Contact investigation of a case of human novel coronavirus infection treated in a German hospital, October-November 2012.

On 24 October 2012, a patient with acute respiratory distress syndrome of unknown origin and symptom onset on 5 October was transferred from Qatar to a specialist lung clinic in Germany. Late diagnosis on 20 November of an infection with the novel Coronavirus (NCoV) resulted in potential exposure of a considerable number of healthcare workers. Using a questionnaire we asked 123 identified contacts (120 hospital and three out-of-hospital contacts) about exposure to the patient. Eighty-five contacts provided blood for a serological test using a two-stage approach with an initial immunofluorescence assay as screening test, followed by recombinant immunofluorescence assays and a NCoV-specific serum neutralisation test. Of 123 identified contacts nine had performed aerosol-generating procedures within the third or fourth week of illness, using personal protective equipment rarely or never, and two of these developed acute respiratory illness. Serology was negative for all nine. Further 76 hospital contacts also tested negative, including two sera initially reactive in the screening test. The contact investigation ruled out transmission to contacts after illness day 20. Our two-stage approach for serological testing may be used as a template for similar situations.

A conceptual evaluation of sustainable variable-rate agricultural residue removal.

Agricultural residues have near-term potential as a feedstock for bioenergy production, but their removal must be managed carefully to maintain soil health and productivity. Recent studies have shown that subfield scale variability in soil properties (e.g., slope, texture, and organic matter content) that affect grain yield significantly affect the amount of residue that can be sustainably removed from different areas within a single field. This modeling study examines the concept of variable-rate residue removal equipment that would be capable of on-the-fly residue removal rate adjustments ranging from 0 to 80%. Thirteen residue removal rates (0% and 25-80% in 5% increments) were simulated using a subfield scale integrated modeling framework that evaluates residue removal sustainability considering wind erosion, water erosion, and soil carbon constraints. Three Iowa fields with diverse soil, slope, and grain yield characteristics were examined and showed sustainable, variable-rate agricultural residue removal that averaged 2.35, 7.69, and 5.62 Mg ha, respectively. In contrast, the projected sustainable removal rates using rake and bale removal for the entire field averaged 0.0, 6.40, and 5.06 Mg ha, respectively. The modeling procedure also projected that variable-rate residue harvest would result in 100% of the land area in all three fields being managed in a sustainable manner, whereas Field 1 could not be sustainably managed using rake and bale removal, and only 83 and 62% of the land area in Fields 2 and 3 would be managed sustainably using a rake and bale operation for the entire field. In addition, it was found that residue removal adjustments of 40 to 65% are sufficient to collect 90% of the sustainably available agricultural residue.

Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction.

We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5­6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation.

MYCN/c-MYC-induced microRNAs repress coding gene networks associated with poor outcome in MYCN/c-MYC-activated tumors.

Increased activity of MYC protein-family members is a common feature in many cancers. Using neuroblastoma as a tumor model, we established a microRNA (miRNA) signature for activated MYCN/c-MYC signaling in two independent primary neuroblastoma tumor cohorts and provide evidence that c-MYC and MYCN have overlapping functions. On the basis of an integrated approach including miRNA and messenger RNA (mRNA) gene expression data we show that miRNA activation contributes to widespread mRNA repression, both in c-MYC- and MYCN-activated tumors. c-MYC/MYCN-induced miRNA activation was shown to be dependent on c-MYC/MYCN promoter binding as evidenced by chromatin immunoprecipitation. Finally, we show that pathways, repressed through c-MYC/MYCN miRNA activation, are highly correlated to tumor aggressiveness and are conserved across different tumor entities suggesting that c-MYC/MYCN activate a core set of miRNAs for cooperative repression of common transcriptional programs related to disease aggressiveness. Our results uncover a widespread correlation between miRNA activation and c-MYC/MYCN-mediated coding gene expression modulation and further substantiate the overlapping functions of c-MYC and MYCN in the process of tumorigenesis.

Evaluation of glycosylation and malonylation patterns in flavonoid glycosides during LC/MS/MS metabolite profiling.

