survival(DFS), and overall survival(OS) between the two groups were estimated. SPSS 19.0 software was used to analyze the data. Results: Group A enrolled twenty-one patients, and group B enrolled forty-three patients.The follow-up of all patients were 4-55 months, median follow-up time was 22 months. In study group, two-year OS and DFS were 80.9% and 68.3%, respectively. In control group, two-year OS and DFS were 90.7% and 67.1%, respectively. There was no significant difference in gender(P=0.215), age(P=0.828), primary tumor site(P=0.927), LR(P=0.126), DFS(P=0.710), and OS(P=0.402) between the two groups, while the RR(χ(2)=5.640, P<0.05) and distant metastasis(χ(2)=10.363, P<0.01) showed significant differences between the two groups. Conclusion: The ICT+ PND+ CCRT treatment model has benefit on regional control of locally advanced head and neck squamous cell carcinoma.

Genetic comparison of the rhabdoviruses from animals and plants.

There are more than 160 viral species in the Rhabdovidae family, most of which can be grouped into one of the six genera including Vesiculovirus, Lyssavirus, Ephemerovirus, Novirhabdovirus, Cytorhabdovirus, and Nucleorhabdovirus. These viruses are not only morphologically similar but also genetically related. Analysis of viral genes shows that rhabdoviruses are more closely related to each other than to viruses in other families. With the development of reverse genetics, the functions of many cis- and trans-elements important in the process of viral transcription and replication have been clearly defined such as the leader, trailer, and the intergenic sequences. Furthermore, it has been shown that there are two entry sites for the RNA-dependent RNA polymerase: 3' entry for leader synthesis and RNA replication, and direct entry at the N gene start sequence for transcription of the monocistronic mRNAs.

Production of a functional monoclonal antibody recognizing human colorectal carcinoma cells from a baculovirus expression system.

The light and heavy chain cDNA of a murine monoclonal antibody (MoAb) with specificity for human colorectal carcinoma cells have been expressed separately, together, and as a dual construct in insect cells infected with recombinant baculoviruses. High levels of the MoAb were expressed under the control of the polyhedrin promoter. The antibody maintained its specific binding to human colorectal carcinoma cells and mediated lysis of these cells by human lymphocytes, monocytes, and murine macrophages, as determined in antibody-directed cellular cytotoxicity (ADCC) assays. The recombinant immunoglobulin (Ig), like its ascitic counterpart, did not mediate lysis by either human or rabbit complement. The expression of a recombinant antibody exhibiting both functional binding site and Fc region capacities shows that the baculovirus system could be employed in the production of therapeutic Ig.

Detection of Borna disease virus-reactive antibodies from patients with affective disorders by western immunoblot technique.

Borna disease (BD) virus is a partially characterized neurotropic agent with a predilection for neurons and astrocytes in the limbic system and cerebrum of infected hosts. Although it usually causes a fatal encephalitis, some laboratory animals which have been experimentally inoculated can develop a persistent non-fatal infection characterized by a neuro-behavioral syndrome akin to human manic-depression. Using immunofluorescent techniques, we previously observed BD virus-specific antibodies in the sera of 4.5% of affectively ill patients, with the highest titers present in bipolar patients. More recently, we have developed a sensitive Western blot assay for the detection of anti-BD virus antibodies to a 38/40 kDa and 24 kDa protein in human serum. In the present study, we screened 138 affectively ill patients and 117 healthy controls and observed a significantly great proportion of patients with antibodies to the 38/40 kDa protein (P < 0.0001), the 24 kDa protein (P < 0.05) and both the 38/40 kDa and 24 kDa proteins (P < 0.025). These data extend prior reports on the presence of BD virus-specific antibodies in psychiatric patients, and suggest that a BD virus-like agent may be associated with affective illness in humans.

Group A rotavirus excretion patterns in naturally infected pigs.

Cross-sectional and cohort studies were conducted in a piggery to investigate the excretion pattern of group A rotavirus in pigs. The cross-sectional survey revealed that 47 (9 per cent) of 521 pigs sampled were excreting rotavirus in the faeces. No rotavirus antigen was detected in the faeces of pigs either less than one week or over two months old. The prevalence of infection increased with age over the sucking period, and was greatest at five weeks old. Diarrhoea was observed in only eight (17 per cent) of the pigs excreting rotavirus. Sixteen piglets from four litters were selected and faecal samples collected daily from each animal from birth to two months old. All the piglets excreted group A rotavirus and the range of ages at which they first became infected was between 13 and 39 days. The average duration of excretion in individual piglets was 7.4 days. Ten of 13 sucking piglets which excreted rotavirus developed diarrhoea soon after it was first detected.