Flavonoid conjugates constitute several classes of plant phenolic secondary metabolites including many isomeric compounds differing in the hydroxylation pattern and substitution of their rings with different groups such as alkyls, acyls or sugars. These compounds occur in plant tissues mainly as glycosides and in many cases it is necessary to have reliable and detailed information concerning the structure of these natural products. Our results were obtained using leaf extracts of Arabidopsis thaliana and Lupinus angustifolius in which different glycosides of flavones, flavonols and isoflavones are present. Analysis of collision-induced dissociation (CID)/MS/MS spectra of protonated [M + H](+), sodiated [M + Na](+) or deprotonated [M - H](-) molecules recorded during HPLC runs may bring needed information in this respect. However, registration of mass spectra of [M + Na](+) ions with a good efficiency is possible only after post-column addition of a sodium acetate solution to the LC column eluate. The retention of sodium cation on the saccharidic parts of the molecule is observed after the CID fragmentation. In many cases, the location of this cation on the glycan attached to C-3 hydroxyl group of flavonol led to assignment of its structure. Additionally, the determination of the structure of the aglycone and of the sequence of the glycan part was made possible through the CID data obtained from the [M + H](+) and [M - H](-) ions. CID spectra show a different order of sugar elimination from hydroxyl groups at C-3 and C-7 in flavonol glycosides isolated from A. thaliana leaves and give sufficient information to discriminate flavonoid O-diglycosides from flavonoid di-O-glycosides.

Epidemiological features of and public health response to a St. Louis encephalitis epidemic in Florida, 1990-1.

A St. Louis encephalitis (SLE) epidemic in Florida during 25 weeks in 1990-1, resulted in 222 laboratory-diagnosed cases, an attack rate in the 28 affected counties of 2.25/100,000. Disease risk rose with advanced age, to 17.14/100,000 in persons over 80 years, and all 14 fatal cases were in persons over 55 years (median, 70 years). Community serosurveys in Indian River County, the epicenter of the outbreak (attack rate 21/100,000), showed acute asymptomatic infections in 3.6% of the persons surveyed, with higher rates in persons with outdoor occupational exposure (7.4%) and in clients of a shelter for the indigent (13.3%). A matched case-control study found that evening outdoor exposure for more than 2 h was associated with an increased risk for acquiring illness (odds ratio [OR] 4.33, 95% CI 1.23-15.21) while a number of recommended personal protective measures were protective. Four SLE patients were dually infected with Highlands J virus, the first reported cases of acute infection with this alphavirus. The case-control study provided the first evidence that a public education campaign to reduce exposure had a protective effect against acquiring the disease.

Standardization of immunoglobulin M capture enzyme-linked immunosorbent assays for routine diagnosis of arboviral infections.

Immunoglobulin M antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) is a rapid and versatile diagnostic method that readily permits the combination of multiple assays. Test consolidation is especially important for arthropod-borne viruses (arboviruses) which belong to at least three virus families: the Togaviridae, Flaviviridae, and Bunyaviridae. Using prototype viruses from each of these families and a panel of well-characterized human sera, we have evaluated and standardized a combined MAC-ELISA capable of identifying virus infections caused by members of each virus family. Furthermore, by grouping antigens geographically and utilizing known serological cross-reactivities, we have reduced the number of antigens necessary for testing, while maintaining adequate detection sensitivity. We have determined that a 1:400 serum dilution is most appropriate for screening antiviral antibody, using a positive-to-negative ratio of >/=2.0 as a positive cutoff value. With a blind-coded human serum panel, this combined MAC-ELISA was shown to have test sensitivity and specificity that correlated well with those of other serological techniques.

Genotoxicity of p-nitrocinnamaldehyde and related alpha, beta-unsaturated carbonyl compounds in two bacterial assays.