Natural transmission of group A rotavirus within a pig population.

Five litters of piglets born within two days of each other, together with their dams, were investigated for faecal excretion of group A rotavirus antigen from birth to two months old. All the 50 piglets in these litters became infected with the virus between 19 and 35 days old. Rotavirus excretion was first seen in one litter which was housed with other litters not included in this study. Two days later, piglets of the second litter in another farrowing room began to excrete rotavirus, and then infection spread to the other three litters in the same room. Within each of these litters, one or two piglets were infected early and thereafter infection spread to other piglets. It took four to 10 days for rotavirus to infect every piglet within a litter, and 16 days in total before all piglets in the five litters were infected. No rotavirus antigen was demonstrated in faeces from sows during the investigation period.

Pre-weaning supplementary feed and porcine post-weaning diarrhoea.

Attempts were made to induce an intestinal hypersensitivity response to weaner diet by feeding pigs with small quantities of this material before weaning. In two trials using different weaner diets piglets subjected to this regimen showed no significant differences in small intestinal structure, in ability to absorb xylose, in bodyweight gain, in incidence of diarrhoea or excretion of enteropathogens after weaning compared with pigs not given any of the diet before weaning, or fed with a different diet before weaning. When post-weaning diarrhoea occurred it was associated with an earlier, more prolonged and greater proliferation of enterotoxigenic Escherichia coli in the small intestines than occurred in healthy pigs after weaning. The greater proliferation in pigs which developed diarrhoea could not be attributed either to an excessive dietary intake after weaning, or to a specific proliferation of rotaviruses.

Transfer of maternal antibody against group A rotavirus from sows to piglets and serological responses following natural infection.

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of antirotaviral antibody in sera and faeces from pigs and used to study the dynamics of antirotaviral antibody responses in three cohorts of pigs. Piglets acquired antirotaviral antibody by sucking their dams soon after birth. Antirotaviral antibodies of IgA and IgG classes were detected in both colostrum and milk of all sows tested but IgM class antibodies were not. The antibody levels in colostrum were eight to 32 times higher than those in milk which was collected 18 days post partum. The levels of antibody in piglets' sera were comparable to those in colostrum but declined quickly to low levels by one month old. Maternal antibody was also detected in the faeces of piglets up to 18 days old. Natural rotavirus infection occurred in each of these cohorts when the geometric mean ELISA titres of maternal antibody in their sera declined to 1/1600 (by days 21, 25 and 30 for cohorts 1, 2 and 3, respectively). However, a positive correlation was not obtained between the levels of antirotaviral antibody and protection in individual litters within each of the cohort groups. In each of the cohorts, rotavirus infection usually occurred in one or two piglets first and then spread to other piglets in the same cohort. It is therefore suggested that maternally derived antibody is protective against rotavirus infection in piglets only for the first one or two weeks. Following natural infection with rotavirus, increases in serum antibodies were detected in two of the three cohorts by 20 to 30 days after the average time of onset of faecal shedding of virus.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of human monoclonal antibodies to rabies.

A total of nine human monoclonal antibodies (MAbs) to rabies virus were generated from peripheral B lymphocytes of subjects immunized with human diploid cell rabies vaccine by somatic cell hybridization. The MAbs were analyzed for their antigen-binding specificities using ELISA, Western blot, and immunoprecipitation assays. The different assays made it possible to identify MAbs directed to the surface glycoprotein, nucleoprotein, nominal phosphoprotein, and matrix protein. One of the MAbs that recognized the surface glycoprotein neutralized rabies virus.

Near-work activity and myopia in rural and urban schoolchildren in China.