Seventeen cinnamaldehydes, cinnamic acids, 2-furylacroleins and related compounds were tested in the Salmonella preincubation reversion assay and in the SOS chromotest. Of eight compounds containing nitrogroups, seven were clearly mutagenic in the presence of S9 mix and six in its absence; whereas none of the parent compounds not containing a nitrogroup and none of the congeners containing chlorine, methoxy or amino groups were mutagenic. Metabolic epoxidation was excluded in additional experiments using SKF525, an inhibitor of mono-oxygenases, and trichloropropene oxide, an inhibitor of epoxide hydrolases. Less or no mutagenicity was found in the nitroreductase deficient strains Salmonella typhimurium TA100NR or TA98NR and in the O-acetyltransferase deficient strains TA100/1,8-DNP6 or TA98/1,8-DNP6 except with 5-nitro-2-furylacrolein which exhibited decreased mutagenicity in TA100NR when compared with TA100 but the highest mutagenicity in TA100/1,8-DNP6. Less or no genotoxic activity was found in the SOS chromotest when using the nitroreductase deficient Escherichia coli strain PQ253 whereas all seven compounds tested were positive in strain PQ37. The results demonstrate the importance of the nitro group and that the compounds are activated either by bacterial nitroreductase or by the nitroreductase in the S9 mix. A chemical activation of the acrolein moiety by the negative inductive effect of the nitro group is unlikely. The genotoxicity of the cinnamyl compounds is dependent on the position of the nitro group in the phenyl ring. The genotoxicities of the p-nitro compounds were about two orders of magnitude higher than those of the ortho and meta congeners. The comparison between the Ames test and the SOS chromotest showed good agreement.

All that wheezes.

New onset wheezing in the young child can present an interesting differential diagnostic challenge, especially when there is an atypical presentation of a foreign body lodged in the airway. A thorough history and physical examination helps, but one must remember that a foreign body in the trachea or esophagus can masquerade as a respiratory illness. The chest x-ray is a useful part of the evaluation process. A high degree of suspicion is necessary on the part of the physician to remember that "all that wheezes is not asthma," even in the absence of a history of aspiration of a foreign body.

Western equine encephalitis in avian populations in North Dakota, 1975.

The involvement of wild birds in western equine encephalitis (WEE) and St. Louis encephalitis (SLE) virus activity in the Red River valley area of North Dakota (USA) during a WEE epidemic was investigated in August 1975. Free-ranging birds were captured with mist nets and nestlings by hand. Virologic and serologic results indicated that a similar rate of WEE virus activity occurred throughout Richland County and between permanent and summer resident birds. The rate of SLE virus activity in the birds of Richland County was lower than for WEE virus, but the SLE antibody prevalence was greater in rural areas than within urban locations. Seven of the nine WEE virus isolations were from nestling birds of four different species; the remaining two from adults of two different species. Overall prevalence of neutralizing (N) antibody against WEE virus was 5% in nestling and 14% in adult birds but was the opposite for N antibody against SLE virus, 17% in nestling and 5% in adult birds. Differences between the two viruses in the presence and persistence of maternal N antibody or differential mortality in nestling birds may have caused the disparity in antibody prevalences.

Borderline defenses and countertransference: research findings and implications.

Among Main's (1957) several cogent insights about the nature of defensive and countertransferential reactions to those so-called "special" patients who ungraciously refuse to improve - patients who in today's parlance would most assuredly be diagnosed as borderline - is his hypothesis that some of us may flee some of the time into research activities to avoid the frustrations and disappointments of clinical work. Writing from the dual perspectives of researchers and psychotherapists, and with the interest of furthering the integration of these often split-off enterprises, we offer some observations on the amassing body of empirical data about the borderline patient, observations which bear upon and lend support for Main's speculation regarding the detachment of researchers from the emotionally charged dilemmas facing the therapist in working with the borderline patient. We also present some findings from our ongoing investigations of borderline dynamics in groups and organizations, which underscore the role of defensive and countertransferential processes in the psychotherapeutic treatment of borderlines. Finally, based upon our observations of the extant data as well as our own findings, we offer a suggestion about the direction of future research on borderline pathology, proposing a shift away from the largely descriptive-level diagnostic studies and toward the investigation of the therapeutic relationship, with a particular focus on countertransferential dynamics.

Rapid and specific serodiagnosis of western equine encephalitis virus infection in horses.

Paired sera from 28 nonvaccinated horses with serologically confirmed western equine encephalitis (WEE) virus infections were evaluated for immunoglobulin (Ig)M and IgG directed against WEE virus, by use of enzyme immunoassay. Twenty-one of the horses developed greater than or equal to 4-fold increases or decreases in serum IgM titers in paired serum samples, confirming the diagnosis of WEE in these horses. Of the remaining 7 horses, 1 had stable IgM titers, 1 had a 2-fold increase in IgM titer between paired sera, 2 had 2-fold decreases in IgM titer, and for 3 horses adequate volumes were not available for both sera of the pair. Twenty-nine of 56 blood samples collected from these 28 horses had been collected within the first 3 days after clinical disease was recognized; all 28 horses and 48 of 53 available serum samples had IgM antibody to WEE virus. Immunoglobulin M also was detected in sera of 27 of 45 other nonvaccinated horses that had illnesses clinically compatible with WEE. Sera with IgM did not have cross-reacting IgM against eastern equine encephalitis virus. Therefore, the sensitivity, specificity, and lack of persistence of IgM was useful in the rapid diagnosis of WEE virus infections in horses.

Serodiagnosis of La Crosse virus infections in humans by detection of immunoglobulin M class antibodies.

Sera from 92 humans with illnesses clinically compatible with those caused by California serogroup virus infections were tested for antibody to La Crosse (LAC) virus by using the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC ELISA), the IgG ELISA, and the hemagglutination inhibition (HI), complement fixation and serum dilution-plaque reduction neutralization tests. On the reported day of onset of illness in 18 individuals, 94% had IgM antibody, 50% had neutralization antibody, 33% had HI antibody, and 11% had IgG antibody. Neutralization, HI, and IgG antibody prevalence rates increased thereafter, whereas IgM antibody prevalence remained high (92% 2 or more weeks after the onset of illness). It was concluded that the MAC ELISA is a sensitive test for the presence of antibody to LAC virus. The sensitivity of the MAC ELISA and the rapidity with which it can be performed appear to provide a powerful tool for the clinically relevant serodiagnosis of LAC virus infections in humans.

Distribution of Bunyamwera serogroup viruses in North America, 1956-1984.

We attempted to tabulate all Bunyamwera serogroup (family Bunyaviridae, genus Bunyavirus) isolates from North America. By summarizing information from the laboratories of the Centers for Disease Control, data generously shared by other laboratories, and the published literature, we were able to accumulate data regarding 1,372 Bunyamwera serogroup viruses. These were: Tensaw (664, including 8 from vertebrates), Cache Valley (396, including 6 from vertebrates), Main Drain (160, including 14 from vertebrates), Lokern (69, including 8 from vertebrates), Northway (13, including 5 from vertebrates), Tlacotalpan (7), Santa Rosa (2), Santa Cruz (1 from a horse), and 60 of undetermined serotype. Virus isolation rates by month of collection were correlated with collection efforts, but associations of viruses and arthropod vectors varied by location, vertebrate host, and arthropod distribution. Tensaw virus was isolated principally from Anopheles crucians mosquitoes (466/656 isolates from arthropods) in the southeastern United States; Cache Valley virus principally from An. quadrimaculatus (94), Coquillettidia perturbans (59), Culiseta inornata (45), Aedes sollicitans (30), Psorophora columbiae (23), An. punctipennis (18), and Ae. vexans and trivittatus (18 each) mosquitoes (total = 305/382 isolates from arthropods from all of the United States and Canada, except the southeastern United States); Main Drain virus from Culicoides variipennis (31), Culicoides (Selfia) sp. (65), and Psorophora (23) and Aedes (21) species mosquitoes in the western United States; Lokern virus from Culicoides species (55/61 isolates from arthropods) in the western United States. Relationships between vector and vertebrate host distributions are discussed briefly in regard to geographic distribution of the Bunyamwera serogroup viruses.

Specificity of immunoglobulin M and G antibody responses in humans infected with eastern and western equine encephalitis viruses: application to rapid serodiagnosis.