PURPOSE: To examine the prevalence of myopia in rural and urban schoolchildren in Xiamen, China, and to assess the impact of environmental factors on rates of myopia. METHODS: Second-grade children attending either a city (n=119) or rural (n=91) school in Xiamen, China, were examined using cycloplegic autorefraction and biometry. Detailed questions on socioeconomic status, near-work activity, reading and writing habits, and family histories of myopia were asked in a face-to-face interview using a standard questionnaire. RESULTS: The prevalence of myopia was 19.3% (95% confidence interval [CI], 12.3, 29) in the city and 6.6% (95% CI, 2.4, 14.3) in the countryside. The average hours per day children spent reading and writing outside of school was 2.2 hours in the city compared with 1.6 hours in the countryside (P<.0001). In both schools, the odds ratio for total reading and writing, adjusted for parental history of myopia, was 2.2 (95% CI, 1.1, 4). CONCLUSION: These data suggest the prevalence of myopia is higher in the city than in the countryside. One possible explanation for these different rates could be that schoolchildren in the city spend more time reading and writing outside of school compared with children in the countryside. Myopic children in both the city and the countryside spent more time reading and writing compared with nonmyopic children. This increased near-work activity may contribute to the prevalence of myopia.

Detection and survival of group A rotavirus in a piggery.

Samples of dust, faeces and effluent were collected from a piggery and examined for group A rotavirus, using a commercial ELISA test, electron microscopy and inoculation of MA-104 cells. Rotavirus antigen was demonstrated in samples collected from farrowing and weaner rooms but not from fattener and sow houses. Rotavirus antigen was also detected in samples collected from a weaner room which had been free of piglets for three months. A cytopathic porcine rotavirus (British isolate SW20/21) was kept at room temperature for four months; it survived with titres reduced by 2 log10. These observations suggest that the environment of commercial piggeries is an important source of rotaviral infection for young piglets.

Detection of antibodies against Borna disease virus in sera and cerebrospinal fluid of horses in the USA.

Sera from 295 horses in the USA were examined by an indirect immunofluorescence assay and Western blot assays to determine the prevalence of Borna disease virus infection. Eight (2.7 per cent) of the samples were positive in both assays, and 18 (6.1 per cent) were positive only in the Western blot assay. The indirect fluorescence titres ranged from 1:20 to 1:80 of antibodies recognising the virus-specific antigen from Borna disease virus-infected cells. The purified virus-specific proteins isolated from infected rat brains were recognised by positive equine serum samples after immunostaining by a Western blot technique. Information obtained from the owners about the history of the seropositive horses revealed that they were either clinically normal or had a pathological diagnosis of disease unrelated to Borna disease. This is the first report of the detection of antibodies to Borna disease virus in horses in the USA. The disease may be more widespread in a subclinical form, with very long incubation periods, and may not necessarily be restricted to historically endemic areas.

An enzyme-linked immunosorbent assay (ELISA) for measurement of antibodies against equine herpesvirus 2 in equine sera.

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of antibodies against equine herpesvirus type 2 (EHV-2) in equine sera. The optimal conditions of antigen concentration, and serum and conjugate dilutions were established by chequerboard titrations. When the standard ELISA test was used for titration of test sera, it was found to give titres approximately 1500 times higher than those obtained in the virus neutralization (VN) test, and a correlation coefficient of 0.815 was obtained between these two tests on 42 equine sera. All the positive serum samples by the VN were also positive by the ELISA, and one negative serum in the former test was found to be positive in the latter. Under field conditions, the test also detected increases in antibody titres against EHV-2 in 13 out of 14 foals soon after these animals excreted the virus.

Expression of the rabies virus glycoprotein in transgenic tomatoes.

We have engineered tomato plants (Lycopersicon esculentum Mill var. UC82b) to express a gene for the glycoprotein (G-protein), which coats the outer surface of the rabies virus. The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus, including the signal peptide, under the control of the 35S promoter of cauliflower mosaic virus. Plants were transformed by Agrobacterium tumefaciens-mediated transformation of cotyledons and tissue culture on selective media. PCR confirmed the presence of the G-protein gene in plants surviving selection. Northern blot analysis indicated that RNA of the appropriate molecular weight was produced in both leaves and fruit of the transgenic plants. The recombinant G-protein was immunoprecipitated and detected by Western blot from leaves and fruit using different antisera. The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 62 and 60 kDa as compared to the 66 kDa observed for G-protein from virus grown in BHK cells. Electron microscopy of leaf tissue using immunogold-labeling and antisera specific for rabies G-protein showed localization of the G-protein to the Golgi bodies, vesicles, plasmalemma and cell walls of vascular parenchyma cells. In light of our previous demonstration that orally administered rabies G-protein from the same ERA strain elicits protective immunity in animals, these transgenic plants should provide a valuable tool for the development of edible oral vaccines.