Paired sera from 20 humans with eastern equine encephalitis (EEE) virus infections and from 17 humans with western equine encephalitis (WEE) virus infections, all with previously demonstrated fourfold or greater rises or falls in hemagglutination-inhibiting, complement-fixing, or neutralizing antibody titers, were tested for immunoglobulin M (IgM) and IgG antibodies by an enzyme immunoassay. All individuals with EEE and 14 of 17 individuals with WEE had IgM antibody, some as early as 1 day after onset. Two of the three persons with WEE who did not develop IgM antibody died. IgM antibody declined but persisted for at least 3 months after the onset of illness in one individual each with EEE and WEE. IgG antibody was not detected until the middle of week 2 after onset. The sensitivity of the IgM antibody capture enzyme immunoassay described and the specificity, as shown by the absence of heterologous alphavirus reactivity, indicate that this is the test of choice for the rapid diagnosis of human infections caused by EEE and WEE viruses.

Sensitive enzyme immunoassay for detecting immunoglobulin M antibodies to Sindbis virus and further evidence that Pogosta disease is caused by a western equine encephalitis complex virus.

An antibody capture enzyme immunoassay (EIA) was adapted for the detection of immunoglobulin M (IgM) antibody to Sindbis (SIN) virus. Sera from humans with a febrile illness characterized by rash and arthralgia in eastern Finland (Pogosta [POG] disease) and Sweden (Ockelbo disease) and from humans with western equine encephalitis (WEE) virus infection in the United States were tested for IgM antibodies by EIA. Seroconversions were documented in patients with POG disease and with WEE virus infections by using SIN virus as antigen and rabbit anti-SIN virus immunoglobulin; this confirms previous observations that POG disease is caused by a virus closely related to SIN virus and that IgM antibodies to WEE complex alphaviruses are not type specific. This IgM EIA provided a sensitive diagnostic and research tool applicable to epidemiologic problems posed by POG disease.

Immunoglobulin M antibody capture enzyme-linked immunosorbent assay for diagnosis of St. Louis encephalitis.

Sera from patients with St. Louis encephalitis were tested with an immunoglobulin M (IgM) antibody capture enzyme immunoassay (MAC ELISA). The assay used five reagents: antihuman IgM, test serum, sucrose-acetone-extracted mouse brain antigen, broadly cross-reactive flavivirus monoclonal antibody conjugated to alkaline phosphatase, and substrate (p-nitrophenyl phosphate). MAC ELISA endpoint titers correlated (r = 0.893) with the absorbance value of a 1:100 dilution of patient serum. Significant (fourfold or greater) changes in the endpoint titers between paired sera corresponded to a critical ratio (ratio of absorbance values at the 1:100 dilution) of greater than or equal to 1.3. IgM antibodies were detected in 71% of patients bled at 0 to 3 days after the onset of illness, in 99% bled at 4 to 21 days, and in 91% bled at 22 to 67 days. Thereafter, the IgM seropositivity rate declined; however, 29% of sera were still positive at 115 to 251 days after the onset of illness. MAC ELISA titers were significantly correlated with hemagglutination inhibition (r = 0.258) and neutralization (r = 0.711) titers. Because IgM antibodies appeared early and waned rapidly, a diagnosis was made on the basis of a decrease in titer more often by MAC ELISA than by hemagglutination inhibition, complement fixation, or neutralization tests. IgM antibodies generally showed a high degree of specificity; heterologous cross-reactions were, however, present in 4 of 14 sera examined. The MAC ELISA is useful for the rapid, early diagnosis of St. Louis encephalitis.

Antigenic relationships among Turlock serogroup Bunyaviruses as determined by neutralization tests.

Antigenic relationships between the five recognized Turlock serogroup viruses (family Bunyaviridae, genus Bunyavirus) were determined by serum dilution-plaque reduction neutralization tests. Results indicated that Turlock , Umbre , M' Poko and Lednice viruses are distinct from each other and that Yaba -1 virus is a subtype of M' Poko virus.